Abstract:
A system for collecting and analyzing maximized amounts of fluorescent radiation using frequency scattering ports or waveguides that absorb a desired size of wavelengths.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. provisional patent application Ser. No. 61/736,749, filed Dec. 13, 2012; and U.S. provisional patent application Ser. No. 61/755,165, filed Jan. 22, 2013, each of which are incorporated herein in their entirety by reference. 
    
    
     TECHNICAL FIELD 
     The present novel technology relates to the field of laser physics, and, more particularly, to a laser-based cytometer flow measurement system. 
     BACKGROUND 
     In the field of cytometry, collection and analysis of fluorescent radiation is important. Cells or particles of interest are bound with various fluorescent tags and generally sent via fluidic transport through an interrogation point, where the particles are then illuminated such as by a laser or other light source. Given an appropriate tag that will interact with the incident wavelength of light, the particles will then radiate a fluorescent signal that indicates a particular trait held by the particles in interest. This signal is then processed through an optical train, typically consisting of a combination of lenses, fibers and/or dichroic mirrors to relay the fluorescent signal to a final detector to be captured. Overall, flow cytometers collect a relatively small amount of the omitted fluorescent signal. This weak signal necessitates the use of photomultiplier tubes (PMTs) for the direct measurement of the fluorescent signal, necessitating yet further amplification of the PMT&#39;s output for subsequent sample characterization. The desire to increase the amount of fluorescent radiation collected is very great, and has generated considerable work and various approaches to reaching this elusive goal. Simply stated, increasing the amount of fluorescent radiation collected for analysis will allow lower threshold levels of radiation to be measured, thereby boosting the number of particles in interest observed and increasing the probability of detecting sporadic or rare events held within the sample set. Conventional commercial flow cytometers typically utilize high numerical aperture optics to collect the fluorescent radiation for analysis. This technique limits the amount of radiation that can be collected by constraining the volume of the fluorescence emitted for observation to that of the numerical aperture optics used. If there was a way to collect and analyze a greater amount of the fluorescent radiation signal, it would be possible to better track the presence of certain cells in health or cancer research, or increase accuracy when conducting research into biomarkers, and gain better feedback for protein engineering, and the like. Thus, a need persists for a more effective technique for capturing and utilizing a greater portion of the generated fluorescent signal during cytometric analysis. The present novel technology addresses this need. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic side sectional view of the internally reflective chamber for fluorescent radiation collection and concentration, according to the first embodiment of the present novel technology. 
         FIG. 2  is a schematic front sectional view of the internally reflective chamber for fluorescent radiation collection and concentration, of  FIG. 1 . 
         FIG. 3  is a schematic side sectional view of a solid externally reflective flow cell for the collection and concentration of fluorescent radiation, according to the second embodiment of the present novel technology. 
         FIG. 4  is a diagram of the solid externally reflective flow cell for the collection and concentration of fluorescent radiation. 
         FIG. 5  is a schematic view of a spherical florescent radiation detector cell, of the present novel technology. 
         FIG. 6  is a schematic diagram of a multispectral wavelength selective detector, for use with the present novel technology. 
         FIG. 7  is a schematic diagram of the pixelated architecture of the multispectral wavelength selective detector of  FIG. 6 . 
     
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     For the purposes of promoting an understanding of the principles of the novel technology and presenting its currently understood best mode of operation, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the novel technology is thereby intended, with such alterations and further modifications in the illustrated device and such further applications of the principles of the novel technology as illustrated therein being contemplated as would normally occur to one skilled in the art to which the novel technology relates. 
     In traditional cytometer flow cell architecture, a very small percentage of the fluorescent radiation generated by the sample being investigated is collected usefully. The novel technology presented herein relates to a method and apparatus for the collection of most or virtually the entire radiation signal generated by fluorescing a sample material and/or maximizes the collected signal to increase detection capabilities. The novel technology operates to encase the point of fluorescence in a hollow reflective chamber wherein the emitted radiation is redirected and/or concentrated for measurement and analysis. The basic configuration from an optical component manufacturing or light manipulation standpoint is typically spherically based, more typically an integration sphere or partial sphere to encase the point of fluorescence. To concentrate and direct the fluorescent radiation emitted during cytometry, the integration sphere is typically truncated to become a highly reflective hemisphere and then conjoined with a high collection, non-imaging reflective optical component with a singular circular output. To increase collection and light concentration efficiency, the profile of this non-imaging reflective component is typically determined to be a hyperboloid of revolution, as this shape lends itself to useful radiation concentration. As an added benefit, the light emitted from the hyperboloid of revolution is very well-behaved and lends itself nicely to manipulation and transmission. This design and functionality applies to either the hollow core or optically transparent solid core versions of the flow cell chamber. 
