Abstract:
A flow cytometry system having a flow channel defined through the thickness of a substrate is disclosed. Fluid flowing through the flow channel is illuminated by a first plurality of surface waveguides that are arranged around the flow channel in a first plane, while a second plurality of surface waveguides arranged around the flow channel in a second plane receive light after it has interacted with the fluid. The illumination pattern provided to the fluid is controlled by controlling the phase of the light in the first plurality of surface waveguides. As a result, the fluid is illuminated with light that is uniform and has a low coefficient of variation, improving the ability to distinguish and quantify characteristics of the fluid, such as cell count, DNA content, and the like.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to biotechnology in general, and, more particularly, to flow cytometry. 
       BACKGROUND OF THE INVENTION 
       [0002]    Flow cytometry is a technique in which a fluid-flow system organizes cells within a stream of fluid such that the cells pass in single-file through a detection zone. As the cells pass through the detection zone, they are illuminated by laser light, which scatters from each cell in a manner that depends on its structure. Most modern flow cytometry approaches also employ numerous fluorochrome-labeled antibodies that selectively bind with specific cellular features, such as cell-associated molecules, proteins or ligands. When excited by light at their respective excitation wavelengths, each fluorochrome emits a characteristic fluorescence signal, indicating the presence of that fluorochrome-specific feature. The scattered light and fluorescence signals are detected and analyzed to classify and/or count the cells according to a set of parameters of interest. In some cases, once classified, the cells are sorted into sub-populations based on their particular characteristics. 
         [0003]    Flow cytometry enables simultaneous multi-parameter analysis of individual cells in a fluid stream, such as analysis of cell surfaces and intracellular molecules, characterization and definition of different cell types in mixed cell populations, assessing the purity of isolated subpopulations, and analyzing cell size and volume. Flow cytometers are used in many clinical and biological applications, such as the diagnosis of blood cancers, basic research, clinical practice, and clinical trials. 
         [0004]    Historically, fluid-flow systems in conventional flow cytometers have been of a stream-in-air configuration, in which the fluid stream is forced through a nozzle system so the cells pass in single file through a detection zone in open air. Other prior-art flow cytometers employ a flow cell configuration, wherein a sheath fluid hydrodynamically focuses the sample fluid into the core of an open stream that traverses the detection zone. Unfortunately, in each case, such prior-art flow cytometers have some significant disadvantages: (1) they are quite expensive; (2) they have a large footprint; (3) they are not easily portable; and (4) they require extensive time, expertise, and expense to use and maintain. In addition, systems having an open-flow design are difficult to adapt for use with infectious disease or pathogenic microbiological samples because of the risk of exposure. 
         [0005]    To mitigate some of these disadvantages, microfluidics-based flow cytometers have been developed in which the sample fluid passes through the detection zone in an enclosed flow channel. The adoption of microfluidics approaches also enables increased on-chip functionality, such as filtering, cell sorting, and overall flow control. 
         [0006]    Microfluidics-based flow cytometers are disclosed, for example, in U.S. Patent Publication No. 2009/0051912, which describes a flow cytometer system that is smaller and more portable than an open-flow system. In operation, the fluid-flow system is held under a microscope objective, which functions as an external optics system that provides the light used to interrogate the cells and collect light scattered or emitted from the detection zone. 
         [0007]    In fact, most conventional flow cytometers rely on external optics for illuminating the detection zone and/or detecting the scattered light signals. Unfortunately, this limits how small and portable a flow cytometer can be made. In addition, careful alignment between the fluid-flow system and the external optics is critical for realizing precise and accurate measurements, and this alignment must be maintained during use to ensure proper system operation. Further exacerbating these issues, the use of several fluorochromes usually gives rise to a need for multiple lasers at different excitation wavelengths to excite the pallet of fluorochromes employed. Still further, numerous wavelength-filtered detectors are required to effectively discriminate between the resultant fluorescence signals. As a result, the use of external optics can add significant cost to a flow cytometry system. 
