Patent Publication Number: US-11639888-B2

Title: Microfluidic system and method with focused energy apparatus

Description:
CROSS-REFERENCES TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 17/496,469 filed Oct. 7, 2021 and of U.S. patent application Ser. No. 17/496,614 filed Oct. 7, 2021, both of which are continuations of U.S. patent application Ser. No. 16/279,430, filed Feb. 19, 2019, which is a continuation-in-part of U.S. patent application Ser. No. 15/387,034, filed Dec. 21, 2016, now abandoned, which is a continuation of U.S. patent application Ser. No. 15/033,001, filed Aug. 24, 2016, now U.S. Pat. No. 10,928,298, which is a 371 of International Application PCT/IB2014/001425, filed Jun. 18, 2014, which claims priority to U.S. Provisional Patent Application No. 61/897,743, filed Oct. 30, 2013, the specifications of which are herein incorporated by reference in their entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to a microfluidic system with an interrogation apparatus that detects and interrogates objects in a sample fluid mixture of a microfluidic chip, and a focused energy apparatus that performs an action that affects the objects. In one embodiment, the interrogation apparatus interrogates the objects to determine their identity, and the focused energy apparatus is an apparatus that acts on target objects. In one embodiment, the focused energy apparatus is used to damage, kill, alter, disable, or destroy the targeted objects. 
     BACKGROUND OF THE INVENTION 
     In the separation of various particles or cellular materials—for example, the separation of sperm into viable and motile sperm from non-viable or non-motile sperm, or separation by gender—the process is often a time-consuming task, with severe volume restrictions. Thus, current separation techniques cannot, for example, produce the desired yield, or process volumes of cellular materials in a timely fashion. 
     Photo-damaging laser systems have utilized lasers to photodamage or kill undesired cellular objects. However, the prior art has required flow cytometers using nozzles, to interrogate and arrange the individual objects in droplet flow, and to attempt to separate and photodamage the objects as they fall into various containers—which has been difficult to achieve. 
     Thus, there exists a present need for a method and apparatus which identifies and discriminates between target objects, is continuous, has high throughput, is time and cost-effective, and causes negligible or minimal damage to the various target objects. In addition, such an apparatus and method should have further applicability to other biological and medical areas, not just in sperm discrimination; but in the discrimination of blood and other cellular materials, including viral, cell organelle, globular structures, colloidal suspensions, and other biological materials. 
     BRIEF SUMMARY OF THE INVENTION 
     The present invention relates to a microfluidic system with an interrogation apparatus that detects and interrogates objects in a sample fluid mixture of a microfluidic chip, and a focused energy apparatus that performs an action that affects the objects. In one embodiment, the interrogation apparatus interrogates the objects to determine their identity, and the focused energy apparatus is an apparatus that acts on target objects. In one embodiment, the focused energy apparatus is used to damage, kill, alter, disable, or destroy the targeted objects. 
     In one embodiment, an apparatus that identifies objects includes: a microfluidic chip in which are disposed a plurality of channels; including; a main fluid channel into which a sample fluid mixture of objects to be identified is introduced; a plurality of sheath fluid channels into which sheath fluids are introduced, the sheath fluids which orient the objects in the main fluid channel in a predetermined direction while still maintaining laminar flow in the main fluid channel; an interrogation apparatus which detects and interrogates the oriented objects in the main fluid channel; and a focused energy apparatus which performs an action on the objects. 
     In one embodiment, the interrogation apparatus detects and interrogates the objects to determine information about the objects. 
     In one embodiment, the information about the objects determines whether the objects are targeted by the focused energy apparatus. 
     In one embodiment, the action of the focused energy apparatus acts on the targeted objects or a region surrounding the targeted objects. 
     In one embodiment, the action on the targeted objects is to damage, disable, alter, kill or destroy the targeted objects. 
     In one embodiment, the apparatus further includes at least one output channel leading from the main fluid channel, the at least one output channel which removes the objects from the microfluidic chip. 
     In one embodiment, the at least one output channel removes both targeted and non-targeted objects from the microfluidic chip. 
     In one embodiment, the apparatus further includes a plurality of side output channels leading from the main fluid channel, the plurality of side output channels disposed on either side of the at least one output channel, the plurality of side output channels which remove the sheath fluids from the microfluidic chip. 
     In one embodiment, the plurality of sheath fluid channels includes: a first plurality of sheath fluid channels which intersect the main fluid channel at a first intersection, such that the sheath fluids compress the sample fluid mixture on at least two sides, such that the sample fluid mixture becomes a relatively smaller, narrower stream, bounded by the sheath fluids while maintaining laminar flow in the main fluid channel. 
     In one embodiment, the plurality of sheath fluid channels further includes: a second plurality of sheath fluid channels which intersect the main fluid channel at a second intersection downstream from the first intersection, such that the sheath fluids from the second plurality of sheath fluid channels compress the sample fluid mixture in one of the at least two sides, or in two sides opposite from the at least two sides, such that the sample fluid mixture is further compressed while still maintaining laminar flow in the main fluid channel. 
     In one embodiment, when the second set of sheath fluid channels compresses the sample fluid mixture from the at least two sides, the plurality of sheath fluid channels further comprises: a third sheath fluid channel disposed vertical to the main fluid channel at a third intersection, and disposed downstream from the second intersection, the sheath fluid from the third sheath fluid channel which further compresses the sample fluid while still maintaining laminar flow in the main fluid channel. 
     In one embodiment, the plurality of sheath fluid channels hydrodynamically focuses the objects such that the objects are oriented in a predetermined direction and disposed in a restricted core volume as the objects flow through the main fluid channel. 
     In one embodiment, the apparatus further includes an action chamber in which the interrogation apparatus interrogates the hydrodynamically focused objects in the sample fluid mixture, the action chamber disposed in the microfluidic chip downstream from at least one of the second intersection or the third intersection. 
     In one embodiment, the interrogation apparatus includes; a light source that emits a light beam into the action chamber, to illuminate and excite the objects in the sample fluid mixture. 
     In one embodiment, the light beam excites fluorescence in the objects such that the targeted objects are distinguished from the non-targeted objects. 
     In one embodiment, the light source is a laser. 
     In one embodiment, the apparatus further includes an optical signal detector which detects the light beam and converts it into an electronic signal; and a controller, which analyzes the electronic signal to determine whether the objects are to be targeted or non-targeted. 
     In one embodiment, the focused energy apparatus is a laser. 
     In one embodiment, the microfluidic chip contains one or more structural layers or planes. 
     In one embodiment, the main fluid channel is disposed in a different structural layer or plane from the plurality of sheath channels. 
     In one embodiment, the at least one of the sample input channel and the plurality of sheath channels are disposed in-between the structural layers or the planes of the microfluidic chip. 
     In one embodiment, the first plurality of sheath channels is disposed in a different structural layer or plane from the second plurality of sheath channels. 
     In one embodiment, the action chamber includes a first opening cut through at least one of the structural layers or the planes in the microfluidic chip, the first opening which is configured to receive a first transparent covering. 
     In one embodiment, the action chamber includes a second opening cut through the at least one of the structural layers or the planes on an opposite side of the microfluidic chip from the first opening, the second opening which is configured to receive a second transparent covering. 
     In one embodiment, the microfluidic chip contains at least one functional layer which includes the plurality of sheath fluid channels and the main fluid channel, and a top layer that contains holes to access the at least one functional layer. 
     In one embodiment, a size of one of the second plurality of sheath fluid channels is different from another of the second plurality of sheath channels. 
     In one embodiment, the size of the second plurality of sheath channels is different from a size of the first plurality of sheath channels. 
     In one embodiment, the apparatus further includes a first output disposed at an end of the at least one output channel. 
     In one embodiment, the apparatus further includes a plurality of outputs disposed at an end of each of the plurality of side output channels. 
     In one embodiment, the apparatus further includes at least one notch disposed in the microfluidic chip, the at least one notch provided between outputs. 
     In one embodiment, a size of the plurality of side output channels increases from a size of the main fluid channel. 
     In one embodiment, the main fluid channel tapers at an entry point into the first intersection in the microfluidic chip. 
     In one embodiment, the main fluid channel tapers into the action chamber. 
     In one embodiment, the second plurality of sheath channels tapers before joining the main fluid channel. 
     In one embodiment, the second plurality of sheath channels includes at least a first vertical portion which joins the main fluid channel from approximately a right angle above the main fluid channel. 
     In one embodiment, the second plurality of sheath channels includes a second vertical portion which joins the main fluid channel from approximately a right angle below the main fluid channel. 
     In one embodiment, the internal ramps are disposed in at least the main fluid channel prior to the first intersection. 
     In one embodiment, the internal ramps are disposed in the main fluid channel prior to the second intersection. 
     In one embodiment, the internal ramps are disposed in at least one of the second plurality of sheath channels. 
     In one embodiment, the objects are cells. 
     In one embodiment, the cells to be acted upon by the focused energy apparatus include at least one of viable or motile sperm from non-viable or non-motile sperm or sperm discriminated by gender or other sex discrimination variations. 
     In one embodiment, the cells to be acted upon by the focused energy apparatus include: stem cells discriminated from cells in a population; one or more labeled cells discriminated from unlabeled cells; cells discriminated by desirable or undesirable traits; cells discriminated based on surface markers; cells discriminated based on membrane integrity or viability; cells having genes which are discriminated in nuclear DNA according to a specified characteristic; cells discriminated based on potential or predicted reproductive status; cells discriminated based on an ability to survive freezing; cells discriminated from contaminants or debris; healthy cells discriminated from damaged cells; red blood cells discriminated from white blood cells and platelets in a plasma mixture, or any cells discriminated from any other cellular objects into corresponding fractions. 
     In one embodiment, the laser is one of a 349 or 355 nm pulsed laser. 
     In one embodiment, the laser is a pulsed Q-switch laser able to deliver 15 ns or shorter energy pulses to the objects at a rate of over 1,000 pulses per second. 
     In one embodiment, the laser is a 532 nm laser. 
     In one embodiment, the pulsed Q-switch laser preferably delivers 10 ns energy pulses to the objects at a rate of over 200,000 pulses per second. 
     In one embodiment, the focused energy apparatus acts upon the objects a predetermined amount of time after the interrogation of the objects. 
     In one embodiment, the focused energy apparatus acts upon the objects prior to interrogation of the objects by the light source. 
     In one embodiment, the focused energy apparatus acts upon the objects when the objects leave the at least one output prior to being collected in a container. 
     In one embodiment, the apparatus further includes a container that collects both the targeted and the non-targeted objects. 
     In one embodiment, the apparatus further includes a pumping apparatus that pumps at least one of the sample fluid mixture or the plurality of sheath fluids into the microfluidic chip. 
     In one embodiment, the pumping apparatus pumps the at least one of the sample fluid mixture or the plurality of sheath fluids into the microfluidic chip using external tubing. 
     In one embodiment, the apparatus further includes: at least one external reservoir which holds at least one of the sample fluid mixture or the plurality of sheath fluids. 
     In one embodiment, the apparatus further includes: a microfluidic chip holder on which the microfluidic chip is mounted, the microfluidic chip holder which includes openings through which the external tubing accesses the microfluidic chip from the at least one external reservoir. 
     In one embodiment, the apparatus further includes: a controller which controls the pumping of the at least one of the sample fluid mixture or the plurality of sheath fluids into the microfluidic chip. 
     In one embodiment, the apparatus further includes a plurality of microfluidic chips disposed in parallel, the plurality of microfluidic chips containing a plurality of sample fluid mixtures; wherein a single interrogation apparatus is used for each of the plurality of microfluidic chips. 
     In one embodiment, a computer system identifies objects, including: at least one memory which contains at least one program which includes the steps of: controlling a flow of a sample fluid mixture containing objects to be identified, through a main fluid channel of a microfluidic chip; controlling an introduction of a plurality of sheath fluid channels into the microfluidic chip, the plurality of sheath fluids which orient the objects in the main fluid channel in a predetermined direction while still maintaining laminar flow in the main fluid channel; and analyzing an interrogation of the oriented objects in the main fluid channel using an interrogation apparatus; and controlling an action on the objects using a focused energy apparatus; and a processor which executes the program. 
     In one embodiment, a non-transitory computer-readable medium containing instructions to identify objects includes: controlling a flow of a sample fluid mixture containing objects to be identified, through a main fluid channel of a microfluidic chip; controlling an introduction of a plurality of sheath fluid channels into the microfluidic chip, the plurality of sheath fluids which orient the objects in the main fluid channel in a predetermined direction while still maintaining laminar flow in the main fluid channel; analyzing an interrogation of the oriented objects in the main fluid channel using an interrogation apparatus; and controlling an action on the objects using a focused energy apparatus. 
     In one embodiment, an apparatus that identifies objects includes: a microfluidic chip in which are disposed a plurality of channels, including: a main fluid channel into which a sample fluid mixture of objects to be identified is introduced; and a plurality of sheath flow channels which perform at least a three-step hydrodynamic focusing process on the objects, such that the objects are oriented in a predetermined direction as the objects flow through the main fluid channel. 
     In one embodiment, the plurality of sheath fluid channels includes: a first plurality of sheath fluid channels which intersect the main fluid channel at a first intersection to accomplish a first step of the at least three hydrodynamic focusing steps, such that the sheath fluids compress the sample fluid mixture on at least two sides, such that the sample fluid mixture becomes a relatively smaller, narrower stream, bounded by the sheath fluids while maintaining laminar flow in the main fluid channel. 
     In one embodiment, the plurality of sheath fluid channels further includes: a second plurality of sheath fluid channels which intersect the main fluid channel at a second intersection downstream from the first intersection to accomplish a second step of the at least three hydrodynamic focusing steps, such that the sheath fluids from the second plurality of sheath fluid channels further compress the sample fluid mixture in the at least two sides, such that the sample fluid mixture is further compressed while still maintaining laminar flow in the main fluid channel. 
     In one embodiment, the plurality of sheath fluids further includes: a third sheath fluid channel disposed vertical to the main fluid channel at a third intersection, and disposed downstream from the second intersection to accomplish a third step of said at least three hydrodynamic focusing steps, the sheath fluid from the third sheath fluid channel which compresses the sample fluid while still maintaining laminar flow in the main fluid channel. 
     In one embodiment, the apparatus further includes: an interrogation apparatus that detects and interrogates the oriented objects in the main fluid channel; and a focused energy apparatus that performs an action on the objects. 
     In one embodiment, a method of identifying objects flowing in a sample fluid mixture includes: flowing a sample fluid mixture containing objects to be identified, through a main fluid channel of a microfluidic chip; introducing a plurality of sheath fluid channels into the microfluidic chip, the plurality of sheath fluids which orient the objects in the main fluid channel in a predetermined direction while still maintaining laminar flow in the main fluid channel; interrogating the oriented objects in the main fluid channel using an interrogation apparatus; and using a focused energy apparatus on the objects. 
     Thus has been outlined, some features consistent with the present invention so that the detailed description thereof that follows may be better understood, and so that the present contribution to the art may be better appreciated. There are, of course, additional features consistent with the present invention that will be described below and which will form the subject matter of the claims appended hereto. 
     In this respect, before explaining at least one embodiment consistent with the present invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangements of the objects set forth in the following description or illustrated in the drawings. Methods and apparatuses consistent with the present invention are capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract included below, are for the purpose of description and should not be regarded as limiting. 
     As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods, and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the methods and apparatuses consistent with the present invention. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) 
       The objects, features, and advantages of the present invention will be more readily appreciated upon reference to the following disclosure when considered in conjunction with the accompanying drawings, in which: 
         FIG.  1 A  shows an exploded perspective view of an illustrative embodiment of a single layer microfluidic chip with a top “blank” layer, according to one embodiment consistent with the present invention. 
         FIG.  1 B  (a) shows an exploded perspective view of an illustrative embodiment of a two-layer microfluidic chip, with the functional layers on the top of the bottom layer, and on the underside of the top layer, according to yet another embodiment consistent with the present invention. 
         FIG.  1 B  (b) shows a top view of the bottom layer and the underside of the top layer of the embodiment shown in  FIG.  1 B  (a). 
         FIG.  1 C  ( a ) shows an exploded perspective view of an illustrative embodiment of a three-layer microfluidic chip with three functional layers, the top and middle layers having the functional portions on the underside of the layers, according to yet another embodiment consistent with the present invention. 
         FIG.  1 C  ( b ) shows the three-layer illustrative embodiment of  FIG.  1 C  (a), from the opposite perspective, with the layers flipped over, showing the underside, functional layers of the top and middle layers. 
         FIG.  1 D  shows an exploded perspective view of an illustrative embodiment of a four-layer microfluidic chip with the top layer being a “blank” layer, according to yet another embodiment consistent with the present invention. 
         FIG.  2    shows a top view of an illustrative embodiment of a microfluidic chip, according to one embodiment consistent with the present invention. 
         FIG.  3 A  shows a perspective view of the sample, and sheath or buffer channels in a two (functional) layer microfluidic chip, according to one embodiment consistent with the present invention. 
         FIG.  3 B  shows a perspective view of the sample, and sheath or buffer channels in a single layer microfluidic chip, according to another embodiment consistent with the present invention. 
         FIG.  4 A  shows a perspective view of the sample channel, with a taper and internal ramp, entering the intersection of the first hydrodynamic focusing region according to one embodiment consistent with the present invention. 
         FIG.  4 B  shows a perspective view of the main channel, with a taper and internal ramp, entering the second hydrodynamic focusing region, according to one embodiment consistent with the present invention. 
         FIG.  4 C  shows a ramp feature in the sample channel prior to the action chamber, according to one embodiment consistent with the present invention. 
         FIG.  5    shows a cross-sectional internal view of an illustrative interrogation by a light source, of objects flowing in a fluid mixture through the main channel of the microfluidic chip system of  FIG.  1 A , according to one embodiment consistent with the present invention. 
         FIG.  6 A  shows a slanted, schematic side view of the flow of objects in the main channel of a microfluidic chip system, from interrogation to discrimination, with activation of the focused energy device after interrogation, according to one embodiment consistent with the present invention. 
         FIG.  6 B  shows a slanted, schematic side view of the flow of objects in the main channel of a microfluidic chip system, from interrogation to discrimination, with activation of the focused energy device at output channel exit, according to another embodiment consistent with the present invention. 
         FIG.  6 C  shows a slanted, schematic side view of the flow of objects in the main channel of a microfluidic chip system, from interrogation to discrimination, with activation of the focused energy device on a disconnected droplet between output channel exit and collection, according to another embodiment consistent with the present invention. 
         FIG.  6 D  shows a slanted, schematic side view of the flow of objects in the main channel of a microfluidic chip system, from interrogation to discrimination, with activation of the focused energy device prior to interrogation, according to another embodiment consistent with the present invention. 
         FIG.  7    shows a perspective view of the shear force gradient across the microfluidic main channel, according to one embodiment consistent with the present invention. 
         FIG.  8 A  shows a cross-sectional view of the main channel of a microfluidic chip, after the first-step hydrodynamic focusing, with the sample fluid compressed on the sides of the main channel, and the objects offset from the central portion of the main channel. 
         FIG.  8 B  shows a cross-sectional view of the main channel of a microfluidic chip, after second-step hydrodynamic focusing, with the sample fluid compressed from above and below the main channel, such that the objects are substantially centrally located in the main channel. 
         FIG.  9    shows a cross-section of the main channel of the microfluidic chip, with an object in the center thereof, according to one embodiment consistent with the present invention. 
         FIG.  10    is a diagram showing a flow velocity profile across the main channel of a microfluidic chip, according to one embodiment consistent with the present invention. 
         FIG.  11    shows a cross-section of the main channel of a microfluidic chip, with an object offset from the center, according to one embodiment consistent with the present invention. 
         FIG.  12 A  shows the second hydrodynamic focusing step, where sheath or buffer channels parallel the sample channel of a microfluidic chip, from above and below, and enter the sample channel from vertical directions, according to one embodiment consistent with the present invention, 
         FIG.  12 B  shows the second hydrodynamic focusing step of  FIG.  12 A , with one sheath or buffer channel of a microfluidic chip, being smaller than the other channel, according to another embodiment consistent with the present invention. 
         FIG.  13 A  shows a histogram of sperm cells in a center of the main channel of a microfluidic chip, according to one embodiment consistent with the present invention. 
         FIG.  13 B  shows a histogram of sperm cells offset from a center of the main channel of a microfluidic chip, according to one embodiment consistent with the present invention. 
         FIG.  14    shows a perspective, internal and oblique view of objects flowing through the microfluidic chip, and an illustrative operation of two-step hydrodynamic focusing, with a single sheath or buffer fluids entering from an upper portion of the main channel, according to one embodiment consistent with the present invention. 
         FIG.  15    shows a perspective view of the microfluidic chip, and an illustrative operation of two-step hydrodynamic focusing, with sheath or buffer fluid channels entering vertically from both an upper and a lower portion of the sample channel, according to one embodiment consistent with the present invention. 
         FIG.  16    shows a slanted, side schematic view of the microfluidic chip system with interrogation and discrimination apparatus, and implementation of the beam optics from opposite sides of the sample channel, with the collection optics shared with the focused energy device optics, according to one embodiment consistent with the present invention. 
         FIG.  17    shows a slanted, side schematic view of the microfluidic chip system with interrogation and discrimination apparatus, and implementation of the beam optics from a same side of the sample channel, with the interrogation and focused energy beams combined through the same, or separate optics, according to one embodiment consistent with the present invention. 
         FIG.  18    shows a schematic view of multiple microfluidic chip systems disposed in parallel, using a single interrogation apparatus, according to one embodiment consistent with the present invention. 
         FIG.  19    shows a schematic view of the flow control network of the microfluidic chip system, with external individual reservoirs for sample and sheath or buffer fluids, according to one embodiment consistent with the present invention. 
         FIG.  20    shows a schematic view of the flow control network of the microfluidic chip system, with an external single sheath or buffer reservoir and sample reservoir, according to one embodiment consistent with the present invention. 
         FIG.  21    shows a schematic view of a pressure-regulated network of the microfluidic chip system with a single sheath or buffer reservoir, according to one embodiment consistent with the present invention. 
         FIGS.  22 A and  223    show the front and back, respectively, of a microfluidic chip holder having three ports for fluids to a functional multilayer microfluidic chip, according to one embodiment consistent with the present invention, 
         FIGS.  23 A and  233    show the front and back, respectively, of a microfluidic chip holder having four ports for fluids to a single layer microfluidic chip, according to one embodiment consistent with the present invention. 
         FIG.  24    shows the total DNA comparison between a female sperm cell and a male sperm cell. 
         FIG.  25    shows staining of DNA in cells to determine the intensity of emitted light which correlates with the amount of DNA in a cell, wherein 3.8% more light emitted correlates to 3.8% more DNA in female sperm cells. 
         FIG.  26    shows the excitation wavelength and the emission wavelength for cells stained with Hoescht 33342. 
         FIG.  27    shows a multiple station setup to analyze cells with a vanguard laser. 
         FIG.  28    shows beam splitting occurring for multiple stations. 
         FIG.  29    shows that the Stoke shift and optical filter create a peak at 450 nm wavelength—therefore setting our detection wavelength. 
         FIG.  30 A  shows the knife blade shape, which requires tight focusing of a cell stream. 
         FIG.  30 B  shows that a kill pulse is triggered based on a timer when a cell is detected. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Before turning to the figures, which illustrate the illustrative embodiments in detail, it should be understood that the present disclosure is not limited to the details or methodology set forth in the description or illustrated in the figures. It should also be understood that the terminology is for the purpose of description only and should not be regarded as limiting. An effort has been made to use the same or like reference numbers throughout the drawings to refer to the same or like parts. 
     The present invention relates to a microfluidic chip with an interrogation apparatus that detects and interrogates objects in a sample fluid mixture, and a focused energy apparatus that performs an action on the objects or a region around the objects. In one embodiment, the interrogation apparatus interrogates the objects to identify the objects and to determine whether the objects should be targeted by the focused energy apparatus. In one embodiment, the targeted objects are unwanted targeted objects. 
     In one embodiment, the focused energy apparatus is a discrimination apparatus that discriminates between targeted and non-targeted objects by damaging, killing, altering; disabling, or destroying the targeted objects. The present invention is conducted in a flowing, continuous fluid stream within the microfluidic network, where objects are subject to hydrodynamic focusing, positioning, and orientation, and non-targeted objects are allowed to flow through the microfluidic chip undisturbed, and targeted objects may be acted upon, including photodamaged, killed, altered, disabled, or destroyed, by a focused energy apparatus. 
     Applications F: The various embodiments of the present invention provide for the selection of objects in a fluid mixture, such as, for example: selecting viable or motile sperm from non-viable or non-motile sperm; selecting sperm by gender, and other sex selection variations; selecting stems cells from cells in a population; selecting one or more labeled cells from un-labeled cells distinguishing desirable/undesirable traits; selecting cells for desirable characteristics; selecting genes in nuclear DNA in cells, according to a specified characteristic; selecting cells based on surface markers; selecting cells based on membrane integrity (viability), potential or predicted reproductive status (fertility), ability to survive freezing, etc.; selecting cells from contaminants or debris; selecting healthy cells from damaged cells (i.e., cancerous cells) (as in bone marrow extractions); red blood cells from white blood cells and platelets in a plasma mixture; and selecting any cells from any other cellular objects, into corresponding fractions; selecting damaged cells, or contaminants or debris, or any other biological materials that are desired to discriminated. The objects may be cells or beads treated or coated with linker molecules or embedded with a fluorescent or luminescent label molecule(s). The objects may have a variety of physical or chemical attributes, such as size, shape, materials, texture, etc. 
     In one embodiment, a heterogeneous population of objects may be measured, with each object being examined for different quantities or regimes in similar quantities (e.g., multiplexed measurements), or the objects may be examined and distinguished based on a label (e.g., fluorescent), image (due to size, shape, different absorption, scattering, fluorescence, luminescence characteristics, fluorescence or luminescence emission profiles, fluorescent or luminescent decay lifetime), and/or particle position, etc. 
     In addition, the subject matter of the present disclosure is also suitable for other medical applications as well. For example, the various laminar flows discussed below may be utilized as part of a kidney dialysis process, in which whole blood is cleansed of waste products and returned to the patient. Further, the various embodiments of the present disclosure may have further applicability to other biological or medical areas, such as for selection of cells, viruses, bacteria, cellular organelles or subparts, globular structures, colloidal suspensions, lipids and lipid globules, gels, immiscible particles, blastomeres, aggregations of cells, microorganisms, and other biological materials. For example, the object selection in accordance with the present disclosure may include cell “washing”, in which contaminants (such as bacteria) are removed from cellular suspensions, which may be particularly useful in medical and food industry applications. Further, the present invention has the applicability to select non-motile cellular objects from motile cellular objects. 
     The subject matter of the present disclosure may also be utilized to transfer a species from one solution to another solution where separation by filtering or centrifugation is not practical or desirable. In addition to the applications discussed above, additional applications include selecting colloids of a given size from colloids of other sizes (for research or commercial applications), and washing particles such as cells, egg cells, etc. (effectively replacing the medium in which they are contained and removing contaminants), or washing particles such as nanotubes from a solution of salts and surfactants with a different salt concentration or without surfactants, for example. 
     The action of selecting species may rely on a number of physical properties of the objects or objects including self-motility, self-diffusivity, free-fall velocity, or action under an external force, such as an actuator, an electromagnetic field, or a holographic optical trap. The properties which may be selected include, for example, cell motility, cell viability, object size, object mass, object density, the tendency of objects to attract or repel one another or other objects in the flow, object charge, object surface chemistry, and the tendency of certain other objects (i.e., molecules) to adhere to the object. 
     While discussion below focuses on the identification and selection of viable or motile sperm from non-viable or non-motile sperm, or selecting sperm by gender and other sex selection variations, or selecting one or more labeled cells from un-labeled cells distinguishing desirable/undesirable traits, etc., the apparatus, methods, and systems of the present invention may be extended to other types of particulate, biological or cellular matter, which are capable of being interrogated by fluorescence techniques within a fluid flow, or which are capable of being manipulated between different fluid flows into one or more outputs. 
     Sample Preparation: In one embodiment, a concentration of objects  160 , such as cells (i.e., raw semen), is determined using a cell counting device, such as a Nucleocounter. In one embodiment, the appropriate staining volume is obtained (i.e., using staining a calculator worksheet), and the volume of staining TALP and objects  160  (i.e., neat semen) that need to be added for a predetermined cell concentration (i.e., 200×106/ml sperm concentration), are calculated. For example, a 1 ml amount of stained sample is prepared when the neat semen concentration=1500×106/ml. Thus, 200×106/ml/1500×106/ml=0.133 ml neat semen, which is added to (in order) 0.012 ml Hoechst 33342 (5 mg/ml stock solution), and 0.855 staining TALP (pH 7.4), to equal 1 ml total staining volume at 200×106/ml. 
     In one embodiment, staining TALP is prepared by filling a container (i.e., beaker) with Milli-Q water to ⅔ of the total desired volume. A stir bar and stir plate are used to mix the solution as chemicals are added. The chemicals, which are added in the order listed (up to the Gentamicin, which is added later), include: 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Chemical components of staining TALP 
               