     While fluorescence measurement is one useful aspect of flow cytometry analysis, forward scatter (FS) and side scatter (SS) measurements may be equally useful. The design disclosed herein also addresses these measurements as well. Dependent upon the particular version of the chamber selected, ports may be either machined directly into the chamber (hollow core) or machined into the lower index coating material (solid core) such that the traditional methods of FS and SS measurements still apply. 
     One embodiment of the novel technology as illustrated in  FIG. 1  and  FIG. 2  is a flow cytometer  1  having an internally reflective, generally hollow chamber  5  in which a sample may be laser fluoresced with the fluorescent radiation collected and concentrated. The chamber  5  typically either inherently reflective or has the shape of a hemisphere and functions as a high collection, non-imaging reflector. The chamber  5  is typically internally coated  10  with broadband reflective material, the selection of which is typically substrate dependent and of a lower index material. A capillary transport  15  is positioned at one or more predetermined locations within the chamber  5  to supply particles  40  from a flow cell  20 , which is typically positioned outside the chamber  5 , through a focus position  25  of an operationally connected integration laser  30 , likewise typically positioned outside the cell. The focus potion  25  is typically centrally positioned inside the chamber  5 . Frequency scattering ports  35  are typically positioned around the chamber  5  to facilitate laser focus as well as forward and side scatter detection. The ports  35  for laser  30  introduction and the forward scatter measurement typically lie in a line that intersects the focus position  25  of the hemisphere. The side scatter port  35  and focal position  25  of the hemisphere  5  typically lie in a line orthogonal to the line formed by the laser(s) (or light) port, focal point of the hemisphere  5 , and the forward scatter port. Particles emitted from the particle interrogation position  25  are collected by reflection and emitted out of the non-imaging reflector output portion  45 . The new imaging reflector output portion  45  is operationally connected in photonic communication with the hemisphere chamber  5  to guide and concentrate fluoresced signals from the particles to an output port  47 . The output portion  45  typically has the shape of a hyperboloid of revolution, although it may have any convenient shape. The collected particles  40  is then evaluated according to methods commonly used in the art, such as spectrometry or other like means of evaluation or measurement. Fiber optic conduits (not shown) may also be used to transport the input or output of particles  40  of the chamber  5 . Additional observation ports  35  may be added to the chamber  5  to allow further imaging of the interrogation point  25 . 
     A second embodiment of the instant novel technology, as illustrated in  FIG. 3  and  FIG. 4 , is a flow cytometry system  55  having an externally reflective chamber  60 . The solid, optically transparent externally reflective chamber  60  is typically a unitary combination of an integration hemisphere  67  cojoined with a high collection, non-imaging hyperboloid of revolution reflector with material selected for broad wavelength transmission. The chamber  60  is typically externally coated  65  for broadband internal reflection. The chamber  60  includes a generally cylindrical elongated conduit  61  formed there through. A capillary transport  70  extends through the conduit  61  within the chamber  60  to supply particles  90  from a flow cell  75 , which is positioned outside the chamber  60  and in fluidic communication with the capillary transport system  70 , through a focus position  80  of an operationally connected interrogation laser  85 , likewise typically positioned outside the cell. The focus position  80  is typically strategically positioned inside the conduit  61 . Frequency scattering ports  95  through the coating  65  are located at the preselected positions on the hemisphere  67  to allow laser  85  focus as well as forward and side scatter detection. The ports  95  for laser  85  introduction and the forward scatter measurement lie in a line that contains the focal point  80  of the hemisphere  87 . The side scatter port and focal point  80  of the hemisphere  87  typically lie in a line orthogonal to the line intersecting the laser  85  port, focal point  80  of the hemisphere  87 , and the forward scatter port  95 . Photons  90  emitted from the particle interrogation position  80  are collected by reflection and emitted out of the non-imaging reflector output positions  100 . The collected photons  90  may then be evaluated though methods commonly used in the art such as a spectrometer or other means of evaluation or measurement. As with the previous embodiment, fiber optic conduits  50  may also be used to transport the photon  90  input or output of the chamber  60 . An example of a possible coating  10 , 65  material for the first and second embodiment would be aluminum, which may increase reflectivity to at least 60% more typically at least 70%, still more typically at least 80%, and even up to at least 90%, allowing close to the theoretical maximum amount of fluorescent energy to be collected for analysis. 