         [0008]    Integrating optical surface waveguides with microfluidics fluid-flow systems offers some promise for mitigating some of the disadvantages of external optics-based flow cytometers. Examples of a microfluidics-based system having integrated optical surface waveguides are disclosed in U.S. Pat. No. 7,764,374, in which both fluid-flow channels and SU-8-based optical surface waveguides are formed on the top surface of a substrate. One SU-8 surface waveguide emits light into an analysis zone of the fluid-flow channel, while a second SU-8 surface waveguide, located across the fluid-flow channel, collects light after it has passed through the analysis zone. 
         [0009]    In similar fashion, U.S. Patent Publication No. 2013/0083315 discloses flow cytometer arrangements having a first flow channel that includes a detection zone, and a plurality of “surface waveguide channels” that are adjacent to the detection zone. The surface waveguide channels are filled with fluid that laterally guides light captured from the detection zone to other regions of the substrate. 
         [0010]    Unfortunately, such prior-art systems suffer from several disadvantages. It is often necessary to couple several independent light signals into or out of a single region. SU-8-based surface waveguides and fluid-filled surface waveguides require significant chip real estate, however. As a result, forming more than few optical surface waveguides that access the same location can be challenging. 
         [0011]    Further, flow cytometry performance is improved when the detection zone is illuminated with substantially uniform light. Prior-art, microfluidics-based flow cytometers, however, are limited to providing illumination from one side of the fluid channel. As a result, uniform illumination of the sample fluid is precluded and system sensitivity is degraded. 
       SUMMARY OF THE INVENTION 
       [0012]    The present invention enables lab-on-a-chip systems having improved illumination of a fluid stream and/or improved detection of light signals that arise from the fluid stream. As a result, embodiments of the present invention are able to provide better system performance, less measurement variation, and higher sensitivity than prior-art lab-on-a-chip systems. For example, lab-on-a-chip-based flow cytometers in accordance with the present invention can distinguish different subsets of cells with improved precision and can better quantify measurement parameters than flow cytometers known in the prior art. Although the present invention is particularly well suited for use in flow cytometers, it provides advantages in other lab-on-a-chip systems as well, such as spectrometers, and the like. 
         [0013]    An illustrative embodiment of the present invention is a flow cytometry system having a fluid channel formed through the thickness of a substrate, and two sets of surface waveguides disposed on a surface of the substrate. Each set of surface waveguides is arranged such that its end facets form a circular arrangement around the flow channel. Each set of surface waveguides is formed in a different plane that is substantially orthogonal with the direction of fluid flow through the channel. 
         [0014]    A first set of surface waveguides is used to illuminate the detection zone. Light from these excitation waveguides forms a substantially uniform illumination pattern in the flow channel. In some embodiments, the phase of the light in one or more of the excitation waveguides is controlled, thereby enabling control over the shape of the illumination pattern in the detection zone. 
         [0015]    The second set of surface waveguides is used to capture light after it has interacted with the fluid in the detection zone. In some embodiments, the phase of the light in one or more of these collection waveguides is controllable. In some embodiments, at least one of the collection waveguides is optically coupled with a wavelength filter that discriminates spectral information in the light coupled into that collection waveguide. 
         [0016]    In some embodiments, at least one set of surface waveguides is arranged such that their facets form a polygonal arrangement around the flow channel. In some of these embodiments, each side of the polygon includes a plurality of surface waveguide facets. 
         [0017]    An embodiment of the present invention is an apparatus comprising: a substrate that defines a first plane, the substrate comprising a flow channel that is operative for conveying fluid along a first direction that is substantially orthogonal to the first plane, the flow channel being located within a first region of the substrate; a first surface waveguide that is optically coupled with the flow channel, the first surface waveguide being located in a second plane within the first region, wherein the second plane is substantially parallel with the first plane; and a second surface waveguide that is optically coupled with the flow channel in the first region, the second surface waveguide being located in a third plane within the first region, wherein the third plane is substantially parallel with the second plane. 