            
           
           
               
               
               
               
               
            
               
                 CHEMICAL 
                 FORMULA 
                 g/100 ml 
                 g/500 ml 
                 g/1000 ml 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 HEPES 
                 C 6 H 12 N 2 O 4 S 
                 0.952 
                 4.760 
                 9.520 
               
               
                 Magnesium 
                 MgCl 2 *6H 2 O 
                 0.008 
                 0.040 
                 0.080 
               
               
                 Chloride 6- 
               
               
                 Hydrate, crystal 
               
               
                 Sodium 
                 NaCl 
                 0.5518 
                 2.759 
                 5.518 
               
               
                 Chloride 
               
               
                 Potassium 
                 KCl 
                 0.0224 
                 0.116 
                 0.224 
               
               
                 Chloride 
               
               
                 Sodium 
                 Na 2 HPO 4   
                 0.004 
                 0.020 
                 0.040 
               
               
                 Phosphate 
               
               
                 Dibasic/ 
               
               
                 Anhydrous 
               
               
                 Sodium 
                 NaHCO 3   
                 0.084 
                 0.420 
                 0.840 
               
               
                 Bicarbonate 
               
               
                 Pyruvic Acid 
                 Na Pyruvate 
                 0.022 
                 0.110 
                 0.220 
               
               
                 Glucose 
                 C 6 H 12 O 6   
                 0.090 
                 0.450 
                 0.900 
               
               
                 Lactic Acid, 
                 Na Lactate 
                 0.361 
                 1.805 
                 3.610 
               
               
                 60% Syrup (ml) 
               
               
                 Bovine Serum 
                 BSA 
                 0.300 
                 1.500 
                 3.000 
               
               
                 Albumin 
               
               
                 Gentamicin 
                   
                 0.25 ml 
                 1.25 ml 
                 2.50 ml 
               
               
                 Solution 
               
               
                 (10 mg/ml) 
               
               
                 AFTER 
               
               
                 FILTRATION 
               
               
                   
               
            
           
         
       
     