     A third embodiment, illustrated in  FIG. 5 , is a flow cytometry system  101  having a generally spherical reflective chamber  105 . The spherical reflective chamber  105  operates much like the above described first and second embodiments, but the spherical shape allows for possible alternative observation strategies for collection of the forward and side scatter of particles  140  through the input and output ports. Similar to the first and second embodiments, capillary tubing  120  provides the input and output of the flow cell  155  via a capillary  120  channel through the center of the cell. The capillary transport is positioned in the center of the spherical chamber  105 , to supply particles  140  from a flow cell  155 , which is positioned outside the chamber  105 , through a focus position  125  of the integration laser  130 . Frequency scattering ports  135  are typically located at the appropriate positions on the sphere to allow laser  130  focus as well as forward and side scatter detection ports  135 . The ports  150  for light beam introduction and the forward scatter measurement typically lie in a line that contains the focal point  125  of the sphere  137 . The side scatter port  135  and focal position  125  of the hemisphere lie in a line intersecting the laser port  150 , focal point of the hemisphere  125 , and the forward scatter port  135 . Optional ports can be added for direct imaging of the interrogated particles  140 . Particles  140  emitted from the particle interrogation position  125  are collected by reflection and emitted out of the non-imaging reflector output positions  145 . The collected particles  140  can then be evaluated though methods commonly used in the art such as a spectrometer or other means of evaluation or measurement. The surface of the sphere is typically covered with a pixelated multispectral wavelength detector  160  to capture the particles released from the chamber  105 . The pixelated multispectral wavelength detector  160  typically encapsulates the entire spherical chamber  105  in its entirety, but for the side and forward scattering observation ports  135  left clear for observation analysis. Alternatively, the chamber  105  may be coated with a broadband anti-reflective coating  162 , to restrain the radiation  140  in the chamber  105 , if no measuring device is encapsulating the chamber  105  for particle  140  analysis. Fiber optics  50  may also be used to transport the input or output of the chamber  105 . Additional observation ports  135  may be added to the chamber  105  to allow imaging of the interrogation point  125 . 
     In any of the above embodiments, an ultrasonic transducer  300  can be used to generate ultrasonic waves  305  to guide particle  40 ,  90 ,  140  flow such that corresponding ultrasonic wave pressure acts as a gate allowing particles to pass through the focal point  25 ,  80 ,  125  individually. The particles  40 ,  90 ,  140  may be injected into the embodiment  1 ,  55 ,  101  at an input point  310  between the ultrasonic transducer  300  and the focal point  25 ,  80 ,  125 . Once the particles  40 ,  90 ,  140  are aligned past the focal point  25 ,  80 ,  125 , the separated particles  40 ,  90 ,  140  and may be evaluated from the output ports  315  though methods commonly used in the art such as a spectrometer or other means of evaluation or measurement. 
     A fourth embodiment useful for assisting in spectral analysis of fluorescent radiation as generated and connected in the above cytometry systems  1 ,  55 ,  101 , illustrated in  FIG. 6  and  FIG. 7 , is a multispectral wavelength selective detector  250  made of multiple layers of waveguides  290 . The layers of waveguides  290  are dimensionally sized to either allow the transmission of incident wavelengths  255 , to be collected for analysis later, or absorption of a selected wavelength  255  for measurement. Longer wavelengths  255  are measured toward the input  265  of the detector, with shorter wavelengths measured by layers father into the lights path&#39;s penetration into the detector  250 . The layers  290  are composed of alternating insulator  270  layer, N layer  275  then P layer  280  repeatedly stacked in consecutive order. Coatings may be applied to the waveguide  290  layers to increase light transmission into the detector  250 . Overall waveguide  290 /detector  250  construction is of a pixelated architecture  285 . The detector  250  can be used to encapsulate reflective chambers, such as those described by the third embodiment, to analyze the particles released from the chamber. This detector  250  architecture may be applied to other measurement or observation methods for wavelength  225  analyses. 
     It will be appreciated that several of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also, that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. 
     While the claimed technology has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character. It is understood that the embodiments have been shown and described in the foregoing specification in satisfaction of the best mode and enablement requirements. It is understood that one of ordinary skill in the art could readily make a nigh-infinite number of insubstantial changes and modifications to the above-described embodiments and that it would be impractical to attempt to describe all such embodiment variations in the present specification. Accordingly, it is understood that all changes and modifications that come within the spirit of the claimed technology are desired to be protected.