         [0018]    Another embodiment of the present invention is an apparatus comprising: a substrate having a thickness between a first major surface and a second major surface; a first flow channel that is operative for conveying fluid through the thickness; a first plurality of surface waveguides, each of the first plurality of surface waveguides being optically coupled with the flow channel in a first region, the first plurality of surface waveguides being coplanar in a first plane within the first region; and a second plurality of surface waveguides, each of the second plurality of surface waveguides being optically coupled with the flow channel, the second plurality of surface waveguides being coplanar in a second plane within the first region; wherein, the first major surface, the second major surface, the first plane, and the second plane are substantially parallel. 
         [0019]    Yet another embodiment of the present invention is a method comprising: conveying a first fluid along a first direction through a first region; interrogating the first fluid with a first illumination pattern that is based on a first light signal emitted from a first surface waveguide that lies in a first plane that is orthogonal to the first direction in the first region; and coupling a first portion of the first illumination pattern into a second surface waveguide that lies in a second plane that is orthogonal to the first direction in the first region, wherein the first portion is coupled into the second surface waveguide after the first illumination pattern has interacted with the first fluid. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0020]      FIGS. 1A-C  depict examples of prior-art microfluidic systems with integrated surface waveguides. 
           [0021]      FIG. 2  depicts a block diagram of a flow cytometer in accordance with an illustrative embodiment of the present invention. 
           [0022]      FIG. 3  depicts operations of a method for performing flow cytometry in accordance with the illustrative embodiment. 
           [0023]      FIG. 4  depicts a schematic drawing of a cross-sectional view of an optofluidic system in accordance with the illustrative embodiment of the present invention. 
           [0024]      FIG. 5  depicts operations of a method for forming optofluidic system  204 . 
           [0025]      FIG. 6  depicts a top view of optics plate  404 . 
           [0026]      FIGS. 7A-B  depict top and cross-sectional views of region  602  of optics plate  404 . 
           [0027]      FIG. 8  depicts sub-operations suitable for use in forming optics plate  404 . 
           [0028]      FIG. 9A  depicts a schematic drawing of a top view of region  602  after the definition of waveguide cores  706 . 
           [0029]      FIG. 9B  depicts a schematic drawing of a top view of region  602  after the definition of waveguide cores  714 . 
           [0030]      FIG. 10  depict a side view of detection zone  414  during interrogation of a cell  226 . 
           [0031]      FIG. 11  depicts a schematic drawing of a top view of region  602  in accordance with a first alternative embodiment of the present invention. 
           [0032]      FIGS. 12A-C  depict simulated illumination patterns across detection zone  1106  for different wavelengths of light. 
           [0033]      FIGS. 13A-C  depict plots of random phase field distribution across detection zone  1106  for different wavelengths of excitation light. 
           [0034]      FIG. 14  depicts an optics plate in accordance with a second alternative embodiment of the present invention. 
           [0035]      FIGS. 15A and 15B  depict cross-section views of phase-control elements  1402 -E-i and  1402 -C-i, respectively, in accordance with the second alternative embodiment of the present invention. 
           [0036]      FIG. 16  depicts simulation results for the change in effective refractive index for excitation waveguide core  706 - i  as a function of thickness, length, and width of piezoelectric layer  1506 . 
           [0037]      FIG. 17  depicts a picture of a conventional flow cytometer flow cell in accordance with the prior art. 
           [0038]      FIGS. 18A-B  depict a flow cytometry flow cell in accordance with a third alternative embodiment of the present invention. 
       
    
    
     DETAILED DESCRIPTION 
       [0039]      FIGS. 1A-C  depict examples of prior-art microfluidic systems with integrated surface waveguides. 