     In one embodiment, after adequate mixing of the chemicals, the pH is adjusted to 7.4 using NaOH. Additional Milli-Q water is used to bring the solution to a final volume in a container (i.e., volumetric flask). A sterile filter (i.e., 0.22 sterile filter) is used to filter/sterilize the volume. An antibiotic (i.e., Gentamicin solution) is added after filtration, and the volume of staining TALP is stored at 5° C. and can be used for 7-10 days. 
     Thus, after the volume of sample is stained with staining TALP, in one embodiment, the stained samples  120  are placed in containers (i.e., tubes) into a water bath set at 34-35 CC, and incubated for a predetermined time (i.e., 45 minutes). In one embodiment, after incubation, the stained samples are removed from the water bath, and an equal volume of 4.0% egg-yolk TALP with red food dye that has been warmed in the water bath set to 34-35° C., is added. 
     To obtain 4% egg yolk TALP with red food dye, the staining TALP as noted above is prepared, and a desired volume of final solution is determined. The volume of staining TALP and egg yolk required to prepare a 4% egg yolk solution is calculated as follows: 
     The desired volume: (250 ml)×0.04=10 ml egg yolk needed for 4% solution. 240 ml of staining TALP is added to a graduated container (i.e., cylinder), and 10 ml of egg yolk is added. FD&amp;C #40 food dye is added to the container (i.e., cylinder), to obtain: 0.261 ml/100 ml of solution. With a desired 250 ml total volume, 0.261 ml×250 ml/100=0.653 ml of red food dye. The container (i.e., cylinder) is covered with parafilm and carefully inverted until the volume is thoroughly mixed. The volume container is then allowed to sit overnight and cooled in a cool room. The volume container is then carefully decanted into a sterile container, leaving any sediment at the bottom of the volume container. The volume is then filtered/sterilized through a 0.22 .tm bottle top filter, and an appropriate amount of antibiotic solution is added (i.e., 0.250 ml Gentamicin/100 ml of egg yolk TALP). 
     Thus, after the addition of an equal volume of 4% egg-yolk TALP with red food dye to the stained sample  120 , the stained samples  120  are filtered by pouring the samples  120  into a 20-micron filter (i.e., CellTrics filter), that drains into another sterile 5 ml culture container. After a predetermined time of staining (i.e., 45 minutes), an equal volume of 4% egg yolk TALP is added. The stained sample  120  (i.e., cells) are run through a filter (i.e., Partec filter, with 50-micron mesh), and the sample is placed into a sample holder or reservoir  233  for introduction into the microfluidic chip  100  (see  FIGS.  19 - 21   ). 
     In one embodiment, the final sperm concentration=100×106/ml, and the final egg-yolk percentage=2%. A new sample aliquot  120  can be prepared and used every hour if desired. 
     Microfluidic Chip System: The various embodiments of the microfluidic chip, as described below, utilize one or more flow channels, having a plurality of substantially laminar flows, allowing one or more objects to be interrogated for identification by an interrogation apparatus, and to be acted upon by a focused energy apparatus, with the objects exiting the microfluidic chip into one or more outputs. In one embodiment, the objects not targeted by the focused energy apparatus are undisturbed, and the focused energy apparatus photodamages, alters, disables, kills, or destroys targeted objects. 
     The various embodiments of the present invention thereby provide a selection of objects on a continuous basis, such as, within a continuous, closed system without the potential damage and contamination of prior art methods, particularly as provided in sperm separation. The continuous process of the present invention also provides significant time savings in selecting and discriminating objects. 
     While the present subject matter is discussed in detail with respect to a microfluidic chip  100  illustrated in  FIGS.  1 A- 2    and a microfluidic chip holder  200  illustrated in  FIGS.  20 - 21   , it should be understood that this discussion applies equally to the various other embodiments discussed herein or any variation thereof. 
     Microfluidic Chip:  FIG.  1 A  is an illustrative embodiment of a microfluidic chip  100 . The microfluidic chip  100  is manufactured of a suitable material such as glass, or a thermoplastic (e.g., low auto-fluorescing polymer, etc.), or combination of materials, through an embossing process, soft photolithography, or injection molding process, as well known to one of ordinary skill in the art, and is of suitable size. Each layer may be any suitable thickness, for example, the thickness may be within a range of approximately 300-400 μm, and more preferably the thickness may be approximately 400 μm. 
     The microfluidic chip  100  includes one or more structural layers in which are disposed micro-channels which serve as sample input channel(s), sheath or buffer fluid channel(s), output channel(s), etc. The micro-channels are of suitable size to accommodate laminar flow streams containing objects, and may be disposed in any of the layers of the chip  100  in the appropriate length, as long as the object of the present invention is realized. In one embodiment, the dimensions of the microfluidic channels range from 50 microns to 500 microns, with 100-300 microns being preferably used to avoid clogging. 
     The desired flow rate through the microfluidic chip  100  may be controlled by a predetermined introduction flow rate into the chip  100 , maintaining the appropriate micro-channel dimensions within the chip  100 , by pumping mechanisms that pump external fluids into the chip  100 , and by providing narrowing or tapering of the micro-channels at various locations, and/or by providing obstacles, ramps, or dividers within the micro-channels (further discussed below). 
     More specifically, a plurality of inputs is provided into the microfluidic chip  100 , which inputs provide access to the micro-channels/channels. In one embodiment, as shown in  FIGS.  1 A- 2   , a sample input  106  is used for introducing a sample of particles or objects (i.e., cells)  160  in a sample fluid mixture  120  into a main fluid channel  164  of the microfluidic chip  100  from at least one reservoir source (see  FIG.  19   ). 
     The microfluidic chip  100  also includes at least one sheath or buffer input for the introduction of sheath or buffer fluids. In one embodiment, there are two sheath or buffer inputs in the microfluidic chip  100 , which include a sheath or buffer input  107  and sheath or buffer input  108 , both disposed proximate to the sample input  106 , and which both introduce sheath or buffer fluids  163  into the microfluidic chip  100  (see  FIGS.  1 A- 3 B ). 
     In one embodiment, there are three sheath or buffer inputs  107 ,  108 , and  172  (see  FIGS.  1 A and  3 B ) which introduce sheath or buffer fluids into the channel  164  of the microfluidic chip  100 . The location of the sheath or buffer inputs  107 ,  108 ,  172  may vary, and they may access channels in the chip  100  which are in the same or different structural layers. In one embodiment, the sheath or buffer fluids  163  are introduced into inputs  107 ,  108 ,  172  from a common reservoir (see  FIGS.  20 - 21   ), or in another embodiment, from separate reservoirs (see  FIG.  19   ). 
     The sheath or buffer fluids are well known in the art of microfluidics, and in one embodiment, may contain nutrients well known in the art to maintain the viability of the objects  160  (i.e., sperm cells) in the fluid mixture. Commercially available Tris, as sold by Chata Biosystems, is one example, and the sheath or buffer fluid  163  may be formulated to include the following: Water—0.9712 L; Tris—23.88 gg; citric acid monohydrate—11.63 g: D-fructose—8.55 g. The pH is adjusted to 6.80±0.05 with hydrochloric acid, and osmolarity is adjusted, if necessary, to 270-276 mOsm with fructose high purity. The mixture is filtered using a 0.22-micron filter. 
     The microfluidic chip  100  may have one or more structural layers in which the micro-channels are disposed. The channels may be disposed in one or more layers or in-between layers. The following embodiments describe a bonding process, but one of ordinary skill in the art would know how to achieve the various features by using an injection molding process. For example, in injection molding, instead of forming two layers, two molds could be made and joined together, such that an injection is made into the cavity in order to obtain the chip of the present invention. 
     In one embodiment, as shown in  FIG.  1 A , one structural layer  101  is included in the microfluidic chip, with a top, “blank” plastic layer  104  disposed thereon. The top, “blank” layer  104  bonds with the functional layer  101  to form an enclosed microfluidic network, and may have multiple holes to provide access to the lower layer(s) of the chip  100 . For example, the top “blank” layer  104  may have holes corresponding to inputs  106 ,  107 ,  108 ,  172 , etc., or provide holes  145  for pins to secure the layers  101 ,  102 ,  104 , etc., of the chip  100  together. In one embodiment, the top layer  104  of the microfluidic chip  100  includes a plurality of apertures configured to align with the fittings on a microfluidic chip holder  200  (further described below). 
     In another embodiment, as shown in  FIG.  1    B (a), two functional, structural plastic layers  101 - 102  are included in the microfluidic chip  100 , with no top, “blank” layer. In this embodiment, the functional side of the top layer  102  is disposed on the underside of the layer  102 , so that when the layers are put together, the channels  114 ,  115 ,  116 ,  117  are formed (see  FIG.  1    B (b). 
     In yet another embodiment, as shown in  FIG.  1 C  ( a ), three functional, structural layers  101 ,  102 ,  103  are used in the microfluidic chip  100 . As with  FIG.  1 B  (a), layers  102 ,  103  include functional sides on the undersides of the layers  102 ,  103 , with layers  101  and  102  forming channels  116 ,  117  when put together. Layer  103  has channels  114 ,  115  disposed on the underside of the layer  103  (see  FIG.  1 C  ( b )). 
     In yet another embodiment, as shown in  FIG.  1 D , four structural plastic layers  101 - 103  and a top, “blank” layer  104 , are used in the microfluidic chip  100 . In this embodiment, layer  102  includes  114 ,  115 , and layers  101  and  103  each include one of channels  117 ,  116 , respectively. 
     However, one of ordinary skill in the art would know that more or fewer structural layers with functional sides, and with or without “blank” layers, may be used, and the channels may be disposed in any of the structural layers, or in different structural layers, and in any arrangement, with access to those channels through a top “blank” layer, as long as the object of the present invention is achieved. 
     In one embodiment, a sample fluid mixture  120  including objects  160 , is introduced into sample input  106 , and the fluid mixture  120  flows through main channel  164  toward action chamber  129  (see  FIGS.  1 A- 2   ). The sheath or buffer fluids  163  are introduced into sheath or buffer inputs  107 ,  108  (see  FIGS.  1 A- 2   ) in most embodiments, and into sheath or buffer inputs  107 ,  108 , and  172  in another embodiment (see  FIG.  1 A ). The sheath or buffer fluids  163  flow-through channels  114 ,  115  and  116 ,  117 , into the main channel  164 , and towards the action chamber  129  before flowing out through at least output channels  140  and  142 , in laminar flow. 
     In one embodiment, the fluid mixture  120  from main channel  164  joins with the sheath or buffer fluids  163  from channels  114 ,  115  at intersection  161  of the microfluidic chip  100 . In one embodiment, buffer fluids  163  from channels  116 ,  117  join the combined fluid mixture  120  and sheath or buffer fluids  163  from first intersection  161 , downstream at second intersection  162  (see  FIGS.  1 A- 2   ). In one embodiment, sheath or buffer fluids  163  are inputted via input  172  into main fluid channel  164 , downstream from the second intersection  162  (see  FIGS.  1 A and  3 B ). 
     In one embodiment, channels  114 ,  115  are substantially the same dimensions as channels  116 ,  117 , as long as the desired flow rate(s) is achieved to accomplish the object of the present invention, but one of ordinary skill in the art would know that the dimensions may be different as long as they accomplish the desired results (further discussion below). 
     In one embodiment, channels  114 - 117  and  140 - 142  may have substantially the same dimensions, however, one of ordinary skill in the art would know that the size of any or all of the channels in the microfluidic chip  100  may vary in dimension (for example, between 50 and 500 microns), as long as the desired flow rate(s) is achieved to accomplish the object of the present invention. In one exemplary embodiment, the channels  114 ,  115  or  116 ,  117  are disposed in the same structural layer or plane of the microfluidic chip  100  as the layer or plane in which the channel  164  is disposed (see  FIG.  1 A , for example), or may be disposed in a different structural layer or plane (see  FIG.  1 B , for example). In another embodiment, the input channel  164  and the sheath channels  114 ,  115  or  116 ,  117 , may be disposed in-between structural layers or planes of the chip  100 . Thus, one of ordinary skill in the art would know that the channels  114 - 117 ,  164 , and  140 - 142 , etc., can be disposed in any layer or between any two layers. Further, although the channels  114 - 117 ,  164 , and  140 - 142 , etc. are described in exemplary embodiments as shown in the Figures, one of ordinary skill in the art would know that the particular arrangement or layout of the channels on the chip  100  may be in any desired arrangement as long as they achieve the described features of the present invention. 
     In one embodiment, the channels  116 ,  117  are cut through layer  101  (see  FIGS.  1 A and  2   ), and join the fluid mixture  120  in channel  164  in the same plane, via holes cut through the layers. In one embodiment, channels  116 ,  117  substantially parallel input channel  164 , and each join intersection  161  at an angle from channel  164  (see  FIGS.  2 - 3   ). The sheath or buffer fluids from channels  116 ,  117  compress the fluid mixture  120  flow horizontally from the sides, or laterally, such that the objects  160  in the fluid mixture  120  are flattened and/or oriented in a selected or desired direction, while still maintaining laminar flow in channel  164  (i.e., the first in two steps of hydrodynamic focusing, as described further below). 
     Further, in one embodiment, channels  114 ,  115  join the fluid mixture  120  in channel  164  at intersection  162 , with each channel  114 ,  115  at an angle from channel  164  (see  FIG.  1 A ). The sheath or buffer fluids from channels  114 ,  115  compress the fluid mixture  120  flow with respect to channel  164  (see  FIG.  14   ), such that the objects  160  in the fluid mixture  120  are further flattened and/or oriented in the selected or desired direction, while still maintaining laminar flow in channel  164  (i.e., the second in two steps of hydrodynamic focusing, as described further below). 
     Further to this embodiment, a third sheath or buffer fluid input  172  is disposed downstream from intersection  162  (see  FIG.  3 B ), which allows a third hydrodynamic focusing step to take place, where the sample fluid mixture  120  is compressed from above the channel  164  by the sheath or buffer fluids  163  introduced therein. 
     In an alternative embodiment, after the first hydrodynamic focusing step described above, the channels  114 ,  115  join the fluid mixture  120  in channel  164  at intersection  162  from an angle from above (see  FIG.  3 A )—and may be above and below (see  FIG.  12 A )—channel  164 , to compress the fluid mixture  120  from a vertical direction to further flatten and/or orient the objects  160  in the channel  164  (i.e., the second hydrodynamic focusing step). 
     However, one of ordinary skill in the art would appreciate that the depicted configurations, angles, and structural arrangements of the microfluidic chip  100  sheath or buffer inputs, sample input, and sample input channel and sheath or buffer channels, as well as the hydrodynamic focusing steps, may be different as long as they achieve the desired features of the present invention. 
     In one embodiment, as shown in  FIG.  2   , channels  114 ,  115 , and  116 ,  117  are depicted as partially coaxial to one another with a center point defined by the sample input  106 . Thus, in one embodiment, channels  114 ,  115 , and  116 ,  117  are disposed in a substantially parallel arrangement, with the channels  114 ,  115 , and  116 ,  117  being equidistant to main channel  164 . However, one of ordinary skill in the art would recognize that the depicted configuration may be different as long as it achieves the desired features of the present invention. 
     In one embodiment, holes and pins/posts  145  are disposed at various convenient positions in the layers  101 ,  102 ,  103 ,  104 , etc., to fix and align the multiple layers during chip  100  fabrication. 
     In one embodiment, a gasket  105  of any desired shape, or O-rings, may be provided to maintain a tight seal between the microfluidic chip  100  and the microfluidic chip holder  200  (see  FIGS.  1 D and  21 - 22   , for example). In the case of a gasket  105 , it may be a single sheet or a plurality of objects, in any configuration, or material (i.e., rubber, silicone, etc.) as desired. In one embodiment, as shown in  FIG.  1 D , a first gasket  105  is disposed at one end of the microfluidic chip  100  and interfaces or is bonded with layer  104 . A plurality of holes  144  are provided in the first gasket  105  and are configured to align with the sample input  106 , sheath/buffer input  107 , and sheath/buffer input  108 . 
     In one embodiment, a second gasket  143  may be disposed at another end of the microfluidic chip  100  opposite to the first gasket  105  (see  FIG.  1 D , for example), and interfaces or is bonded with (using epoxy) the top structural layer  104  (see  FIGS.  1 D and  21 - 22   ). 
     In one embodiment, O-rings are used instead of gaskets, to assist in sealing, as well as stabilizing the microfluidic chip  100  in the chip holder  200 . 
     However, one of ordinary skill in the art would know that one or more gaskets or O-rings may be applied to the outer layers of the chip  100  in order to protect the chip  100  in a chip holder  200 , during operation thereof. 
     In one embodiment, the channels  114 - 117 , and  140 - 142 , of the microfluidic chip  100 , may not just vary in dimension but may have tapered shapes at entry points to other channels in the chip  100  in order to control the flow of fluid through the channels. For example, main channel  164  may taper at the entry point into intersection  161  (see  FIG.  4 A , taper  166 A), or at the entry point into intersection  162  (see  FIG.  4 B , taper  166 A) to control and speed up the flow of sample  120  into the intersection  161 , and allow the sheath or buffer fluids  163  from channels  116 ,  117  or  114 ,  115 , respectively, to compress the sample fluid mixture  120  in a first direction (i.e., horizontally or laterally) on at least two sides, if not all sides (depending on where the fluid channel  164  enters the intersection  161 ), and in a second direction (i.e., vertically) (see  FIGS.  3 A- 3 B,  4 A- 43   , and tapers  166 A). 
     In another embodiment, ramps may be disposed in channel  164  or channels  114 - 117  to achieve the effect of controlling and speeding up the sample flow through the channels. The ramps may be in addition or instead of tapers. 
     For example, a ramp  1663  may be disposed in channel  164  prior to the sample flow approaching intersections  161  and  162 , respectively, or prior to entering action chamber  129  (see  FIGS.  4 A and  4 B ). 
     Thus, the sample fluid mixture  120  becomes a relatively smaller, narrower stream, bounded or surrounded by sheath or buffer fluids  163 , while maintaining laminar flow in channel  164 . However, one of ordinary skill in the art would know that the main channel  164 , or the buffer channels  114 - 117  may be of any physical arrangement, such as a rectangular or circular-shaped channel, with tapers, ramps, or other internal features, as long as the object of the present invention is obtained. 
     In one embodiment, a plurality of output channels stemming from main channel  164  (see  FIG.  2   ) is provided for removal of fluid flowed through the microfluidic chip  100 , including any targeted or non-targeted objects  160  and/or sheath or buffer fluids  163 . In one embodiment as shown in  FIGS.  1 A- 2   , there are three output channels  140 - 142  which include a left side output channel  140 , a center output channel  141 , and a right side output channel  142 . The left side output channel  140  ends at a first output  111 , the center output channel  141  ends at a second output  112 , and the right side output channel  142  ends at a third output  113 . However, it is possible to have only one output channel  141 , and output  112 . 
     In one embodiment, output channels  140 - 142  depart from channel  164  within chamber  129  to outputs  111 - 113 . In one embodiment, the cross-section and the length of the output channels  140 - 142  should be maintained at a predetermined volume ratio (i.e., 2:1:2, or 1:2:1, etc.) to obtain the desired hydraulic resistance of the output channels  140 - 142 . 
     In one embodiment, the output channels  140 - 142  increase in dimension from the channel  164 , leaving the chamber  129 , such that the output ratio for the objects  160 , is increased through the relevant channel  141 . 
     In one embodiment, instead of a straight edge, where necessary, a plurality of notches or recesses  146  may be disposed at a bottom edge of the microfluidic chip  100  to separate the outputs (i.e., outputs  111 - 113 ) and for the attachment of containers, and external tubing (for recycling the sheath or buffer fluids  163 —see  FIGS.  19 - 21   ), etc. The first output  111 , the second output  112 , and the third output  113  are reached via output channels  140 - 142  which originate from action chamber  129  (see  FIG.  2   ). 
     In one embodiment, a container  188  collects the objects  160  from the second output  112 , although other containers may collect the output from first output  111  and third output  113  (see  FIGS.  6 A- 6 D ). In one embodiment, portions of the first, second, and third outputs  111 - 113  may be characterized electronically, to detect concentrations of objects  160 , pH measuring, cell  160  counts, electrolyte concentration, etc. 
     In one embodiment, the targeted objects  160  are acted upon by the focused energy apparatus  157 , and those objects  160 , as well as non-targeted objects  160 , may be collected as product  165  from the second output  112 . 
     In one embodiment, the product  165  of targeted and non-targeted objects  160  may continue to be processed for storage, for further separation, or for processing, such as cryopreservation (discussed further below). 
     In one embodiment, the microfluidic chip  100  is provided in a sterile state, and may be primed with one or more solutions (i.e., sheath or buffer fluids  163 ), or purged of any fluids or materials by either draining the microfluidic chip  100  or by flowing sheath or buffer fluids  153  or other solutions through the microfluidic chip  100 , according to known methods. 
     Action Chamber: In one embodiment, downstream from intersection  162 , the objects  160  in the fluid mixture  120  flow-through channel  164  into an action chamber  129 , where the objects  160  are interrogated and acted upon. In one embodiment, channel  164  tapers into the chamber  129  (see  FIG.  4 B ), which speeds up the flow of the fluid mixture through the chamber  129 . However, one of ordinary skill in the art would know that the channel  164  need not taper and could be of any dimension and size as long as the present invention performs according to the desired requirements. 
     In one embodiment, an interrogation apparatus  147  is used to interrogate and identify the objects  160  in the fluid mixture in channel  164  passing through the chamber  129 . Further, in one embodiment, the focused energy device  157  also acts upon the objects  160  passing through the chamber  129 . 
     In one embodiment, the chamber  129  includes a relatively small diameter opening or window  150  (see  FIG.  5   ) cut through the microfluidic chip  100  and layers  101 - 102 , through which the objects  160  can be visualized as they pass through channel  164 . 
     Further, a shallow opening with a relatively larger diameter is cut into layer  104  as a top window, and into layer  101  as a bottom window. In one embodiment, the top window is configured to receive a first transparent covering  133 , and the bottom window  152  is configured to receive a second transparent covering  132 . The coverings  133 ,  132  may be made of any material with the desired transmission requirements, such as plastic, glass, or may even be a lens. In another embodiment, instead of a window with coverings, a contiguous plastic sheet can be used. Note that although the relative diameters of the coverings  132 ,  133 , and opening  150  are shown in  FIG.  5   , these may vary according to design or manufacturing considerations. 
     In one embodiment, the above-mentioned first and second coverings  133 ,  132  are configured to enclose the chamber  129 . The windows and coverings  133 ,  132  (see  FIG.  5   ), allow the objects  160  flowing in the fluid mixture  120  in channel  164  through the chamber  129 , to be viewed through opening  150 , and acted upon by a suitable light source  147  and a focused energy apparatus  167  (discussed later). 
     In one embodiment, windows and/or openings are not required due to the structure of the chip layers (i.e., glass), and/or their configuration, or the focused energy device  157  and light source  147  wavelengths and power levels are such that no damage to the chip  100  will occur. 
     Interrogation Apparatus: The interrogation apparatus of the present invention includes a light source  147  which is configured to emit a high-intensity beam  148  with any wavelength that matches excitable objects in the fluid mixture  120  (see  FIG.  5   ). Although a laser  147  is preferred, any suitable other light sources  147  may be used, such as a light-emitting diode (LED), or arc lamp, etc. to emit a beam that excites the objects. 
     In one embodiment, such a high-intensity laser beam  148  from a suitable laser  147  of a preselected wavelength—for example, a 349 nm or 355 nm continuous wave (CW), or quasi-CW pulsed laser  147 —is required to excite the objects  160  in the fluid mixture (i.e., sperm cells). In another embodiment, a 532 nm green laser  147  is utilized. 
     In one embodiment, the laser  147  (see  FIG.  5   ) emits a laser beam  148  through the covering  133  at an uppermost portion of the chip  100 , through opening  150 , to illuminate the objects  160  flowing through channel  164  in chamber  129  of the chip  100 , and then through covering  132  in layer  101  of the chip  100 . 
     In one embodiment, the light beam  148  can be delivered to the objects  160  by an optical fiber that is embedded in the microfluidic chip  100  at opening  150 . 
     The high-intensity beam  148  interacts with the objects  160  (see detailed explanation below), and passes through the first coverings  133 , to exit from the covering  132  at the bottom window, such that the emitted light  151 , which is induced by the beam  148 , is received by an objective lens  153  or other collection optics. The objective lens  153  or other collection optics may be disposed in any suitable position with respect to the microfluidic chip  100 —for example, parallel to the main channel with the optical axis perpendicular to the sample fluid flow  120 . Because the chamber  129  is sealed by the first and second coverings  133 ,  132 , the high-intensity beam  148  does not impinge on the microfluidic chip  100  and damage the layers  101 , 104  (see  FIG.  1 A ). Thus, the first and second coverings  133 ,  132  help prevent damage to the microfluidic chip  100  from the high-intensity beam  148  and photonic noise induced from the microfluidic chip  100  material (i.e., plastic). 
     In one embodiment, the light beam  148  passes through the chip  100  and the emitted light  151  received by the objective lens  153  or other collection optics, is detected by detector  154 , and is converted into an electronic signal by an optical sensor  154 , such as a photomultiplier tube (PMT) or photodiode, etc. The electronic signal can be digitized or processed by an analog-to-digital converter (ADC)  155  and sent to a digital signal processor (DSP) based controller  156  or computer. The electronic controller  156  can be any electronic processor with adequate processing power, such as a DSP, a Micro Controller Unit (MCU), a Field Programmable Gate Array (FPGA), or even a Central Processing Unit (CPU). 
     In one embodiment, the DSP-based controller  156  monitors the electronic signal and based upon predetermined criteria, the focused energy apparatus  157  may be employed when a targeted object  160  is detected. 
     However, in another embodiment, the interrogation apparatus simply interrogates the objects  160  in the sample fluid flow  120  for identity, and it is not connected to the employment of the focused energy apparatus  157  (see  FIG.  6 C ). 
     Focused Energy Apparatus: In one embodiment, in order to deliver a desired energy level to objects  160 , a focused energy apparatus  157  is used to provide focused energy pulses to the objects  160 . The focused energy apparatus  157  may be a thermal, electrical, optical, or electromagnetic device  157 , which would have a desired wavelength, and would deliver high peak power with a very high repetition rate (or pulse frequency), to the target objects  160 . 
     In one embodiment, the focused energy apparatus  157  is triggered a predetermined time (i.e., milliseconds) after activation by the controller  156  (the timing being set based upon the traveling speed of objects  160  through the channel  164 , and is discussed further below), and issues a pulse to the selected or targeted (i.e., unwanted) object  160 . 
     Examples of pulsed lasers  157  include mode-locked, Q-switch, as well as those lasers using both mode-locking and Q-switch techniques. For example, a focused energy device  157  such as an Avia 355-5-100 (made by Coherent, Inc., Santa Clara, Calif.), or the Explorer XP lasers Q-switch laser from Spectra-Physics Inc., is capable of operating in a pulse-on-demand mode, and can deliver 15 ns energy pulses or less, at a rate of over 1000 pulses per second, to the target objects  160 . 
     In one embodiment, pulse energy levels of 0.5-8.0 pJ are used, and in a preferable embodiment, a Q-switch laser  157  in pulse-on-demand mode is used to deliver an average pulse energy of 1.8 pJ with a range for individual pulses of 1.3 pJ to 2.3 μJ. In one embodiment, the pulse width ranges from 3 nanoseconds to 1 microsecond, and preferably, is in a range of 5-9 nanoseconds. However, one of ordinary skill in the art would know that any high power laser existing now, or later developed, with the appropriate high energy pulses and pulse frequency, would be suitable for the present invention in order to achieve the desired target accuracy and/or effect. 
     In one embodiment, the need for a tight action region (i.e., chamber  129 , or space between the chip  100  and container  188 ) in order to deliver the pulsed energy from the focused energy apparatus  157  to target objects  160  or a surrounding region thereof, is important to minimize the potential impact of delivering the energy outside of the targeted objects  160  or region, to otherwise unselected, or non-targeted objects  160 . For example, a focused energy apparatus  157  such as the Explorer XP 355-1 Q-switch laser, is capable of delivering &lt;4% rms, providing high pulse-to-pulse stability when fired at regular uniform intervals. 
     However, for flow cytometric analysis and action systems where objects  160  or cells enter the action region (i.e. chamber  129 , or space between the chip  100  and container  188 —see  FIGS.  6 A-D ) in non-uniform intervals, additional measures are employed to deliver uniform pulse energy  158  to impinge only the targeted objects  160  or cells, or the surrounding region thereof. Such measures include matching laser  157  performance parameters such as pulse length and peak power levels, to enable the system of the present invention to achieve a desired target accuracy (i.e., in one embodiment, photodamage, or kill rate of 95% or higher hit rate on target objects  160 ). 
     In addition, further tuning the pulse-on-demand operation and performance of the laser  157  to deliver extremely high pulse-to-pulse stability when fired at non-uniform intervals, greatly reduces the spatial variability in the area impacted by the pulse  158 . Thus, by reducing pulse-to-pulse variability in the focused energy device  157 , the unintended action, damage, or destruction to non-target objects  160  or cells, is greatly reduced, achieving, for example, an 85% or higher rate of viability for live, non-target objects  160  or cells. 
     In one embodiment, the focused energy apparatus  157  is utilized in an action region  129 , such as chamber  129 , prior to interrogation by the interrogation apparatus  157  (see  FIG.  6 D ), and in another embodiment, the focused energy apparatus  157  is utilized in the action region (i.e., chamber  129 ) after interrogation by the interrogation apparatus  157  (see  FIG.  6 A ). In yet another embodiment, the focused energy apparatus  157  acts upon the sample fluid with objects  160  after it leaves the chip  100  and enters a container  188 —either at the output  112  or in disconnected droplet form  187  before it enters the container  188  (see  FIGS.  6 B-C ). 
     In the embodiment where the focused energy apparatus  157  acts upon the objects  160  after they are interrogated by the interrogation apparatus  147  in the action region  129  (i.e., chamber  129 ), upon determination that the objects  160  are to be targeted, the focused energy apparatus  157  emits a focused energy beam  158  to act upon the objects  160  flowing through channel  164  (see  FIGS.  5  and  6 A- 6 C , for example). 
     In the embodiment, the focused energy apparatus  157  acts upon the objects  160  after interrogation in action region  129 , and after the objects  160  flow through output channel  141 , but before the sample fluid  120  is collected by container  188 . In this embodiment, the focused energy apparatus  157  is utilized as above but is positioned to emit the beam between the chip  100  and the container  188 . In one embodiment the sample fluid  120  simply falls from output  112  through the air into the container  188  in droplet form  187 , and in another embodiment, there may be a transparent enclosure between the chip  100  and the container  188 . 
     The focused energy apparatus  157  can be set to damage, alter, disable, kill or destroy the targeted or unwanted object  160  in the sample fluid  120  with a pulse, or to activate one of several mechanisms in the object  160  or cell, such that cell damage or death ensues. 
     However, depending on the desired arrangement (see further below for various embodiments), the targeted or selected objects  160  may be wanted objects  160 , in which case the focused energy apparatus  157  is not activated or triggered, or the targeted or selected objects  160  may be unwanted objects  160 , where the focused energy apparatus  167  is activated act upon the objects  160 , such as to damage, alter, disable, kill or destroy the selected, unwanted objects  160 . However, these are not the only embodiments, and the various embodiments are discussed further below. 
     In one embodiment, when the selected object  160  is damaged, altered, disabled, killed, or destroyed by the focused energy device  157 , the object  160  continues to flow through the main channel  164  to the center output channel  141 , and to the second output  112 , and into container  188 , along with any non-targeted objects  160 . The sheath or buffer fluids  163  proceed in laminar flow through output channels  140 ,  142 , to outputs  111 ,  112 , respectively. 
     However, in one embodiment, as noted above, the objects  160  in channel  164  may flow out from the chip  100  through output channel  141  and single output  112 . 
     Accordingly, in one embodiment, the present methods and apparatus are capable of producing a discriminated product  165  (see  FIGS.  6 A- 6 D ) of objects  160  in container  188 , including a high viability of non-target or wanted objects  160  or cells, and a high percentage of photodamaged, altered, disabled, destroyed, or dead target objects  160 . 
     Beam Shaping and Optics: In order to achieve satisfactory signal repetition and efficient damaging, altering, disabling, killing, or destruction of objects  160 , it is advantageous to use beam shaping optics for both the interrogation beam  148  and the focused energy beam  158  (see  FIGS.  16 - 17   ). As used herein, the phrase “beam spot” refers to a cross-section of either beam  148 , or beam  158 . 
     In one embodiment, the focused energy apparatus  157  is disposed downstream from a light source  147 , and on the same side as the light source  147  (see  FIG.  17   ), but the focused energy apparatus  157  may also be disposed downstream and on an opposite side of the light source  147  (see  FIG.  16   , for example). 
     In the embodiment of  FIG.  16   , a beam shaping optics for the interrogation beam  148  is disposed on one side of chip  100 . The interrogation beam  148  from light source  147  passes through the action region (i.e., chamber  129 ) and is received by objective lens  153 . 
     In one embodiment, the beam  148  is expanded by the beam shaping optics  181 , which may include a plurality of lenses, which arrangement would be well known to one of ordinary skill in the art. For example, the beam shaping optics  181  may include a pair of prisms and a pair of cylindrical lenses with appropriate focal lengths or may include other lenses, with or without prisms, which would be available to one of ordinary skill in the art. The beam expansion enables the final spot size at the focal point in the interrogation region  129 . In one embodiment, the circular beam  148  spot is expanded using a beam expander  180 . The beam expansion also reduces the influence on the downstream optics, limiting damage and extending lifetime. However, in one embodiment, no beam expander is utilized. Alternatively, if the source beam has too large of a diameter, optics could be used to reduce that diameter to a suitable size. 
     In one embodiment, the beam shaping optics  181  include two perpendicular cylindrical lenses to alter the beam shape  148  into an ellipse perpendicular to the direction of sample fluid  120  flow, and along the direction of sample fluid  120  flow, when focused at the center thereof. This elliptical beam  148  spot serves to excite the objects  160  passing through the channel  164  of the microfluidic chip  100 , and provides maximum uniform illumination at a center area of the beam  148  spot, to compensate for minor fluctuations in the flow of objects  160  through the channel  164 . Further, in one embodiment, the ellipse of the beam shape having a wider dimension perpendicular to the sample fluid  120  flow, helps to reduce variation in the fluorescence signal coming from the objects  160  (i.e., sperm cells) that are not perfectly centered within the sample fluid  120  flow stream. The narrow dimension keeps the beam  148  at a high enough intensity to adequately excite the fluorescent dye for interrogation of the objects  160  (i.e., sperm cells). While an elliptical beam  148  spot is preferred, in other embodiments of the present invention, a different shaped beam may be utilized. The power of the interrogation beam can be adjusted as well to assist in the interrogation and to limit the impact on the interrogated objects 
     In one embodiment, the focused energy beam  158  is also shaped by beam shaping optics  180 . The shape of the focused beam  158  spot of the focused energy device  157  influences the desired target accuracy and potential for impacting non-target objects  160  in the channel  164 , and can be varying beam shapes, as the application requires. In a flow-based system, the beam width along the direction of flow of the sample fluid  120  should be adjusted to be sufficiently narrow such that only the target object  160  is affected, and sufficient beam intensity concentration is achieved. The length of the beam  158  spot across the fluid channel  164  can be intentionally adjusted to compensate for any slight instability and variability in the focused flow of the sample fluid  120 . Desired beam shaping can easily be achieved by one skilled in the art. 
     In one embodiment, as shown in  FIG.  16   , beam shaping optics  180  for the focused energy beam  158  is utilized to focus the beam  158  down to a much smaller size to increase a laser flux in the action region  129 . In one embodiment, the beam  158  is expanded by beam expander optics  180  (see  FIG.  16   ) which may include a plurality of lenses or prisms with appropriate focal lengths, which would be readily known to one of ordinary skill in the art. 
     In one embodiment, the beam  158  passes through a pair of anamorphic prisms, for example, to shape the beam  158 . The beam  158  is further focused and compressed in the horizontal and vertical directions by an optical object due to a Gaussian beam property. In one embodiment, the optical object may be, for example, detector optics  153  such as a microscope objective or a focusing lens with a short focal length. The beam spot provides a combination of energy concentration for efficient action (i.e., killing, etc.) and sufficient width to compensate for minor fluctuations in a flow of the objects  160  through the microfluidic channel  164 . In one embodiment, a pair of cylindrical lenses is used to expand the beam in the vertical dimension, before a spherical focusing lens or objective lens is used to focus the beam to an elliptical beam spot of a minor diameter of 2 .tm and a major diameter of 20 gm. 
     In alternative embodiments, a different shape and/or dimension(s) may be used for the beam  158 . Note that other major and minor diameters are attainable by one skilled in the art and can be applied to the same process. 
     In another embodiment, the focused energy device  157  is implemented from the opposite side of the interrogation beam  148 , as shown in  FIG.  17   . The configuration shown is advantageous because it is easily implemented and efficiently uses the free space at a photo detector side of the system. 
     In one embodiment, dichroic mirrors are used to split off specific wavelengths or to integrate specific wavelengths into the optical path. Further, although a mirror and dichroic mirror/beam splitter may be used, one of ordinary skill in the art would know that more than one mirror and/or dichroic mirror/beam splitter may be utilized in the present system. 
     In one embodiment, collection optics  153 , including a microscope objective, collect the fluorescence emission from the objects  160  in the chip  100 , and a dichroic mirror passes the fluorescence emission from the collection optics  153  towards the optical signal detector  154 . In one embodiment, the focused energy device  157  emits a beam  158  which passes through beam shaping optics  180  (as described above), and which is directed by a mirror and also reflected by dichroic mirror through collection optics  153  onto the chip  100 . Specifically, in one embodiment, the objective lens of the collection optics  153  focuses the focused energy beam  158 , which enters the back aperture of the objective lens, into a tight spot on the objects  160  just slightly downstream from the interrogation/excitation point in action region  129 . However, one of ordinary skill in the art would know that the focused energy beam  158  may be disposed below the output  112  of the chip  100 , or slightly upstream from the interrogation/excitation region  129 . 
     In one embodiment, a distance between the beam spot of the interrogation beam  148  and the beam spot of the focused energy beam  158 , is adjustable. 
     In one embodiment, a dichroic mirror or any beam splitting device may split a small bit of light off to a camera  182  (see  FIG.  5   ), allowing the user to visually examine alignment. 
     In one embodiment, the camera  182  provides a visual image of the microfluidic flow environment for general alignment purposes. The camera can be used to determine location and timing for firing of the focused energy device  157 . 
     In another embodiment, the focused energy beam  158  is implemented from the same side as the interrogation beam  148  (see  FIG.  17   ). With this approach, the focusing lens for the focused energy apparatus  157  is not shared with the detection side, so it is more flexible for beam shaping, and it eliminates the need for a microscope objective that is rated for high power at the action (i.e., photodamage, killing) wavelength, which can reduce the system cost. 
     In this embodiment, the focused energy device  157  emits a beam  158  from the same side as the interrogation apparatus  147 , and the beam  158  is shaped by beam shaping optics  180  (described above), to be directed and aligned by a mirror and dichroic mirror, to be focused onto the objects  160  in channel  164  of the chip  100 . In one embodiment, beam focusing optics  181  (as described above), are disposed between a dichroic mirror and the chip  100 , to focus the beam  158 . 
     As stated above, in the embodiment, the beam  158  is focused into a tight spot on the objects  160  just slightly downstream from the interrogation/excitation point in action region  129 . However, one of ordinary skill in the art would know that the focused energy beam  158  may be disposed below the output  112  of the chip  100 , or slightly upstream from the interrogation/excitation region  129  (see  FIGS.  6 A-D ). 
     Further, as stated above, in the embodiment, a distance between the beam spot of the interrogation beam  148  and the beam spot of the focused energy beam  158 , is adjustable. 
     Object Focusing and Orientation: In conventional flow cytometry systems, since objects or cells, especially with asymmetric shapes, tend to orient as they flow close to a solid surface, the function and improvement of object or cell orientation relies on complex nozzle designs, such as orienting baffles or an offset structure inside the nozzle. To avoid the complex design of nozzle-based flow cytometry systems, and their high fabrication cost, the microfluidic chip  100  design of the present invention focuses, positions, and orients the objects  160 , in order to optimize its analytical capability. Thus, in one embodiment, the objects  160  (i.e., cells) with non-spherical shapes are aligned into a restricted core volume in channel  164  and maintained in a similar and desired orientation when they pass through the interrogation/detection beam  148 . As a result, more uniform scattering and detection signals will be obtained, thus, helping to increase the system&#39;s  100  sensitivity and stability. 
     In one embodiment, the orientation of objects  160  can be realized by positioning the sample core stream  120  offset with respect to the center of the central plane of the channel  164  cross-section, using hydrodynamic focusing. 
     Two-Step Hydrodynamic Focusing: The following describes two-step hydrodynamic focusing that takes place during fluid flow in one embodiment of the microfluidic chip  100  (see  FIGS.  1 B- 1 D ). 
     In one embodiment, the first hydrodynamic focusing step of the present invention is accomplished by inputting a fluid sample  120  containing objects  160 , including biological samples such as sperm cells  160 , etc., through sample input  106 , and inputting sheath or buffer fluids  163  through sheath or buffer inputs  107 ,  108 . In one embodiment, the objects  160  are pre-stained with dye according to known methods (e.g., Hoechst dye), in order to allow fluorescence and imaging thereof. 
     In one embodiment, objects  160  in the sample fluid mixture  120  flow through main channel  164 , are surrounded and shaped by the fluid flow, and have random orientation and position (see  FIGS.  3 A and  6 A ). At intersection  161 , the sample mixture  120  flowing in main channel  164  is surrounded and shaped by the sheath or buffer fluids  163  from channels  116 ,  117 , and compressed in a first direction (i.e., at least horizontally, on at least both sides of the flow, if not all sides depending on where the main channel  164  enters the intersection  161 ), when the sheath or buffer fluids  163  meet with the sample mixture  120 . This compression is termed hydrodynamic focusing (three-dimensional (3-D)) and is used to align the objects  160  in the channel  164  into a restricted core volume that may approximate a single file formation. The hydrodynamic focusing takes advantage of significantly large sheath or buffer flow in channel  164  to accelerate the travelling velocity of the objects  160  through the planar microfluidic channel  164 . In one embodiment, the sample core stream  120  may also be offset from the central plane by a ramp  166 B or taper  166 A structure in the channel  164  which is prior to the junction  161  of the channel  164  and the first-step sheath or buffer channels  116 ,  117 . 
     As a result, the objects  160  are speeded up and the spacing between the objects  160  in the microfluidic channel  164  also can be stretched. The velocity of the objects  160  is dependent upon the sample  120  flow rate and the ratio of the value to total sheath or buffer  163  flow rate. This function is useful to avoid clogging issues and object  160  clumping with highly concentrated object samples  120 . 
     However, as shown in  FIG.  8 A , at this stage, the resulting sample  120  core stream across the main channel  164  still shows overlapped objects  160  or cells along the channel  164  depth direction or vertical axis. In particular, the objects  160  are focused around the center of the channel  164 , and may be compressed into a thin strip across the depth of the channel  164 . Thus, at intersection  161 , as the sample fluid  120  is being compressed by the sheath or buffer fluids  163  from channels  114 ,  115 , toward the center of the channel  164 , the objects  160  (i.e., sperm cells) move toward the center of the channel  164  width. 
     In one embodiment, the present invention includes a second focusing step, where the sample mixture  120  containing objects  160 , is further compressed by sheath or buffer fluids  163  from a second direction (i.e., the vertical direction, from the top and the bottom) entering from channels  114 ,  115  at intersection  162  (see  FIG.  14   ). The intersection  162  leading into channel  164 B is the second focusing region. Note that although the entrances into intersection  162  from channels  114 ,  115  are shown as rectangular, one of ordinary skill in the art would appreciate that any other suitable configuration (i.e., tapered, circular) may be used. 
     In one embodiment, the sheath or buffer fluids  163  in the channels  114 ,  114  enter from the same plane (see  FIGS.  3 A and  6 A ), or from different planes into the channel  164  (see  FIG.  15   , where channels  114 ,  115  are disposed above and below main channel  164 , entering channel  164  vertically), to align the objects  160  in the center of the channel  164 B by both width and depth (i.e., horizontally and vertically) as they flow along channel  164 B. Then, the resulting flow in the main channel  164  is subsequently compressed and repositioned by the second-step sheath flow via microfluidic channels  114 ,  115 . 
     Thus, in the second focusing step of the present invention, the sample mixture  120  is again compressed by the vertical sheath or buffer fluids  163  entering at channels  114 ,  115 , and the sample  120  stream is focused at the center of the channel  164  depth, as illustrated in  FIG.  8 B , and the objects  160  flow along the center of the channel  164  in a restricted core volume that may approximate a single file formation in a particular orientation. 
     Accordingly, after these two subsequent hydrodynamic focusing steps, a restricted core volume of objects  160  or cells is obtained and the position of the stream also can be adjusted to a desired location along the vertical axis (see  FIG.  8 B ). Thus, the objects  160  introduced into sample input  106 , undergo two-step hydrodynamic focusing, which allows the objects  160  to move through the channel  164 B in a restricted core volume that may approximate a single file formation, in a more uniform orientation (depending on the type of objects  160 ), which allows for easier interrogation of the objects  160 . 
     Three-Step Hydrodynamic Focusing: In one embodiment, three-step hydrodynamic focusing is performed on the objects  160  in the chip  100 . In this embodiment, as shown in  FIGS.  1 A and  3 B , first two hydrodynamic focusing steps are accomplished by horizontally compressing the sample fluid stream  120  at intersections  161  and  162 , and then in a third step, the sample fluid stream  120  is vertically compressed in the channel  164 . Sheath or buffer channels  114 ,  115 , and  116 ,  117  enter the channel  164  from a horizontal direction, at an angle of 45 degrees or less for each channel. 
     More specifically, in the first hydrodynamic focusing step, sample  120  flow enters into the first intersection  161 , and the sheath or buffer fluids  163  from channels  116 ,  117  surround the sample  120  flow and immediately compress it into a thin sample  120  stream in channel  164 . In one embodiment, the sample fluid channel  164  is tapered with an internal ramp prior to intersection  161 , where sheath or buffer fluid channel  116 ,  117  enter the channel  164  (see  FIG.  3 B ). Meanwhile, as the channel  164  is shallower (i.e., smaller in dimension) than the first sheath channels  116 ,  117 , the sample  120  stream is lifted by the sheath or buffer fluid  163  from channels  116 ,  117 , to flow to the top of the main channel  164 . 
     In the second hydrodynamic focusing step, the sheath or buffer fluids  163  are introduced from channels  114 ,  115  into channel  164 —the channel  114 ,  115  which are disposed close to the top of the main channel  164  (see  FIG.  3 B ). In one embodiment, as shown in  FIG.  33   , sheath or buffer channels  114 ,  115  join channel  164  horizontally, and may be of a smaller dimension from that of sheath or buffer channels  116 ,  117 . Thus, since the depth of the channels  114 ,  115  are shallower than the main channel  164 , the sheath or buffer fluids  163  further compress the sample  120  stream along the channel  164  width, to constrain the width of sample  120  stream. This sheath or buffer fluid  163  flow significantly improves the signal measurement sensitivity. 
     Without a third hydrodynamic focusing step, the lack of vertical compressing at intersections  161  and  162  may result in multiple objects  160  or cells simultaneously entering the detection region  129 , thus, reducing the detection sensitivity or causing measurement errors, especially for a high throughput flow cytometry application. 
     However, three-dimensional hydrodynamic focusing is an effective way to align the objects  160  in a restricted core volume that may approximate a single file formation in channel  164 . Further, the consistent positioning of the object  160  in the channel  164  results in minimum variability in velocity from object to object in the parabolic flow. 
     In microfluidic-based flow cytometry, the flow velocity profile is parabolic along the rectangular cross-section of the micro-channel.  FIG.  7    illustrates the profile of shear force on the cross-section of the microfluidic channel  164 . The shear force gradient shown in  FIG.  7    has minimum shear force at the tapered tip and the larger region at the rear has the maximum shear value. Thus, the hydrodynamic shear force gradient is formed across the channel  164  cross-section. The large shear force close to the channel  164  wall helps to orient asymmetric objects  160 . 