         [0040]      FIG. 1A  depicts a portion of a Micro Total Analysis System (JAS) that includes flow channel  102 , illumination waveguide  104 , and collection waveguide  106 , all of which are formed on the top surface of substrate  108 . System  100  is in accordance with lab-on-a-chip (LOC) systems disclosed by J. Hubner, et al., in U.S. Pat. No. 7,764,374, issued Jul. 27, 2010, which is incorporated herein by reference. 
         [0041]    System  100  is an example of an absorption spectroscopy system. In operation, illumination waveguide  104  emits light signal  120  into analysis zone  118 , which is defined by the area between illumination waveguide  104  and collection waveguide  106 . Fluid  124  absorbs certain characteristic wavelengths of the light based on the constituents of the fluid. Some of the light not absorbed by the fluid is captured as light signal  122  by collection waveguide  106 , which carries the light to a wavelength dispersion system (not shown) that enables its spectral analysis. 
         [0042]    Illumination waveguide  104  and collection waveguide  106 , as well as flow channel  102 , are formed on the top surface of substrate  108 . Substrate  108  typically comprises silicon, on which a layer of silicon dioxide (i.e., lower cladding  110 ) is formed as a lower cladding layer for the waveguides. Substrate  108  defines substrate plane  114 , which is aligned with the x-y plane. 
         [0043]    Each of the waveguides comprises a core region of SU-8 that is surrounded by layer  112 , which acts to laterally confine light signals  120  and  122  in the waveguides. Layer  112  is typically a layer of silicon dioxide disposed on lower cladding layer  110 . It should be noted that layer  112  defines plane  116 , which is parallel to substrate plane  114 . Flow channel  102  and waveguides  104  and  106  are all coplanar in plane  116 . 
         [0044]    To complete the waveguide structures and enclose flow channel  102 , a second substrate (not shown for clarity) having a third layer of silicon dioxide is bonded to layer  112 . 
         [0045]    While mitigating some of the drawbacks related to microfluidics-based analytical systems discussed above, system  100  still has some significant drawbacks. For example, by forming all surface waveguides and flow channels such that they are coplanar, optical access to analysis zone  118  is limited to primarily only one surface waveguide pair. As a result, simultaneous interrogation of analysis zone by more than one light signal travelling along diverse paths is precluded. In order to interrogate fluid  110  with multiple light signals, therefore, multiple pairs of illumination and collection waveguides are required, which leads to increased chip real estate for system  100  and commensurately higher cost. Further, in applications where it is desirable to collect light scattered by material in analysis zone  118 , only forward-scattered light can be collected via a collection waveguide. Surface waveguides formed at positions to capture side-scattered light would, in general, be separated by a relatively large distance, making it difficult for a surface waveguide to capture sufficient light for a reliable measurement. Further, the use of SU-8 in system  100  can lead to degradation over time, particularly when the system is used for short wavelengths and/or high intensities, due to absorption of the light. 
         [0046]      FIGS. 1B-C  depicts schematic drawings of a top and cross-section view, respectively, of another example of a lab-on-a-chip system having integrated microfluidics and surface waveguides. System  126  is an example of a portion of a partially integrated flow cytometer. System  126  includes flow channel  128 , surface waveguides  130 - 1  through  130 -N, and lasers  132 - 1  through  132 -N. System  126  is in accordance with flow cytometers described by C. Vannahme, et al., in “Plastic lab-on-a-chip for fluorescence excitation with integrated organic semiconductor lasers,” Optics Express, Vol. 19, No. 9, pp. 8179-8186 (2011), which is incorporated herein by reference. 
         [0047]    System  126  includes flow channel  128 , surface waveguides  130  and laser  132 , all of which are monolithically integrated on substrate  138 . Like system  100  described above, all flow channels and surface waveguides are coplanar in substrate plane  150 . 
         [0048]    Substrate  138  is a poly(methyl methacrylate) (PMMA) substrate into which flow channel  128  and depressions  140  are formed using conventional plastic imprinting techniques. Depressions  140  are formed such that the bottom of each depression is characterized by a nascent grating structure  142 , which is later coated with a thin film of organic semiconductor tris(8-hydroxyquinoline) aluminum (Alq 3 ) to form organic semiconductor lasers  132 . 