     In the present embodiment, after the first two steps of hydrodynamic focusing, the sample  120  flow shrinks into a very thin stream in both horizontal and vertical directions. The ratio of independently controlled sheath or buffer fluids  163  from channels  116 ,  117  and  114 ,  115 , each determines the size of the resulting sample  120  stream in channel  164 . After the compression caused by sheath or buffer fluid  163  from channels  114 ,  115 , the objects  160  may spread a little along the channel  164  depth, but objects  160  still follow the sample  120  stream, which is very close to the top ceiling of the main channel  164  (note: with the chip described as horizontally disposed for ease of reference). Therefore, it is necessary to have a third sheath or buffer fluid  163  flow from channel  172  to further compress the sample  120  stream along the vertical direction, and confine the objects  160  well inside the thin, sample  120  stream. 
     Additionally, since the sheath or buffer fluid  163  flow is introduced perpendicularly to the pre-confined sample  120  stream from sheath or buffer channel  172 , it helps to position the sample  120  stream to a location along the cross-section of the channel  164  (i.e., achieves an end result as shown in  FIG.  8 B ). By fine-turning the flow rate of sheath or buffer fluid  163  from channel  172 , the objects&#39;  160  positions can be precisely controlled when they pass through the detection region  129 . 
     In one embodiment, the sheath or buffer fluid  163  is introduced by channel  172  from external sheath tubing (see  FIG.  20   ) instead of by micro-channels running through the microfluidic chip  100 . Thus, an external flow controller is required to provide a constant and stable flow rate through the input channel  172  (see  FIGS.  19 - 21   ). 
     The design of the present invention allows the core sample stream  120  to orient flat-shaped objects, position the objects  160  in the channel  164  in a physical arrangement approaching uniformity, all of which improves the downstream precision action of the focused energy apparatus  157 . 
     Although three hydrodynamic focusing steps are disclosed above, one of ordinary skill in the art would know that the configuration and number of the sheath or buffer channels may change, as long as they achieve the desired features of the present invention, with respect to the orientation and focusing of the objects in the sample fluid  120 . 
     Flow control methods: To realize the exemplary three-dimensional hydrodynamic focusing methods described above, both sample fluid  120  and sheath or buffer fluids  163  are required to be precisely delivered so that a constant flow can be streamed through the microfluidic chip  100 . After being compressed by the sheath or buffer fluid flows  163 , the objects  160  or cells have been accelerated and the average spacing between the objects  160  or cells in the sample  120  core stream is also stretched significantly therefrom. The ratio of the total sheath or buffer fluid  163  flow rate and the sample  120  flow rate can be adjusted between 100:1 and 1000:1. Preferably, the ratio of 200-400:1 is used in the microfluidic chip  100  of the present invention. The overall fluid flow rate in the microfluidic chip  100  is about 2-4 ml/min. The introduced sheath or buffer fluid flows  163  have to be constant and pulse-free to ensure a stable traveling speed of the objects  160  during interrogation and signal detection, and between the detection/interrogation position and the position of the action of the focused energy apparatus  157  (see  FIG.  6 A ). This facilitates an accurate signal reading and action on the target object  160  by the focused energy apparatus  157 . With the precise control of fluid flow through the main channel  164 , the overall flow rate variation is less than 1% of the set flow rate, and the traveling speed of target objects  160  for potential action by the focused energy apparatus  157  varies less than 1% from the position where interrogation and detection of objects  160  takes place, to the position where the focused energy apparatus  157  acts on the objects  160  (see  FIG.  6 A ). 
     Orientation of Objects: One of the challenging issues in the detection of flat-shaped objects  160  (i.e., sperm cells) is to constrain the objects  160  in a uniform orientation when passing through the interrogation beam  148 . Thus, an approximately uniform positioning of objects  160  and a corresponding orientation of objects  160  in the channel  164 , helps to increase the sensitivity of the system. With the aforementioned hydrodynamic focusing strategy, the position of objects  160  along the channel  164  can be manipulated in a controlled way. Thus, by adjusting the ratio between the sheath or buffer fluid  163  flows from channels  116 ,  117 , and  114 ,  115 , and  172 , a position of the focused sample  120  stream offset from the center of the channel  164  by about 5-20 microns (based on a channel  164  cross-section of 150-micron width; and 100-micron heights, for example), is preferred for the detection of flat-shaped objects  160 . Generally, an adjustment of 0-100 microns bias position of the objects  160  can be achieved. 
     Specifically; in order to align objects  160  in the channel  164  to improve their orientation, the high aspect ratio of the microfluidic channel  164  is taken advantage of to induce the shear force to turn the flat surface of the object  160  (i.e., sperm cell) facing the channel  164  wall. Further, the sheath or buffer fluid  163  flow can be actively employed to compress and position the objects  160  in the channel  164 . These methods are described below in more detail. 
     Passive Method: In one embodiment, an asymmetric geometry structure may be utilized to position the focused objects  160  in the channel  164 , by one of: a) placing an asymmetric ramp  166 B in the sample main channel  164  to lift the sample flow  120  (see explanation above regarding  FIG.  4 A ); and b) placing an asymmetric ramp  166 B in the main channel  164  prior to the action chamber  129  to lift up the focused sample stream  120  (see explanation above regarding  FIG.  4 B ). The above asymmetric features can be used individually or in an appropriate combination of two or more. However, one of ordinary skill in the art would know that these features are not necessary to the achievement of the position of the objects  160  in the channel  164 . 
     In one embodiment, as noted above, ramps may be used in the channels  114 - 117  to lift the sample flow  120 , although they are not necessary. The placement of ramps in the channels is dependent upon the direction in which the objects  160  of the sample core stream  120  are required to be offset from the center to improve object  160  or cell orientation. However, the above passive method has less flexibility to vary the object  160  position in the main channel  164 . 
     Active Method: In an alternative method to offset the sample  120  core stream in channel  164 , an asymmetric sheath flow  163  is introduced to adjust the positions of objects  160  or cells in the channel  164 . There are several methods of realizing asymmetric sheath or buffer fluid flow  163 , two embodiments of which are described below. 
     One embodiment is to introduce a single sheath or buffer fluid flow  163  which forms a 90 degree angle with the main channel  164  wall, as shown in  FIG.  3 A  (in a two-step hydrodynamic focusing method) or  FIG.  3 B  (in a three-step hydrodynamic focusing method). In the two-step hydrodynamic focusing embodiment, the introduced sheath or buffer fluid flow  163  at the second-step hydrodynamic focusing intersection  162 , further compresses the sample core stream  120  subsequent to the first-step hydrodynamic focusing at intersection  161 . 
     In the alternative three-step hydrodynamic focusing embodiment, this compressing of the sample core stream  120  occurs at the third-step hydrodynamic focusing intersection where channel  172  joins main channel  164 . Thus, the final hydrodynamic focusing step positions the objects  160  or cells to a desired location along the vertical axis. By controlling the ratio of the hydrodynamic focusing flow rates, a desired position of the objects  160  can be obtained to achieve the optimum orientation. 
     In a second embodiment using the two-step hydrodynamic focusing method, two second-step sheath or buffer channels  114 ,  115 , merge in an angle to the main channel  164  wall, and parallel main channel  164  from above and below, as shown in  FIGS.  12 A- 12 B . The angle of the second-step hydrodynamic focusing channels  114 ,  115 , and main channel  164  may vary and is dependent upon the fabrication methods. Preferably, a 90-degree angle is selected (see  FIGS.  12 A- 12 B ). Different flow rates of sheath or buffer fluids  164  may flow via the two channels  114 ,  115 , which are capable of repositioning the sample core stream  120  in the main channel  164 . After the objects  160  are offset from the central plane of the channel  164 , orientation of the objects  160  is improved. In a specific embodiment such as sperm cells  160 , the sperm cells  160  tend to turn their flat sides to the channel  164  wall in the vertical axis. 
     As can be seen from the embodiment of  FIG.  12 B , the dimensions of the channels  114 ,  115  are not necessarily identical (as shown in  FIG.  12 A ), in order to obtain different hydraulic resistances. Thus, the same flow rate of the second-step sheath or buffer fluid flows  164  in the channels  114 ,  115  will also generate bias fluid flow as well. 
     To summarize, both of the passive and active methods above can help to optimally position the objects  160  and improve their orientation in the channel  164 . 
     In one embodiment, pancake-shaped sperm cells  160  are taken as an example of the objects  160 . Because of their pancake-type or flattened teardrop-shaped heads, the sperm cells  160  will re-orient themselves in a predetermined direction as they undergo the second, or third (depending on the embodiment) focusing step—i.e., with their flat surfaces perpendicular to the direction of light beam  148  (see  FIG.  6   ). Thus, the sperm cells  160  develop a preference on their body orientation while passing through the hydrodynamic focusing process. Specifically, the sperm cells  160  tend to be more stable with their flat bodies perpendicular to the direction of the compression. Hence, with the control of the sheath or buffer fluids  163 , the sperm cells  160  which start with random orientation, now achieve uniform orientation. Thus, the sperm cells  160  are not only disposed in a restricted core volume at the center of the channel  164 B, but they also achieve a uniform orientation with their flat surface normal to the direction of compression in the last hydrodynamic focusing step. 
     The above methods improve the sperm cells&#39;  160  orientation and the capability to differentiate the DNA content of X- and Y-sperm chromosomes (and thereby distinguish between X and Y sperm). The histograms of  FIGS.  13 A and  13 B  show the sperm cells  160  in the center of the channel  164 , and offset from the center of the channel  164 , respectively. The circles on the left and right of the histogram in  FIG.  13 A  shows the populations of mal-orientated sperm cells  160 . The left hump of the major population weakens the capability to differentiate X- and Y-sperm populations and contributes to the asymmetric distribution of X- and Y-sperm populations. 
     Operation of Microfluidic Chip System 
     Interrogation of Objects: In one embodiment, the interrogation light source  147  is an excitation laser  147  (see  FIG.  16   ), having 350 mW power, 355 nm wavelength, 12 ps pulse width. 
     In one embodiment, further downstream from the hydrodynamic focusing steps, in channel  164 , the objects  160  are detected in the action chamber  129  at opening  150  through covering  133 , using the light source  147 . Light source  147  emits a light beam  148  (which may be via an optical fiber) which is focused at the center of the channel  164  at opening  150 . 
     In one embodiment, the objects  160  are sperm cells  160 , which are oriented by the hydrodynamic focusing steps, such that the flat surfaces of the sperm cells  160  are facing toward the light beam  148 . In addition, all objects  160  or sperm cells  160  are moved into a restricted core volume that may approximate a single file formation, by the hydrodynamic focusing steps, as they pass under light beam  148 . As the objects  160  pass under light source  147  and are acted upon by light beam  148 , the objects  160  emit the fluorescence which indicates the identity of the desired objects  160 . 
     The light source  147  provides the fluorescence excitation energy for detection of objects  160  in the action region  129 . In one exemplary embodiment with respect to the objects  160  being sperm cells  160 , X chromosome cells fluoresce at a different intensity from Y chromosome cells (based on DNA content, as is well known in the art) (note: 355 nm is selected for the Hoescht 33342 dye used on the DNA). Further, in other embodiments, objects  160  which are cells carrying one trait may fluoresce in a different intensity or wavelength from cells carrying a different set of traits. In addition, the objects  160  can be viewed for shape, size, or any other distinguishing indicators. 
     Thus, in the embodiment of sperm cells  160 , the illumination to the flat surface and the edge of the cells  160  is quite different with sperm cells  160  as compared to other cells. The fluorescence signal derived from the edge of the sperm cells  160  is significantly stronger than that from the flat surface, which increases the difficulty for the digital processor  156  to deal with the stronger signal from the edge, and the normal lower signal from the X- and Y-flat surface of the cells  160 . Thus, turning the flat surface of the sperm cell  160  to face the laser illumination (i.e., light beam  148 ) helps to reduce the orientation variability and increase the capability of the system to differentiate X- or Y-sperm cells  160 . 
     In the embodiment of beam-induced fluorescence, the emitted light beam  151  (in  FIG.  5   ) is then collected by the objective lens  153 , and subsequently converted to an electronic signal by the optical sensor  154 . The electronic signal is then digitized by an analog-digital converter (ADC)  155  and sent to an electronic controller  156  for signal processing. 
     As noted above, in one embodiment, the DSP-based controller  156  monitors the electronic signal, and when a particular signal is noted, a focused energy apparatus  157  may be employed to act upon a target object  160  (see  FIGS.  6 A- 6 C ). However, in an alternative embodiment, the interrogation apparatus interrogates the objects  160  after they are acted upon by the focused energy device  157  (see  FIG.  6 D ). 
     In one embodiment, interrogation of the sample  120  containing objects  160  (i.e., biological material), is accomplished by other methods. Thus, portions of, or outputs from, the microfluidic chip  100  may be inspected optically or visually. Overall, interrogation methods may include direct visual imaging, such as with a camera, and may utilize direct bright-light imaging or fluorescent imaging; or, more sophisticated techniques may be used such as spectroscopy, transmission spectroscopy, spectral imaging, or scattering such as dynamic light scattering or diffusive wave spectroscopy. 
     In some cases, the optical interrogation region  129  may be used in conjunction with additives, such as chemicals that bind to or affect objects  160  of the sample mixture  120 , or beads which are functionalized to bind and/or fluoresce in the presence of certain materials or diseases. These techniques may be used to measure cell concentrations, to detect disease, or to detect other parameters which characterize the objects  160 . 
     However, in another embodiment, if fluorescence is not used, then polarized light back scattering methods may also be used. Using spectroscopic methods, the objects  160  are interrogated as described above. The spectrum of those objects  160  which had positive results and fluorescence (i.e., those objects  160  which reacted with a label) are identified for selection by the focused energy apparatus  157 . 
     In one embodiment, the objects  160  may be interrogated and identified based on the reaction or binding of the objects  160  with additives or sheath or buffer fluids  163 , or by using the natural fluorescence of the objects  160 , or the fluorescence of a substance associated with the object  160 , as an identity tag or background tag, or meet a selected size, dimension, or surface feature, etc. 
     In one embodiment, upon completion of an assay, selection may be made, via computer  182  (which monitors the electronic signal and employs the focused energy apparatus  157 ) and/or operator, of which objects  160  to discard and which to collect. 
     Applications for Focused Energy Apparatus: The focused energy apparatus  157  of the present invention may carry out a number of actions on the objects  160  in channel  164 , or between chip  100  and container  188 . 
     In one embodiment, the focused energy device  157  acts to photodamage or destroy the objects  160  in a number of ways. 
     Specifically, the focused energy apparatus  157  acts to kill objects  160  (i.e., cells). For example, the target objects  160  may be unwanted cells  160 , and upon action by the focused energy apparatus  167 , cellular death may be caused by overheating of the intracellular environment, which may promote, but is not limited to, protein denaturation or reduction in enzyme activity. 
     In another method, the action of the energy dosage  158  from the focused energy apparatus  157  is strong enough to cause rupture of the plasma membrane and leaking of the cellular contents out of the cell  160  and into the surrounding environment (i.e., sheath or buffer fluid  163 ). 
     In another method, object  160  or cell death can be caused by the formation of radical oxygen species (ROS) due to adsorption of energy from the focused energy pulses  158  from the focused energy apparatus  157 , which will cause, among other things, DNA and protein damage. 
     In another embodiment, the focused energy apparatus  157  can temporarily or permanently disable target objects  160 , such as cells  160 , using focused energy pulses  158  from the focused energy apparatus  157 . 
     For example, exposing sperm cells  160  to focused energy pulses  158  such as those produced by a laser or LED  157  generates photo-activation within the cells  160  and results in temporary or permanent disablement of cellular mechanisms responsible for sperm  160  motility. After disabling target sperm cells  160 , the resulting sample  120  contains motile sperm  160  and immotile (target) sperm  160 , where the immotile sperm are unable to fertilize oocytes naturally. 
     In another embodiment, it may be desirable to use focused energy pulses  158  to make sperm  160  infertile through the dimerization of nucleotides in the DNA. Dimerization occurs when cells  160 , such as sperm cells  160 , are exposed to UV light, causing bonds between pyrimidine bases, and resulting in a type of “cross-linking”, which, if not repaired, inhibits replication and transcription. Thus, although the target sperm cells  160  are still alive as evidenced by their motility, the fertility of the targeted sperm cells  160  is greatly reduced. 
     In addition to sperm cells  160 , one can use high-powered focused energy sources  157  such as LEDs or lasers  157  to photobleach fluorescence in objects  160 , such as cells or colloids  160 , which express a predetermined level of fluorescence. For instance, in many self-assembly object formulations, a wide size range of objects  160  are formed that are difficult to separate from each other. To produce an enhanced sample  120  of objects  160  having a desired size, one can fluorescently label all objects  160  using methods known in the art, and one can use optical interrogation to determine object  160  size, and photobleach objects  160  possessing the predetermined level of fluorescence. 
     In a specific example, a semen sample  120  may contain contaminants such as bacterial or viral cells, which are the target of photobleaching. Another example may include sperm cells  160  containing a given trait of the cell  160  or DNA and labeled with the fluorophore for quantitative and/or qualitative measurement. Sperm cells  160  containing the trait may be targeted by the focused energy apparatus  157 . In another embodiment, sperm cells  160  which do not contain the trait may be targeted for photobleaching. Specific cells  160  in other cell mixtures such as blood are also candidates for photobleaching treatments to reduce viability. In yet another embodiment, photobleached cells/objects  160  can be undetectable downstream and therefore, are not subject to subsequent processing steps (i.e., can bypass subsequent processing steps). 
     In another embodiment, in contrast to the disabling of objects  160 , focused energy pulses  158  can be used to activate materials such as caged molecules or compounds within the objects  160 . 
     In one application, the caged compounds represent but are not limited to, fluorescent markers or cell responsive molecules. In these applications, focused energy pulses  158  are used to cause photo-activation of the caged molecules or compounds which alters cell  160  signaling kinetics for ex-vivo therapies. 
     In another embodiment, focused energy pulses  158  can activate photo-polymerization events which disable cells or colloids  160  by altering internal properties of the object  160 . 
     In another embodiment, with respect to intracellular signaling pathways, focused energy pulses  158  are used to activate heat shock proteins or induce mitochondrial biogenesis or activation within objects  160  or cells, including germ cells, to enhance cellular viability and functionality. Additional intracellular pathways may also be activated to repair damage done by either the interrogation/detection device  147  or a number of other factors that are too numerous to list (i.e., environmental, heat, chemical, etc.) 
     In one embodiment, one skilled in the art can generate photo-polymerization through focused energy pulses  158  to temporarily encapsulate or permanently contain target cells  160 , colloids, or other objects, using multi-armed PEG-acrylate/PEG-vinyl/etc., which promotes encapsulation of the target objects  160  or cells. Sperm cells  160  can be encapsulated to enhance the preservation of viability and fertility through commercial storage and delivery processes. 
     In another embodiment, focused energy pulses  158  are used to cause photo-polymerization events on the surface of targeted cells or objects  160  which increase the size or density of the encapsulating material in order to alter the size or density of the target object  160  or improve properties and performance of the encapsulating material. 
     In another embodiment, a photopolymerizable sequence in the hydrophobic portion of the vesicle may be used to permanently seal the desired molecule or object  160  by encapsulation therein. 
     In another method, the focused energy apparatus  157  may be used to act on externally or alter the environment around the target objects  160 . 
     In one method, focused energy pulses  158  are used to heat the local environment around target cells or objects  160  so that the thermal enhancement is sufficient to cause toxicity to target cells  160 . 
     In another embodiment, focused energy pulses  158  are used to promote rupture of analytes containing delivery vehicles (such as vesicles) which are in close proximity to target cells  160 . The delivery vehicles carry molecules such as sodium fluoride (NaF) which causes temporary immobility of sperm cells  160 , or heparin which promotes capacitation of spermatozoa  160 . When the concentration of analytes or activating agents are increased locally, target sperm cells  160  or other objects respond to the local signals without activating similar responses in non-target cells  160 . 
     In another embodiment, when objects  160  or cells are attached to a surface, the surrounding environment is modified with focused energy pulses  158  to vary the modulus of elasticity of the surface or release cell responsive chemicals from the surface of surrounding objects  160  or cells. 
     In one embodiment, heat production through absorption of light/EM waves  158  causes a temperature change which kills the objects  160  or cells. 
     In another embodiment, focused energy pulses  158  are used to form predetermined chemical bonds or break chemical bonds in the attachment material, thus, directing the differentiation of objects  160 , such as stem cells  160 , into differentiated cell lines. 
     In one embodiment, for some applications, it is desirable to use focused energy pulses  158  to promote cellular uptake or adhesion onto target objects  160 . 
     In one embodiment, cellular uptake of antibodies, cellular probes, or DNA is enhanced through local heating. 
     In one embodiment, with said local heating, when the temperature increase is optimized also to maintain object  160  or cell viability, the internalization of objects  160  into target viable cells  160  is selectively promoted. 
     In one embodiment, the object  160  to be delivered is attached to an object that when targeted with a light source  147 , may cause a brief microbubble. For instance, an oligonucleotide may be conjugated to a gold nanoparticle. When the gold is heated with a light source  147  at an optimized wavelength, a microbubble is briefly formed. Upon cavitation of the microbubble, the gold nanoparticles are broken apart and the pieces of the nanoparticle and the object attached to it are permeabilized through a cell membrane. 
     In another embodiment, the temperature increase of the target object  160  is not sufficient to cause the formation of a microbubble. The localized temperature increase is optimized to maintain cell viability and selectively promote the internalization of objects  160  into target viable cells. 
     Similarly, in another embodiment, by using focused energy pulses  158  to adhere materials onto colloids or objects, object  160  geometry or object  160  properties is altered, thus enabling additional separation techniques, such as magnetic or electric fields to separate materials that are normally not susceptible to such forces. 
     Operation of Focused Energy Apparatus: Generally, flow cytometric analysis and action systems that use an electromagnetic radiation source such as a focused energy apparatus  157  or laser, to act on selected objects  160 , typically desire to deliver a controlled energy level to individual objects  160 . In one embodiment, such systems can kill, alter, damage, or destroy targeted objects  160  or cells using the focused energy apparatus  157 . In other embodiments, such systems can, among other methods, activate targets in selected objects  160  or cells or in the fluid, media, or matrix surrounding selected objects  160 , as described above. 
     In the above methods which utilize focused energy pulses  158 , radiation can be applied by methods of either targeted firing or continuous elimination, such that the desired objects  160  are unaffected, and the unwanted, altered, killed, destroyed, or damaged objects  160  are discriminated from the sample  120 . Similar considerations as noted above, are given when selecting laser wavelength and laser power for targeted firing and continuous firing modes. 
     Targeted Firing: More specifically, in targeted firing, the focused energy apparatus  157  is employed for targeted objects  160 . Specifically, an object  160  in a sample  120  fluid mixture, may pass through an interrogation/detection area in chamber  129 , for example, where specified characteristics of the object  160  are evaluated by one or more of the above methods. 
     Thus, in a flow-based system, for example, the focused energy apparatus  157  action area  129  is downstream from the optical interrogation area using light source  147  for interrogation. Alternatively, the focused energy apparatus  157  is utilized prior to interrogation further downstream. In one embodiment, the focused energy apparatus  157  acts on the objects in action chamber  129 . The distance between the optical interrogation region and the action region may be adjusted to accommodate different timings. 
     Based upon predetermined criteria, a decision is made to either keep, discard, or act upon the selected object  160 . Objects  160  marked for action are hit with a triggered pulse of energy  158  from the focused energy apparatus  157  (see  FIGS.  6 A- 60   , for example). 
     When the object  160  or cell is not targeted, it remains unaffected, and flows through the chamber  129 , via channel  164  to output channel  141  and container  188 , which collects target and non-target objects  160  as a discriminated product  165 . 
     In one embodiment, the laser pulse  158  has a short duration and can selectively target individual objects  160  or cells while exerting no intended impact on non-target objects  160  or cells which may be nearby, thus, avoiding “overspray” to non-target objects  160  or cells. Pulse energy is selected to impart the desired effect while avoiding undesired disturbances to the surrounding media, or for example, in flow systems, does not cause unintended cavitation or bubble formation. A variety of laser wavelengths can be used; however, the flux requirement may be different depending on the characteristics of the target object  160 , dye, and environment. 
     Laser units  157  have limited power, especially those compact models that operate at high pulse frequency (&gt;100 kHz typically), and it may be preferable to choose a laser wavelength that minimizes the required flux. For example, matching the laser  157  wavelength to the absorbance of dyes, other targets, or objects  160  (i.e., molecules) used in the action process greatly improves efficiency and effectiveness. Additionally, pulse energy  158  is selected to impart the desired effect while avoiding undesired disturbances to the surrounding media, or for example, in flow systems, not causing unintended cavitation or bubble formation. 
     In one specific example related to sperm cells  160  as the objects  160 , a 355 nm laser  157  was used to take advantage of the dye (i.e., Hoechst 33342 dye) used for the cell staining process. In a similar example, a 349 nm laser  157  may be used. In such examples, when unwanted sperm cells  160  are photodamaged, destroyed or killed, pulse energy levels of 0.5-8.0 pJ are used.  FIG.  26    shows the excitation wavelength and the emission wavelength for cells stained with Hoescht 33342. 
     Continuous Firing: In continuous firing, such as in a flow-based system, the focused energy pulse  158  is constantly employed and only interrupted for the passage of the non-target objects  160  (i.e., wanted objects  160  that are not to be damaged, destroyed, altered, or killed), or debris or contaminants which do not require action thereon. 
     As stated above, based upon predetermined criteria, a decision is made to either keep or discard an object  160 , or act upon, including photodamage, kill, alter, disable, or destroy an object  160 . The focused energy apparatus  157 , such as a continuous wave (CW) or rapidly pulsed laser or LED, delivers a continuous stream of focused energy  158  to the objects  160 , and is used to act upon (i.e., including, photodamage, alter, disable, destroy, or kill) every object  160  passing through a particular location in the flow stream of the sample  120  fluid flow in an exemplary flow system. When non-target objects  160  are encountered in the action region, the laser beam  158  is shut off, deflected, or otherwise interrupted for a short period of time, to allow the non-target objects  160  (in some cases, discarded items), to pass through unaffected. The objects  160 —target or non-target flow through channel  141  into container  188 . 
     Methods for interrupting or diffusing the beam  158  include mechanical (shutters, choppers, galvanometer mirror), optical (acoustic optic deflector, acoustic optic modulator, spatial light modulator, digital micromirror device, polarization modification, liquid crystal display), electronic (pulse conditioning, dropping, or alteration of a Q-switch laser), or acoustic. Any other known or future suitable methods or techniques may be utilized to interrupt the focused energy beam  158 . 
     With either targeted or continuous methods, the energy pulse  158  has a short duration and the focused energy apparatus  157  can selectively target only a single object  160  and not impact other objects  160  which are nearby in the sample  120  fluid flow (i.e., can limit “collateral damage”). The energy pulse  158  is selected to be sufficient to achieve the desired action on the object  160  (i.e., damage, alter, kill or destroy the object  160 ), per user requirements. The pulsed energy  158  from the focused energy apparatus  157  should fall within a range where it will not cause a disturbance to the sample  120  fluid due to cavitation, bubble formation, or method of energy absorbance. 
     Other technologies for reducing unintended action, damage, and destruction to non-target objects  160  include those which absorb a significant portion of the pulse energy  158 , alter the direction of the beam  158 , or discharge excess energy by firing pulses  158  into the flow stream  120 . Specifically, these can include mechanically moving a mirror or lens so as to defocus or deflect excess laser pulses  158  into an energy absorptive device, lenses altered electronically in order to change the laser&#39;s  157  propagation angle, and sophisticated triggering technologies which coordinate pulse energy data from the laser  157  with data about the object-to-object timing of objects  160  in the immediate flow stream. 
     Pulse Timing: The timing between actions by the focused energy device  157  on the objects  160  is not uniform and follows a Poisson distribution where many short and extremely long intervals occur. Because a laser-based action system  157  includes inherent limiting factors, it is preferable to include a short “recharge time” between laser pulses  158 . The latency time (inherent in the focused energy apparatus  157 ), plus the “charge/recharge” time, is the minimum time that the focused energy apparatus  157  can react (deliver a pulse) and still provide the required energy level to the targeted object  160 . 
     In one embodiment, charge time should range from 0.1 Its to 1 second, and preferably should be from 0.1 μs to 4 ms. Pulse-to-pulse variability in energy levels affects the rate of producing the desired effect on target objects  160  or cells, and the potential for impacting non-target objects  160  or cells. When fired at non-uniform intervals, pulse-to-pulse stability should be high. In one example, a Q-switch laser  157  in pulse-on-demand mode was used to deliver average pulse energy of 1.8 μJ with a range for individual pulses of 1.3 μJ to 2.3 μJ. 
     In a flow based system, the action region  129  may be located downstream from the optical interrogation region in the chamber  129 , or upstream thereof, and the distance between the optical interrogation region and the action region may be adjusted to accommodate different timings. To accommodate sufficient change time for a pulsed laser  157 , the minimum timing between interrogation of the objects  160  or cells, and action on selected objects  160  or cells, should be no less than 1 μs. 
     The focused energy apparatus  157  operates successfully, for substantial periods, at action rates up to 5,600 objects per second, with accuracy rates which can be selected, and range for example, from 75-95%. In systems where spacing between objects  160  in the flow stream is controlled, the system  157  can operate at action rates up to the repetition rate of the laser  157 . 
     Selection of Objects: In one embodiment, the focused energy apparatus  157  is employed prior to interrogation of the object  160 . However, in another embodiment, in order to determine which objects  160  are selected for action by the focused energy apparatus  157 , as noted above, a histogram, or any graphical representation of the measured/calculated characteristics of the objects  160  after interrogation, can be used in order to make the decision for action thereof on the population of objects  160 . In one embodiment, after interrogation is accomplished, the plot of the span (i.e., transit time through the interrogation region of the chamber  129 ) can reflect the relative size of the sample  120  core stream under different flow conditions, object  160  or cell distribution across the main channel  164 , and object  160  traveling velocity, as well as the variation of object  160  velocity within a particular chip  100  design. 
     As noted above, the high aspect ratio of the main channel  164  is important to object  160  or cell hydrodynamic focusing, migration, and orientation. In one embodiment, less than one for the high aspect ratio for the microfluidic channel  164  is used in the present invention. Preferably, ⅔ high aspect ratio is used for the main channel  164 . The value of the span itself roughly indicates the object  160  velocity. Large span value indicates that objects  160  pass the interrogation light beam  148  slowly. The tight size of the span indicates that the sample  120  core stream is closer to the channel  164  central plane and there is less object  160  velocity variation. 
     In one embodiment, to precisely act on the selected objects  160  whether flowing through chip  100  or departing from output  112 , less velocity variation of objects  160  is allowed to ensure that the focused energy apparatus  157  can precisely target the selected objects  160  or cells. Thus, based on the above-described positioning and orientation methods (i.e., active, passive methods), the objects  160  are positioned close to the center of the cross-section of the channel  164  to reduce the velocity variation of the objects  160 . 
     In one embodiment, for flat-shaped object  160  or cells, such as live sperm cells  160 , both orientation and velocity variation need to be taken into consideration. Thus, sperm cells  160  pushed offset from the central plane of the channel  164  along the vertical axis (see  FIG.  9 B ), tend to obtain a better resolution (e.g., the differentiation of X- and Y-sperm cells  160  is more than 50% separated on the histogram obtained after interrogation), and a less mal-oriented cell  160  population. Thus, resolution and target (i.e., photodamage, killing) efficiency are balanced, with, in one exemplary embodiment, the sample  120  core stream being preferably shaped into about 10 microns in width, and 5-10 microns in height of the main channel  164  across the cross-section of the main channel  164  in the interrogation/detection region of chamber  129 , and offset by about 2-10 microns from the central plane of the main channel  164 , preferably by controlling the hydrodynamic focusing steps. 
     In one embodiment, where the objects  160  are sperm cells, a target sperm cell  160  may be a male-bearing sperm cell (i.e., a Y chromosome-bearing sperm cell) and a non-target sperm cell  160  may be a female-bearing sperm cell (i.e., an X chromosome-bearing sperm cell). In another embodiment, a target sperm cell  160  may be a female-bearing sperm cell (i.e., an X chromosome-bearing sperm cell) and a non-target sperm cell  160  may be a male-bearing sperm cell (i.e., a Y chromosome-bearing sperm cell). 
     In one embodiment, the objects  160  are acted upon prior to interrogation, by utilizing localized heat shock to incorporate molecules such as DNA or other probes through protective outer layers and into objects—i.e., through protective membranes and into cells (see  FIG.  6 D ). Traditional methods for incorporating molecules using the high voltages requires for successful electroporation to permeabilize membranes may be desirable, as evidenced by the high cell mortality rates. Localized heat shock represents a more gentle procedure and therefore, may be more desirable for maintaining viability of the cells. The focused energy apparatus  157 , is used to generate a localized rise in temperature, thus achieving heat shock. The localized heat shock results in permeabilizing the objects  160 , thus, facilitating incorporation of the desired molecules. An interrogation apparatus  147  is used thereafter, to detect and interrogate the objects  160  from which the interrogation apparatus  147  determines the number or proportion of objects  160  for which incorporation of the molecules has been attained. This method may be particularly desirable in biodetection, cellular engineering, targeted therapeutics, and drug/gene delivery. 
     Action Zone: In one embodiment; after interrogation is performed; and after an acceptable histogram is obtained (i.e., with acceptable resolution and relatively small span distribution), then the decision is made to employ the focused energy apparatus  157  to act on the selected objects  160  or cells. One of the more important parameters is the timing setting for the pulse from the focused energy apparatus  157  (i.e., delay or time interval for the object  160  between the interrogation/detection beam  148  and the focused energy beam  158 ). 
     The focused energy apparatus  157  action zone is the area in the cross-section of the main channel  164  where the selected objects  160  or cells can be effectively acted upon (i.e., photodamaged, altered, disabled, killed, destroyed, etc.), as shown in  FIG.  16   . Based on a predetermined energy level and beam shape of the focused energy apparatus  157 , and on the microfluidic channel  164  design and flow conditions; the action zone can be estimated by an action percentage (e.g., &lt;97%). The energy level of the focused energy apparatus  157  is dependent upon the current and charging time of the focused energy apparatus  157 . The larger overall flow rate in the main channel  164  means that the objects  160  are traveling faster. Thus, the transit time of the objects  160  through the focused energy beam  158  is shorter. For example, for an energy level of the focused energy apparatus  157  of 2.3 tJ with a certain beam shape (e.g., 2.5 microns×15 microns), and object  160  traveling velocity around 7.5 m/s, the focused energy apparatus  157  action zone is estimated to be about 20 microns in the Y-axis direction, and about 16 microns in the vertical direction. 
     Within the action zone, the percentage of affected (i.e., damaged, altered, or killed) target objects  160  also relies on the shape and the position of the sample  120  core stream. By adjusting the flow rates of the sheath or buffer hydrodynamic focusing flows, the size and position of the sample  120  core stream can be tailored to accommodate the action zone in the horizontal and vertical directions. Eventually, at the desired energy level of the focused energy apparatus  157 , flow conditions for the present microfluidic chip  100  can be determined. Thus, the shape of the sample  120  stream can be preferably constrained to around an exemplary 10 microns in width and 5-10 microns in height. 
     In other embodiments, the action zone as substantially described above is disposed prior to interrogation, or after the sample fluid  120  leaves the chip  100  for container  188 . 
     Operation on Sperm Cells: As discussed above, in one exemplary embodiment using sperm cells  160  as the objects  160 , the live sperm cells  160  (i.e., bovine sperm cells, with approximately 50-50 X-chromosome and Y-chromosome cells) are introduced into sample input  106 , and pass through hydrodynamic focusing steps, to reach the interrogation region  129 . In the interrogation region  129 , the dye (i.e., Hoechst 33342 dye) is excited by an interrogation beam  148  from a light source  147 , such as a laser  147 , which generates fluorescence in the cells  160  which is captured by an optical signal detector  154  after passing through an objective lens  153 . 
     Based on characteristics of the fluorescence signal, for example, a difference in reflective properties, the controller  156  can individually identify and distinguish target sperm cells  160  from non-target sperm cells  160 . If the sperm cell  160  is a wanted sperm cell (i.e., one of X- or Y-chromosome sperm cell), a determination is made to allow the wanted or non-target sperm cell  160  to pass through the microfluidic channel  164  to collection apparatus  188  unaffected. However, if the sperm cell is an unwanted sperm cell (i.e., the other of X- or Y-chromosome sperm cell), the focused energy apparatus  157  will be employed to act upon the target sperm cell  160  after a predetermined delay time, to allow for the unwanted/target sperm cell to reach the action zone (which may be in the chamber  129 , or between output  112  and collection apparatus. 
     The present invention allows for jitter in the system of the present invention, in order to have the most effective operation of the focused energy device  157  in the action zone, after the predetermined delay time. Depending on the travelling velocity of the target sperm cell  160 , the target sperm cell  160  will be in the action zone for about 2 .ts and the focused energy beam  148  is most effective when aimed at the center of the target sperm cell  160 . Thus, it is preferable to limit jitter to within 1 is or less. 
     As noted above, the target sperm cells  160  may be altered, photodamaged, killed, altered, disabled, or destroyed by either: 1) targeted or “pulse-on-demand” mode, or 2) a “continuous firing” mode, of the focused energy apparatus  157 . As noted above, it is preferable to choose a laser wavelength for the focused energy device  157  that minimizes the required flux. For example, matching the laser wavelength to the absorbance of the dye used in the sperm cell staining can improve efficiency and effectiveness. For example, if Hoechst 33342 dye is used for the staining process, a 355 nm laser wavelength for focused energy apparatus  157 , is optimal. 
     In one embodiment, the target sperm cells are killed, or destroyed. In another embodiment, the target sperm cells  160  are sufficiently disabled such that they are no longer capable of performing a defined function. For example, a tail of the target sperm cell  160  may be disabled such that it no longer exhibits progressive motility. Thus, the target, disabled sperm cell  160  will be prevented from fertilizing an egg. 
     In one embodiment, sophisticated software for the controller  156  can be designed for a high power laser  157  meeting the requirements described above that allows single laser pulses  158  to be fired. Thus, the targeted firing or “pulse-on-demand” mode delivers consistent laser pulses  158  whenever requested by the focused energy apparatus  157 . The targeted firing mode is preferably used for samples  120  that contain mostly non-target sperm cells  160 , and only a relatively small number of target sperm cells  160  that need to be eliminated. However, with high speed pulse-on-demand systems commercially available, the targeted firing mode can be implemented for samples having other ratios of non-target to target sperm cells. 
     In an alternative embodiment, when target sperm cells  160  largely outnumber the non-target sperm cells  160 , the focused energy apparatus  157  may not be able to generate laser pulses  158  fast enough to act upon (i.e., kill or disable) all of the target sperm cells  160 . Thus, the targeted firing mode becomes less effective. In this case, the “continuous firing” mode becomes more favorable. 
     In the continuous firing mode, as discussed above, the focused energy apparatus  157  is a continuous wave (OW) or quasi-OW, or rapidly pulsed laser  157 , used to act on (i.e., kill or disable) every sperm cell  160  passing through a certain location in the microfluidic channel  164  without discriminating between target and non-target sperm cells  160 . The focused energy beam  158  is shut off, deflected, or otherwise interrupted for a short period of time to allow non-target sperm cells (i.e., one of X-chromosome or Y-chromosome cells) to pass through unaffected when a group of non-target sperm cells  160  is identified. The group can be any predetermined number of non-target sperm cells  160 . After the non-target sperm cells  160  pass the action zone, the continuous firing mode is re-started. 
     In both the targeted and continuous firing modes of operation, sperm cells  160  that are too close to each other or that overlap one another in the sample fluid  120  stream are both killed, or both disabled, even if one of the sperm cells  160  is a non-target sperm cell  160  instead of a target sperm cell  160 . As used herein, the term “too close” refers to the presence of two or more sperm cells within range of the focused energy beam  158  such that both objects  160  are sufficiently acted upon by the focused energy beam  158  that the desired action occurs in both cells. In addition, sperm cells  160  that are unable to be effectively identified as either a non-target or target sperm cell  160 , are killed or disabled by the focused energy beam  158 , to ensure overall sample purity for the discriminated product  165 . Reasons that the discriminating system of the present invention may be unable to effectively identify a non-target sperm cell  160 , may be due to flow issues within the microfluidic channel  164 , staining problems, doublets, etc. Because the system of the present invention errs on the side of killing or disabling target sperm cells  160 , or any sperm cells  160  that cannot be effectively identified as either target or non-target, more pulses  158  may be used than the total number of actual target sperm cells  160  present in the sample  129 . 
     In one embodiment, as noted above, it is preferable to include a short “recharge/charge time” between laser pulses  158  of the focused energy apparatus  157 . First, the spacing between two consecutive sperm cells  160  is not uniform, but instead follows a Poisson distribution. In the sample fluid flow  120 , there is a percentage of sperm cells  160  that are relatively close to each other. To employ the focused energy apparatus  157  at the events that have closer spacing (i.e., shorter elapsed time), less focused energy beam  158  “recharge/charge time” would be allowed. Further, if two sperm cells  160  are too close to each other such that their fluorescence signals interfere with each other, and no clear identification of whether the sperm cell  160  is a target or non-target cell, the focused energy apparatus  157  will have to fire multiple laser pulses  158  in a short period of time to kill or disable all of the unidentifiable sperm cells  160  in order to maximize sample purity. Thus, to achieve a higher throughput, generally a shorter average elapsed time between pulses  158  is required. 
     As noted above, the discriminated sample  120 , after being acted upon by the focused energy apparatus  157 , is collected in a collection device  188 . Thus, in one embodiment, the collected product  165  contains both the non-target sperm cells  160 , and the target (i.e., killed, altered, destroyed, disabled) sperm cells  160 , still in the same gender ratio as the original sample  120 . The collection in a single container  188  does not affect an overall sample  120  quality. Thus, in one embodiment, the final product  165  that is used for eventual fertilization includes both the non-target and target sperm cells  160 . Alternatively, subsequent product  165  separation techniques (i.e., flow cytometry, electrostatic plates, holographic optical trapping, etc.) may be used to, for example, separate the sperm cells  160  of the product  165  into live or dead/disabled sperm cells  160 , or centrifugation may be used for removing unwanted debris such as the remains of the killed or disabled target sperm cells  160 . 
     In one embodiment, the microfluidic chip system of the present invention is used in concert with a separation or isolation mechanism, such as the exemplary piezoelectric actuator assembly device described in pending U.S. patent application Ser. No. 13/943,322, filed Jul. 16, 2013, or an optical trapping system as described in in U.S. Pat. Nos. 7,241,988, 7,402,131, 7,482,577, 7,545,491, 7,699,767, 8,158,927 and 8,653,442, for example, the contents of all of which are herein incorporated by reference in their entirety. 
     In one embodiment, as shown in  FIGS.  6 B-C , the main channel  164  is shortened past the action chamber  129 , such that the discriminated objects  160  leave the output channel  141  and output  112  in droplet  187  form, before falling toward a collection apparatus  188 . The operation of the focused energy apparatus  157  is the same, except that the objects  160  are acted upon as they leave the exit of output channel  112  (see  FIG.  6 B ), or as the disconnected droplets  187  fall (see  FIG.  60   ), and before they enter the collection apparatus  188 . 
     Post-Collection of the Discriminated Product: In one embodiment, the discriminated objects  160  are collected in a container  188  having 20% Tris (note: commercially available, such as sold by Chata Biosystems), which is disposed below the chip  100  output  112 . In one embodiment, the contents of the container  188  are circulated at predetermined intervals, to ensure mixing of the product  165  therein. In one embodiment, when the container  188  reaches a predetermined volume (i.e., 18 ml), the container  188  may be replaced with a new container  188 . 
     In one embodiment, antibiotics are added to a filled container  188  (i.e., 0.5 ml CSS antibiotics to 30 ml of product  165 ), the time recorded, and a plurality of containers  188  with product  165  are collected in a large container and cooled. In one example, three containers  188  with antibiotic-containing product  165 , at a time, are disposed in a 400 ml plastic container and placed in a cold room. 
     In one embodiment, after a predetermined amount of time in cooling (i.e., 2 hours). Tris B extender (14% glycerol Tris) is added in two aliquots, a predetermined time apart (i.e., 15 minutes), to each tube. After the last sample  120  has cooled for a predetermined time (i.e., 2 hours), and B extender is added, the samples  120  are centrifuged for 20 minutes at 850 xG in a refrigerated centrifuge at 5° C. Supernatant is aspirated from each tube, leaving—0.2 ml with the pellet. All the pellets are combined into a single, pre-weighed container (i.e., tube), and a concentration of combined pellet using commercially available counting protocols (i.e., SpermVision) is determined, and the final concentration calculated to 11.35×106 cells/ml. The final volume is adjusted with a complete Tris A+B+CSS antibiotics extender (i.e., 50:50 ratio of 20% egg yolk Tris+14% glycerol Tris). 
     In the exemplary embodiment of sperm cells  160 , the printed semen straws are filled and sealed, and the straws frozen using liquid nitrogen vapor. 
     In one embodiment, quality control measures post-freeze include bacterial contamination steps, where a frozen straw is thawed in a water bath, and the thawed straw is wiped with alcohol to disinfect it. The straw contents are plated onto blood agar plate (5% sheep&#39;s blood), and incubated at 37° C. for 24 hours to determine the bacterial content. 
     In one embodiment, quality control measures for progressive motility include thawing one frozen straw in a water bath, and expelling the straw contents into a small tube placed in the tube warming stage. Commercial methods such as Motility Analysis or SpermVision are used to determine the number of motile cells per straw. 
     In one embodiment, quality control measures for sample purity include thawing one frozen straw in a water bath, and isolating the live cells by processing them through glass wool. Then FISH analysis is performed on the live cell population to determine the sex chromosome ratio. 
     Multiple Systems: In one embodiment, a plurality of microfluidic chips  100 , optical interrogation apparatuses and focused energy apparatuses, are set up in parallel to increase throughput. 
     Referring now to  FIG.  18   , in one embodiment, a multiple systems layout  400  with, for example, “n” systems  401 ,  402 , etc., each equipped with a focused energy apparatus  157  is illustrated. In one embodiment, the multiple systems layout  400  includes a single interrogation apparatus  147 , and a single interrogation beam  148  which is configured to provide a fluorescence excitation energy for the detection of objects  160 —for example, sperm DNA content. 