         [0049]    Surface waveguides  130  are formed directly in the PMMA material by exposing it to deep UV light, which breaks the molecular chains in the PMMA material to locally increase its refractive index. The unexposed PMMA retains its original, lower refractive index enabling it to serve as cladding material for the waveguides. 
         [0050]    After the surface waveguides have been defined, PMMA cover  144  is joined to substrate  138  to complete the fabrication of system  126 . 
         [0051]    In operation, lasers  132  are optically pumped to generate light signals  146 - 1  through  146 -N, which couple into surface waveguides  130 - 1  through  130 -N, respectively. Light signals  146  are used to excite the different fluorochromes used to stain analytes in fluid  136 . As the cells in the fluid flow sequentially through each of detection zones  134 - 1  through  134 -N, fluorochromes selectively bound to features of the cells fluoresce at their characteristic fluorescence wavelengths as light signals  148 - 1  through  148 -N. 
         [0052]    Fluorescence signals  148  propagate out of plane  150  and are detected via a free-space optics-based detection system. 
         [0053]    The need to provide different detection zones so that multiple excitation signals can be used to excite the full pallet of fluorochromes adds significant complexity to system  126  and its operation. For example, because the fluorochromes are not excited simultaneously, ambiguity can creep into the measurement results. Further, the need for an external free-space detection system mitigates many of the benefits of integrating flow channel  128  and surface waveguides  130 . Still further, as discussed above, multiple detection zones requires more chip real estate, which leads to higher system cost. 
         [0054]    The present invention enables improved flow cytometry by arranging a plurality of surface waveguides in a plane that is not co-planar with the direction in which a flow channel conveys a fluid. As a result, the facets of the surface waveguides can be arranged on different sides of the flow channel. The present invention, therefore, enables greater control over the manner in which the fluid is illuminated. It also improves the ability to collect light from the flow channel by enabling collection of light close to the flow channel even though the light exits the flow channel along different directions. 
         [0055]    It should be noted that, while the present invention is particularly well suited for flow cytometry, it can also provide similar advantages in other microfluidic applications, such as spectroscopy, chemical synthesis, capillary electrophoresis, lab-on-a chip applications, and the like. 
         [0056]      FIG. 2  depicts a block diagram of a flow cytometer in accordance with an illustrative embodiment of the present invention. Flow cytometer  200  includes light source  202 , optofluidic system  204 , detector  206 , and processor  208 . 
         [0057]    System  200  is operative for analyzing cells  226 , which are contained in liquid-phase fluid  224 . In some embodiments, system  200  is operative for other particles contained in a liquid-phase medium. In some embodiments, system  200  is operative for particles and/or cells contained in a gas-phase medium (e.g., air, etc.). In some applications, system  200  is operative for a gas-phase or liquid-phase fluids that are substantially particle-free. 
         [0058]      FIG. 3  depicts operations of a method for performing flow cytometry in accordance with the illustrative embodiment. Method  300  begins with operation  301 , wherein optofluidic system  204  is provided. Method  300  is described herein with continuing reference to  FIG. 2 , as well as reference to  FIGS. 4-10 . 
         [0059]    Optofluidic system  204  is a monolithically integrated system that includes fluid-flow system  212  and detection system  214 . Detection system  214  comprises surface-waveguide-based excitation network  216 , surface-waveguide-based collection network  218 , and a portion of fluid-flow system  212 . Optofluidic system  204  is described in more detail below and with respect to  FIGS. 4-9 . 
         [0060]      FIG. 4  depicts a schematic drawing of a cross-sectional view of an optofluidic system in accordance with the illustrative embodiment of the present invention. Optofluidic system  204  includes channel plates  402 - 1  and  402 - 2 , and optics plate  404 . These plates collectively define each of fluid-flow system  212  and detection system  214 . 