     In one embodiment, the multiple systems layout  400  further includes beam shaping optics  181 , with a series of beam splitters  189 , each beam splitter providing a 50/50 split of the incoming power. This results in nearly equal power beams  158  for each of the multiple systems  401 ,  402 , etc. 
     External Devices 
     Separation Apparatus: In one embodiment, after the focused energy apparatus  157  acts on the objects  160  flowing through the microfluidic chip  100 , instead of flowing into output channel  141 , a separation mechanism (i.e., piezoelectric actuator assembly device (external or internal), optical trapping assembly, electrostatic plates, or other separation mechanism well known to one of ordinary skill in the art, may separate the targeted and non-targeted objects  160 . (See U.S. patent application Ser. No. 13/943,322, filed Jul. 16, 2013, or U.S. Pat. Nos. 7,241,988, 7,402,131, 7,482,577, 7,545,491, 7,699,767, 8,158,927 and 8,653,442, for example, the contents of all of which are herein incorporated by reference in their entirety). 
     Thus, in one exemplary embodiment, targeted objects  160  that have been damaged, killed, disabled, destroyed, or altered by the focused energy apparatus  157 , can be separated from non-targeted objects  160 , with the separation mechanism separating the targeted objects into one of the output channels  140 - 142 , and the non-targeted objects  160  into another of the output channels  140 - 142 . 
     In another embodiment, the targeted objects are separated using a separation mechanism after exiting from output  112 , by use of electrostatic plates—well known in the art, for example. 
     In yet another embodiment, the focused energy apparatus  157  acts on the objects  160  as they exit or drop from output  112 , and the separation mechanism also separates the targeted objects  160  from the non-target objects  160  thereafter, or from product  165 . 
     Pumping Mechanisms: As shown in  FIGS.  19 - 21   , in one embodiment, a pumping mechanism includes a system having a pressurized gas  235  which provides pressure for pumping sample fluid mixture  120  from reservoir  233  (or sample tube  233 ), via tubing  242 , into sample input  106  of the microfluidic chip  100 . 
     A central reservoir  240  (see  FIGS.  20 - 21   ), or individual reservoirs  241  (see  FIG.  19   ), contain sheath or buffer fluids  163 , and are connected to each sheath or buffer input  107 ,  108 ,  172  of the microfluidic chip  100  via tubing, in order to introduce sheath or buffer fluids  163  therein. In one embodiment, the reservoirs are collapsible containers  237  (see  FIGS.  20 - 21   ), which are disposed in pressurized vessels  240 , and the pressurized gas  235  pushes sheath or buffer fluids  163  to the microfluidic chip  100 . In one embodiment, the pressurized vessel  240  pushes sheath or buffer fluids  163  to a manifold  238  (see  FIG.  21   ) having a plurality of different outputs, such that the sheath or buffer fluids  163  are delivered via tubing  231   a ,  231   b , or  231   c  to the sheath or buffer inputs  107 ,  108 , or  172  respectively, of the chip  100 . Although a three input sheath or buffer fluid arrangement is shown (i.e.,  FIG.  1 D ), one of ordinary skill in the art would know that less or more tubing delivering sheath or buffer fluids to the microfluidic chip  100  is possible. In addition, although the tubing is shown as entering microfluidic chip  100  in  FIGS.  18 - 21   , the disposition of the tubing with respect to the inputs on the chip  100  are not shown in exact order. Further, one of ordinary skill in the art would know that the tubing enters the chip  100  via chip holder  200  (discussed below). 
     In one embodiment, each individual reservoir  241 , or central reservoir  240 , includes a pressure regulator  234  which regulates the pressure of the gas  235  within the reservoir  241 ,  240  (see  FIGS.  19 - 21   ). In one embodiment, a pressure regulator  239  regulates the pressure of gas  235  within the sample vessel  233 . Flow meters  243  and flow valves  244  control the sheath or buffer fluids  163  pumped via tubing  231   a ,  231   b , or  231   c , respectively, into the sheath or buffer inputs  107 ,  108 , or  172 , respectively (via holder  200 ). Thus, tubing  230 ,  231   a ,  231   b ,  231   c , is used in the initial loading of the sheath or buffer fluids  120  into the chip  100  and may be used throughout chip  100  operation to load sample fluid  120  into sample input  106 , or sheath or buffer inputs  107 ,  108  (and  172 ). 
     In one embodiment, the flow meters  243  are used to provide the feedback to flow valves  244  (see  FIGS.  19 - 20   ) placed in the sheath flow routes to achieve a stable flow with constant flow rate in the microfluidic channels. With the precise control of the flow, the overall flow rate variation is less than 1% of the set flow rate, and the traveling speed to target objects  160  varies less than 1% during the detection and between interrogation/detection spot and action spot. 
     In one embodiment, sheath or buffer fluid  163  is pumped through a vacuum chamber (not shown) that is disposed in between the pressure canister and the manifold, so as to remove dissolved gas in the sheath or buffer fluid. Inside the vacuum chamber, a segment of gas permeable tubing is disposed between the input port and output port. While the sheath or buffer fluid travels inside the vacuum chamber via the gas permeable tubing, the dissolved gas passes through the wall of the tubing, while the liquid remains inside the tubing. The applied vacuum helps gas permeate through the tubing wall. In one embodiment, the gas-permeable tubing is made of a hydrophobic porous material, for example expanded polytetrafluoroethylene (EPTFE), which has a high water entry pressure for liquid but is permeable for gas. 
     Computer Control: In one embodiment, the user interface of the computer system  156  includes a computer screen that displays the objects  160  in a field of view acquired by a CCD camera  182  over the microfluidic chip  100 . 
     In one embodiment, the computer  156  or a controller  156  controls any external devices such as pumps (i.e., pumping mechanisms of  FIGS.  19 - 20   ), if used, to pump any sample fluids  120 , sheath or buffer fluids  163  into the microfluidic chip  100 , and also controls any heating devices which set the temperature of the fluids  120 ,  163  being inputted into the microfluidic chip  100 . 
     In accordance with an illustrative embodiment, any of the operations, steps, control options, etc. may be implemented by instructions that are stored on a computer-readable medium such as a computer memory, database, etc. Upon execution of the instructions stored on the computer-readable medium, the instructions can cause a computing device  156  to perform any of the operations, steps, control options, etc. described herein. 
     The operations described in this specification may be implemented as operations performed by a data processing apparatus or processing circuit on data stored on one or more computer-readable storage devices or received from other sources. A computer program (also known as a program, software, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, declarative or procedural languages, and it can be deployed in any form, including as a stand-alone program or as a module, object, subroutine, object, or other unit suitable for use in a computing environment. A computer program may, but need not, correspond to a file in a file system. A program can be stored in a portion of a file that holds other programs or data (e.g., one or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files that store one or more modules, sub-programs, or portions of code). A computer program can be deployed to be executed on one computer or on multiple computers that are located at one site or distributed across multiple sites and interconnected by a communication network. Processing circuits suitable for the execution of a computer program include, by way of example, both general and special purpose microprocessors, and any one or more processors of any kind of digital computer. 
     Microfluidic Chip Holder: In one embodiment, the microfluidic chip  100  is loaded on a chip holder  200  (see  FIGS.  22 A- 23 B ). The chip holder  200  is mounted to a translation stage (not shown) to allow positioning of the holder  200  with respect to the interrogation apparatus and focused energy apparatus. The microfluidic chip holder  200  is configured to hold the microfluidic chip  100  in a position such that the light beam  148  from the interrogation apparatus may intercept the objects  160  in the above described manner, at opening  150 , and the focused energy apparatus  157  may act upon the objects  160 . 
     The mechanisms for attachment of the chip  100  to the holder  200  are described below along with their method of operation, but one of ordinary skill in the art would know that these devices may be of any configuration to accommodate the microfluidic chip  100 , as long as the objectives of the present invention are met. 
     As illustrated in  FIGS.  22 A- 23 B , in one embodiment, a microfluidic chip holder  200  is made of a suitable material, such as aluminum alloy, or other suitable metallic/polymer material, and includes main plate  201  and fitting plate  202 . The main plate  201  and fitting plate  202  may be of any suitable shape, but their configuration depends on the layout of the chip  100  and the requirements for access thereto. 
     In one embodiment, the main plate  201  has a substantial L-shape, but the shape of the holder  200  depends on the layout of the chip  100  (see  FIGS.  22 A- 23 B ). One leg of the L-shaped main plate  201  includes a plurality of slots that are configured to receive mounting screws  219 , which are used to mount the holder  200  on the translation stage. Any number of slots of any suitable shape and size, may be included in the main plate  201 . In one embodiment, four screws  219  are used to mount the holder  200  on the translation stage and adjust the position of the holder  200 . However, one of ordinary skill in the art would know that any number of any size slots and mounting screws may be used. 
     In one embodiment, a switch valve  220  is attached to the main plate  201  above the mounting screws  219  on the one leg of the L-shaped main plate  201  (see FIGS.  22 A- 23 B). In addition, in the other leg of the L-shaped main plate  201 , a pair of linear actuators, such as pneumatic cylinder actuators  207 A and  207 B, are disposed on the same side of the main plate  201  as the switch valve  220  (see  FIGS.  22 A- 23 B ). 
     In one embodiment, a fitting plate  202  is attached to the main plate  201 , on the other side and along the other leg of the L-shaped main plate  201  (see  FIGS.  22 A- 23 B ). The fitting plate  202  includes a plurality of apertures that accommodate a plurality of fittings  204 - 206  (see  FIGS.  22 A- 22 B , for example) configured to receive and engage with external tubing (see  FIGS.  19 - 23 B ) for communicating fluids/samples to the microfluidic chip  100 . In one embodiment, the fitting plate  202  includes three fittings  204 - 206 , which are used to align with the sheath or buffer inputs  107 ,  108 , and sample input  106 , respectively (see  FIGS.  1 B and  22 A- 22 B ). In another embodiment, four fittings  204 - 206  and  216 , are used to align with the sheath or buffer inputs  107 ,  108 , sample input  106 , and sheath or buffer input  172 , respectively (see  FIGS.  1 A and  23 A- 23 B ). However, one of ordinary skill in the art would know that the fittings would be arranged in any way by number and position, so as to align with the number of inputs in the microfluidic chip  100  and allow transmission of fluids from one or more reservoirs via tubing (see  FIGS.  19 - 21   ). 
     In one embodiment, a pair of slot washers  203 A,  203 B affix the fitting plate  202  and pneumatic cylinder actuators  207 A,  207 B to the main plate  201  (see  FIGS.  22 A- 23 B ). Thus, the fitting plate  202  is easily removed from the main plate  201  without disassembling the switch valve  220  and pneumatic cylinder actuators  207 A,  207 B from the main plate  202 . 
     In one embodiment, the pneumatic cylinder actuators  207 A,  207 B each include a piston (not shown) having a piston rod coupled thereto and extendable and retractable relative to the cylindrical body portion. In one embodiment, air is supplied through port  209  into the switch valve  208 , and exits through ports  210 ,  211  to ports  212 ,  213 , respectively, in pneumatic cylinder actuator  207 A. The air is further supplied from ports  214 A,  214 B in pneumatic cylinder actuator  207 A, to ports  215 A,  215 B, respectively, in pneumatic cylinder actuator  208 A. 
     The toggle switch  220  of switch valve  208  opens and closes, to allow or prevent, respectively, the air from the air supply entering through port  209  and to pneumatic cylinder actuators  207 A,  207 B. When the air is provided to the pneumatic cylinder actuators  207 A,  207 B, the piston rods of the pneumatic cylinder actuators  207 A,  2073  are extended outwardly to an open position (not illustrated), and fitting plate  202  is pushed outwardly away from the microfluidic chip  100  to an open position to allow a user to load (or unload) the microfluidic chip  100  (see  FIGS.  22 A- 23 B ). When the air supply is closed, the piston rods of the pneumatic cylinder actuators  207 A,  207 B are retracted inwardly to a closed position (as illustrated in  FIGS.  22 A- 23 B ), and the fitting plate  202  is drawn towards the chip  100  and pressed against the chip  100  forming a liquid seal between the chip  100  and the connections to the tubing for the sheath or buffer and sample fluids. 
     In one embodiment, when the microfluidic chip  100  is in the closed position, O-rings, which are disposed on the surface of the fitting plate  202 , between the fitting plate  202  and the chip  110 , form a substantially leak-free seal to protect the microfluidic chip  100  from damage. However, one of ordinary skill in the art would know that any number and configuration of O-rings or gaskets may be used. 
     In one embodiment, the holder  200  is positioned such that the chip  100  is at a sufficient height to accommodate at least one collection vessel  188  disposed underneath the chip  100 . In another embodiment, collection vessels are disposed between each output  111 - 113 . 
     In another embodiment, the present invention discloses a method of inseminating an animal comprising flowing a stream of a population of sperm cells through a channel, differentiating the sperm cells into at least two subpopulations with one subpopulation comprising X-chromosome containing sperm cells and another subpopulation comprising Y-chromosome containing sperm cells, selecting a desired subpopulation, ablating an undesired subpopulation, and collecting both the desired subpopulation and the ablated undesired subpopulation together, wherein the collected population of sperm cells is used to fertilize an egg. Insemination is the deliberate introduction of sperm into a female animal for the purpose of impregnating or fertilizing the female for sexual reproduction. Insemination is inclusive of introduction of a fertilized egg into a female animal. X chromosome and Y chromosome are both sex chromosomes, and generally, two X chromosomes are present in female cells, and generally, only one X chromosome and one Y chromosome are present in male cells. Fertilization is defined as the fusion of male and female gametes to form a zygote, a fertilized egg. Specifically, a sperm cell unites with an egg. Differentiating sperm cells includes identifying a physical or a genetic characteristic of a sperm cell including, but not limited to, whether a sperm cell has an X-chromosome or a Y-chromosome. Selecting a desired subpopulation includes targeting a subpopulation of cells for a certain action, for instance, ablating the subpopulation of cells while not ablating a different subpopulation of cells. Ablating an undesired subpopulation is defined as rendering sperm cells deficient in fertilizing an egg. It could be further defined as rendering sperm cells incapable of fertilizing an egg. Collecting subpopulations of sperm cells includes gathering both the selected and the unselected subpopulations together, usually in a container, a tube or a straw. It could be further defined as the stream of population of sperm cells are not physically subdivided prior to and subsequent to ablation of a subpopulation of sperm cells and that both subpopulations exit together and are gathered. 
     In a further embodiment, the present invention includes inserting the collected population of sperm cells into the mammal (human or animal) to fertilize an egg. Given that sperm cells outside the body are generally subject to conditions that are unfavorable for their survivability, fertilization should occur as soon as possible following collection. In some embodiments, the insertion occurs within forty-eight hours following the collection. 
     In a further embodiment, the present invention includes fertilizing the egg in vitro with the collected population of sperm cells to create a fertilized egg and implanting the fertilized egg within the animal wherein the implantation occurs within forty-eight hours following fertilization. In vitro fertilization is a process of creating embryos from oocytes (unfertilized egg cells) by fertilizing them with semen outside the uterus. Implantation of the fertilized egg within the mammal (human or animal) includes, at least, the placement of the fertilized egg in any part of the female mammal (human or animal), such as the uterus to allow for the development of the fertilized egg into an embryo according to the genetics of the fertilized egg. 
     In a further embodiment, the present invention includes obtaining genetic information of the sperm cells or the fertilized egg prior to implantation. In some embodiments, at least one, alternatively at least two cells of the fertilized egg for analysis. In other embodiments, a portion of a DNA molecule or an RNA molecule of the fertilized egg is sequenced. In some embodiments, yj DNA or RNA sequencing occurs within forty-eight hours following removal of at least one, alternatively at least two cells of the fertilized egg for analysis. Alternatively, the DNA or RNA sequencing occurs within twenty-four hours following removal of at least one, alternatively at least two cells of the fertilized egg for analysis. Obtaining genetic information, i.e., DNA or RNA sequencing, of the sperm cells or the fertilized eggs includes assessing the genetic profile or a gene or a set of genes to determine the quality of the cells and/or the phenotypic trait(s) of the grown animal. Alternatively, probes, including oligonucleotides could be used to ascertain the genetic sequence or profile of certain genes. DNA or RNA arrays could be used to assess the genetic profile of a cell. All known molecular biological and cellular techniques could be employed to assess the characteristics and the identities of the cells and their likely phenotypic outcome as an animal. Such analyses typically occur within a short period such as within forty-eight hours, alternatively within twenty-four hours to lessen any damage to lessen any damage to the sperm cells or the fertilized eggs. 
     In a further embodiment, implanting the fertilized egg within the mammal (human or animal) occurs within forty-eight hours following obtaining the genetic information of the fertilized egg. The sooner a fertilized egg is implanted within a mammal (human or animal), the better to ensure survivability. 
     With respect to flowing the stream of the population of sperm cells through the channel, a further embodiment includes orienting the sperm cells in the population in a particular position with respect to the channel, aligning the sperm cells wherein the sperm cells are flowing singularly through the channel, and exiting all the sperm cells through an output portion of the channel. With respect to orienting the sperm cells and aligning the sperm cells, some embodiments include compressing the stream by sheath fluid flowing along at least one side of the stream, and wherein the compressing further comprises intersecting at a substantially 90 degree vertical plane by the sheath fluid from above the stream. In other embodiments, the sheath fluid is in laminar flow with the stream of the population of sperm cells during the differentiating and the ablating steps. In an alternative embodiment, orienting the sperm cells and aligning the sperm cells includes compressing the sperm cells by an inertial force directed by a designed structure of the channel. Orienting includes placing the cell in a proper three-dimensional space within the channel to allow for proper determination of characteristics of the cells. Aligning includes placing the cells in proper order within the channel, and in most cases, the proper order is cells in a single file line with consistent orientation. Exiting all the sperm cells through an output portion of the channel includes that the selected and unselected sperms are not separated from each other or their path diverged to a separate path or channel during collection. 
     In another embodiment, differentiating the sperm cells into two subpopulations of X-chromosome containing sperm cells and Y-chromosome containing sperm cells includes illuminating the sperm cells with a beam of light, and detecting a difference between the two subpopulations of sperm cells. In a further embodiment, the beam of light is from a detection laser.  FIG.  27    shows a multiple station setup to analyze cells with a vanguard laser. In another embodiment, the beam from the laser is split into multiple beams, and each beam of the multiple beams is configured to illuminate a different sperm cell. In another embodiment, the beam from the laser is not split into multiple beams and the beam illuminates one sperm cell at a time.  FIG.  28    shows beam splitting occurring for multiple stations. The beam from the laser is emitted as a continuous wave, or it is emitted in pulses. In an alternative embodiment, the beam of light is from an LED light source. 
     In another embodiment, the difference comprises how the sperm cells reflect, refract, diffract, absorb, and/or emit the light. In some instances, a molecule of the sperm cell or a molecule associated with the sperm cell is excited by the beam and the molecule emits a second beam in a Stoke shift.  FIG.  29    shows that the Stoke shift creates a peak at 450 nm wavelength, therefore, creating the detection wavelength. The difference reflects a difference in DNA concentration per cell. As seen in  FIG.  24   , the difference between the subpopulations is at least about 3.8% difference in DNA content. The approximate 3.8% difference reflects the difference between sperm cells with the X-chromosome compared with sperm cells with the Y-chromosome. For bovines, the female sperm cell has the X-chromosome and 29 autosomes and male sperm cell has the Y-chromosome and 29 autosomes.  FIG.  25    shows staining of DNA in cells to determine the intensity of emitted light which correlates with the amount of DNA in a cell, wherein 3.8% more light emitted correlates to 3.8% more DNA in female sperm cells. 
     In another embodiment, selecting the desired subpopulation of particles includes gating each subpopulation based on a physical difference for each subpopulation, and identifying the desired subpopulation. Gating each subpopulation and identifying the desired subpopulation includes correlating a graphic representation for each subpopulation and choosing or marking the desired subpopulation. As the cells travel through the channel, they are graphically captured and their particular characteristic(s) are plotted on a graph. With each cell plotted on a graph, a gating of any particular population is possible wherein a particular subpopulation is selected or designated for certain action, such as laser ablation rendering certain sperm cells incapable of fertilizing an egg. 
     In another embodiment, ablating the undesired subpopulation further includes damaging or killing the undesired subpopulation of sperm cells with a second beam of light in an area of the channel, wherein a damaged or killed sperm cell is rendered unable to fertilize an egg. In some embodiments, the second beam of light is from a laser. In a further embodiment, the laser is configured to provide a pulse of beam. In yet another embodiment, the laser is configured to recharge to damage or kill the next sperm cell that enters the area of the channel. As seen in  FIGS.  30 A and  30 B  in some embodiments, the beam of light is tightly focused with a blade-shape configured to cut across approximately 2 microns in length of the channel to damage or kill the cell. As seen in  FIG.  30 B , there is an initial detection of the cell, which triggers a pulse laser based on a timer, and knowing the distance of travel (e.g., 175 microns), the pulse laser damages or kills the cell. In other embodiments, the damaging or killing is coordinated with the travel time of the sperm cells and wherein the sperm cells are traveling at a known velocity. The damaging or killing is coordinated so that the laser hits any particular cell as it travels through the channel. The coordination is timed so that only certain cells are hit with the laser beam. 
     In another embodiment, the animal is a cow or a heifer. A heifer is a young female cow that has not borne a calf. A cow is one that has previously borne a calf. In a further embodiment, the success rate for insemination of the heifer is at least about 60% on average. In an alternative embodiment, the success rate for insemination of the cow is at least about 37% on average. The success rate for insemination of the heifer is better compared with insemination comprising a sexing process that comprises a physical separation of selected sperm cells from unselected sperm cells. Table 2 depicts conception rates (CR) of cattle herds that have received different kinds of insemination technology: sexing technology of the present invention (IntelliGen Sexed Technology), conventional insemination without sexing, and competitors&#39; sexing technology. Different types of heifers (Holstein and Jersey) were inseminated. Over 6000 recorded insemination were analyzed. IntelliGen Sexed Technology achieved 63.8% conception rate for Holstein heifers and 61.9% conception rate for Jersey heifers. For Holstein (HO) heifers, the CR (61.9%) of insemination using IntelliGen Sexed Technology is comparable to the CR (64.6%) for heifer&#39;s that received insemination of unsexed sperms (conventional) and much better to the CR (54.7%) of insemination from competitors&#39; sexing technology, suggesting that IntelliGen Sexed Technology is less detrimental compared with competitors&#39; sexing processes. Competitors&#39; sexing processes comprise a physical separation of selected sperm cells from unselected sperm cells. In contrast, IntelliGen Sexed Technology does not require physical separation. Both selected and unselected sperm cells are collected together—only that the undesired subpopulation of sperm cells has been rendered incapable of fertilization. For Jersey (JE) heifers, the CR (61.9%) of insemination from IntelliGen Sexed Technology was surprisingly much better compared with the CR (38%) of conventional insemination as well as the CR (54.7%) of insemination from competitors&#39; sexing technology. Table 3 depicts a comparison of three types of insemination of cows are analyzed with respect to CR: IntelliGen Sexed Technology (42.6% for HO; 37.5% for JE); unsexed conventional insemination (40.3% for HO; 44.0% for JE), and competitors&#39; sexing technology (36.3% for HO; 34.2% for JE) wherein the desired sperm cells are physically separated from the undesired sperm cells. Consistently, the CR using the IntelliGen Sexed Technology was comparable to the CR using conventional insemination technique while they were both better compared to the CR using competitors&#39; sexing technology. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Heifer Insemination Records on Farms that used 
               
               
                 all 3 technology types (conventional, Genus Sexed 
               
               
                 Semen (IntelliGen), Other Sexed Technology): 
               
            
           
           
               
               
               
               
               
            
               
                   
                 IntelliGen 
                   
                 Other 
                   
               
               
                   
                 Sexed Tech 
                 Conventional 
                 Sexed Tech. 
               
               
                 Heifers 
                 (N) CR % 
                 (N) CR % 
                 (N) CR % 
                 Farms (N) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 HO 
                 (1109) 63.8% 
                 (398) 64.6 
                 (1785) 54.7% 
                 14 
               
               
                 JE 
                  (155) 61.9% 
                  (50) 38% 
                  (245) 47.8% 
                 1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Cow Insemination records on farms that used all 3 
               
               
                 technology types. The inseminations were from September 
               
               
                 1 and only on 1st and 2nd service on cows: 
               
            
           
           
               
               
               
               
               
            
               
                   
                 IntelliGen 
                   
                 Other 
                   
               
               
                   
                 Sexed Tech 
                 Conventional 
                 Sexed Tech. 
               
               
                 Cows 
                 (N) CR % 
                 (N) CR % 
                 (N) CR % 
                 Farms (N) 
               
               
                   
               
               
                 HO 
                  (665) 42.6% 
                 (3510) 40.3% 
                  (259) 36.3% 
                 7 
               
               
                 JE 
                 (2762) 37.5% 
                 (1037) 44.0% 
                 (2794) 34.2% 
                 5 
               
               
                   
               
            
           
         
       
     
     In another embodiment; the current invention discloses a collection of a population of sperm cells with both a desired subpopulation and an ablated undesired subpopulation according to the method of inseminating an animal that comprises flowing a stream of a population of sperm cells through a channel, differentiating the sperm cells into two subpopulations of X-chromosome containing sperm cells and Y-chromosome containing sperm cells, selecting a desired subpopulation, ablating an undesired subpopulation, and collecting both the subpopulations of sperm cells including the desired subpopulation and the ablated undesired subpopulation together, wherein the collected population of sperm cells is used to fertilize an egg. 
     In another embodiment, the current invention discloses an embryo, wherein the embryo is made from the step of inserting the collected population of sperm cells into the animal to fertilize an egg. In an alternative embodiment, the embryo is made from fertilizing the egg in vitro with the collected population of sperm cells creating a fertilized egg, and implanting the fertilized egg within the mammal (human or animal), and wherein the implantation occurs within forty-eight hours following fertilization. 
     Further aspects and embodiments of the present technology are described in the following paragraphs. 
     A method of producing a sperm cell composition the steps of: flowing a stream of a population of sperm cells through a channel; differentiating the sperm cells into two subpopulations of X-chromosome containing sperm cells and Y-chromosome containing sperm cells; selecting a desired subpopulation; ablating an undesired subpopulation; and collecting both the subpopulations of sperm cells including the desired subpopulation and the ablated undesired subpopulation together. 
     A method of fertilizing one or more eggs comprising the steps of providing at least one egg obtained from a female mammal, providing the sperm cell composition from a male mammal of the same species produced using the method described above as the female mammal, and mixing the one or more eggs with the sperm cell composition. An aspect of the methods described above, wherein the female mammal is a cow or a heifer. An aspect of the methods described above, wherein the male mammal is a bull or boar. An aspect of the methods described above, further comprising implanting the at least one fertilized egg within the female animal within forty-eight hours following fertilization. An aspect of the methods described above, further comprising obtaining genetic information of the sperm cells or the fertilized egg prior to implantation. An aspect of the methods described above, wherein implanting the fertilized egg within the female animal occurs within forty-eight hours following obtaining the genetic information of the fertilized egg. An aspect of the methods described above, wherein obtaining the genetic information of the fertilized egg comprises removing at least one cell of the fertilized egg for analysis. An aspect of the methods described above, wherein obtaining the genetic information of the fertilized egg comprises sequencing a portion of a DNA molecule or an RNA molecule of the fertilized egg. An aspect of the methods described above, wherein obtaining the genetic information of the fertilized egg occurs within forty-eight hours following removing at least one cell of the fertilized egg for analysis. An aspect of the methods described above, wherein obtaining the genetic information of the fertilized egg occurs within twenty-four hours following removing at least one cell of the fertilized egg for analysis. 
     A method of producing an embryo comprising using the sperm cell composition produced using the method described above and an assisted reproductive technique. An aspect of the methods described above, wherein the assisted reproductive technique is selected from the group consisting of in vitro fertilization (IVF), artificial insemination (AI), intracytoplasmic sperm injection (ICSI), multiple ovulation and embryo transfer (MOST), and other embryo transfer techniques. An aspect of the methods described above, wherein a heifer or cow is inseminated using artificial insemination. An aspect of the methods described above, wherein success rate for insemination is better than insemination comprising a physical separation of selected sperm cells from unselected sperm cells. An aspect of the methods described above, wherein success rate for insemination of a heifer is at least about 60% on average. An aspect of the methods described above, wherein success rate for insemination of the cow is at least about 37% on average. 
     An aspect of the methods described above, wherein flowing the stream of the population of sperm cells through the channel comprises orienting the sperm cells in the population in a particular position with respect to the channel; aligning the sperm cells wherein the sperm cells are flowing singularly through the channel; and exiting all the sperm cells through an output portion of the channel. An aspect of the methods described above, wherein orienting the sperm cells and aligning the sperm cells comprise compressing the stream by sheath fluid flowing along at least one side of the stream wherein the compressing further comprises intersecting at a substantially 90-degree vertical plane by the sheath fluid from above the stream. An aspect of the methods described above, wherein the sheath fluid is in laminar flow with the stream of the population of sperm cells during the differentiating and the ablating steps. An aspect of the methods described above, wherein orienting the sperm cells and aligning the sperm cells comprises compressing the sperm cells by an inertial force directed by a designed structure of the channel. An aspect of the methods described above, wherein differentiating the sperm cells into two subpopulations of X-chromosome containing sperm cells and Y-chromosome containing sperm cells is accomplished via the steps of: illuminating the sperm cells with a beam of light; and detecting a difference between the desired subpopulation and the ablated undesired subpopulation. An aspect of the methods described above, wherein the beam of light is from an LED light source. An aspect of the methods described above; wherein the beam of light is from a detection laser. An aspect of the methods described above, wherein the beam of light is split into multiple beams of light; and wherein each beam of light of the multiple beams of light is configured to illuminate a different sperm cell. An aspect of the methods described above, wherein the beam of light is not split into multiple beams and the beam illuminates one sperm cell at a time. An aspect of the methods described above, wherein the beam of light is emitted as a continuous wave. An aspect of the methods described above; wherein the beam of light is emitted in pulses. An aspect of the methods described above, wherein the difference comprises how the sperm cells reflect, refract, diffract, absorb, and/or emit the light. An aspect of the methods described above, wherein a molecule of the sperm cell or a molecule associated with the sperm cell is excited by the beam of light and the molecule emits a second beam of light in a Stoke shift. An aspect of the methods described above, wherein the difference reflects a difference in DNA concentration per cell. An aspect of the methods described above, wherein the difference between the subpopulations is at least about 3.8% difference in DNA content. 
     An aspect of the methods described above, wherein selecting the desired subpopulation comprises gating each subpopulation based on a physical difference for each subpopulation; and identifying the desired subpopulation. An aspect of the methods described above, wherein gating each subpopulation and identifying the desired subpopulation comprises correlating a graphic representation for each subpopulation; and choosing or marking the desired subpopulation. An aspect of the methods described above, wherein ablating the undesired subpopulation further comprises damaging or killing the undesired subpopulation of sperm cells with a second beam of light in an area of the channel, wherein a damaged or killed sperm cell is rendered unable to fertilize an egg. An aspect of the methods described above, wherein the second beam of light is from a laser. An aspect of the methods described above, wherein the laser is configured to provide a pulse of the beam of light and wherein the laser is configured to recharge to damage or kill the next sperm cell that enters the area of the channel. An aspect of the methods described above, wherein the beam of light is tightly focused with a blade shape configured to cut across at least approximately 2 microns in length of the channel. An aspect of the methods described above, wherein the damaging or killing is coordinated with travel time of the sperm cells and wherein the sperm cells are traveling at a known velocity. 
     A process for the production of at least one fertilized egg from a female mammal, the process comprising the steps of a) flowing a stream of a population of sperm cells through a channel; b) differentiating the sperm cells into two subpopulations of X-chromosome containing sperm cells and Y-chromosome containing sperm cells; c) selecting a desired subpopulation; d) ablating an undesired subpopulation; e) collecting both the subpopulations of sperm cells including the desired subpopulation and the ablated undesired subpopulation together; and f) mixing at least a portion of the collected sperm cells with at least one egg from a female mammal to produce a fertilized egg. An aspect of the process described above, further comprising implanting the at least one fertilized egg within the female animal within forty-eight hours following fertilization. An aspect of the process described above, further comprising obtaining genetic information of the sperm cells or the fertilized egg prior to implantation. 
     A process for the production of at least one embryo from a female mammal, the process comprising the steps of a) flowing a stream of a population of sperm cells through a channel; b) differentiating the sperm cells into two subpopulations of X-chromosome containing sperm cells and Y-chromosome containing sperm cells; c) selecting a desired subpopulation; d) ablating an undesired subpopulation; e) collecting both the subpopulations of sperm cells including the desired subpopulation and the ablated undesired subpopulation together; f) inserting at least a portion of the collected sperm cells into a female mammal; and g) fertilizing at least one egg from said female mammal to produce at least one embryo from said female mammal. 
     The construction and arrangements of the microfluidic chip system with a focused energy device, as shown in the various illustrative embodiments, are illustrative only. It should be noted that the orientation of various elements may differ according to other illustrative embodiments, and that such variations are intended to be encompassed by the present disclosure. Although only a few embodiments have been described in detail in this disclosure, many modifications are possible (e.g., variations in sizes, dimensions, structures, shapes and proportions of the various elements, values of parameters, mounting arrangements, use of materials, colors, orientations, etc.) without materially departing from the novel teachings and advantages of the subject matter described herein. Some elements shown as integrally formed may be constructed of multiple parts or elements, the position of elements may be reversed or otherwise varied, and the nature or number of discrete elements or positions may be altered or varied. The order or sequence of any process, logical algorithm, or method steps may be varied or re-sequenced according to alternative embodiments. Other substitutions, modifications, changes and omissions may also be made in the design, operating conditions and arrangement of the various illustrative embodiments without departing from the scope of the present disclosure. All such modifications and variations are intended to be included herein within the scope of the invention and protected by the following claims.