         [0061]      FIG. 5  depicts operations of a method for forming optofluidic system  204 . Method  500  begins with operation  501 , wherein channel plates  402 - 1  and  402 - 2  are formed. 
         [0062]    Each of channel plates  402 - 1  and  402 - 2  is a conventional microfluidic channel plate formed via conventional methods (e.g., reactive-ion etching (RIE), wet-chemical etching, sand-blasting, etc.). Channel plates  402 - 1  and  402 - 2  include channel networks  406 - 1  and  406 - 2 , respectively, each of which is formed in a conventional planar processing substrate. Channel plates  402 - 1  and  402 - 2  also include vias  410  and ports  412  distributed among the channel plates and optics plate  404  to enable interconnection of the channel networks to each other and interconnection of fluid-flow system  212  to external facilities, such as fluid sources, waste containers, etc. 
         [0063]    Typically, for optical systems such as the illustrative embodiment, the channel plate substrates are made of fused silica because it does not exhibit significant autofluorescence. In some applications, however, the channel plate substrates comprise a material other than fused silica. Materials suitable for use in the channel plate substrates include, without limitation, glasses (e.g., silicon dioxide, borofloat glass, quartz, Pyrex, etc.), semiconductors (e.g., silicon, silicon carbide, germanium, GaAs, InP, etc.), metals, ceramics, plastics, composite materials, and the like. 
         [0064]    Each of channel networks  406 - 1  and  406 - 2  is a system of microfluidic channels suitable for, in combination with flow channel  408 , performing conventional fluidic operations on fluid  224 , such as flow separation, filtering, mixing, sorting, etc., as well as forcing the cells in fluid  224  to flow in single-file order through flow channel  408 . The specific arrangement and functionality of channel networks  406 - 1  and  406 - 2  is typically a matter of application-based design. Channel networks  406 - 1  and  406 - 2 , flow channel  408 , vias  410 , and ports  412  collectively define fluid-flow system  212 . 
         [0065]    At operation  502 , optics plate  404  is formed. 
         [0066]      FIG. 6  depicts a top view of optics plate  404 . Optics plate  404  includes flow channel  408 , excitation network  216 , collection network  218 , flow channel  408 , and vias  410 . In some embodiments, optics plate  404  is an element provided as a portion of a conventional flow-cytometer flow chamber to, for example, improve or replace a conventional optical excitation or collection system. 
         [0067]    Each of excitation network  216  and collection network  218  includes a plurality of N surface waveguides (referred to, individually, as excitation waveguide  604 - i  or collection waveguide  606 - i , where 1&lt;i&lt;N). Although N=8 in the illustrative embodiment, it will be clear to one skilled in the art, after reading this Specification, how to specify, make, and use alternative embodiments of the present invention wherein N is equal to any practical number, and can be as small as one. Further, one skilled in the art will recognize that excitation network  216  and collection network  218  can include different numbers of surface waveguides. 
         [0068]    In the illustrative embodiment, collection waveguides  606  are disposed in a plane located above excitation waveguides  604  in region  602 . In some embodiments, collection waveguides  606  are not disposed above excitation waveguides. Further, in some embodiments, excitation waveguides  604  lie in a plane that is above the plane of collection waveguides  606 . Still further, in some embodiments, at least some of excitation waveguides  604  and collection waveguides  606  lie in the same plane. In other embodiments, at least one of the pluralities of excitation waveguides  604  and collection waveguides  606  is distributed among two or more waveguide layers. 
         [0069]      FIGS. 7A-B  depict top and cross-sectional views of region  602  of optics plate  404 . Region  602  provides a detailed view of flow channel  408 , excitation waveguides  604 - 1  through  604 - 8 , and collection waveguides  606 - 1  through  606 - 8 . Within region  602 , excitation waveguides  604  and collection waveguides  606  are formed on substrate  608  such that their end facets are arranged in a substantially circular arrangement about the center of flow channel  408 . One skilled in the art will recognize, after reading this Specification, that the region of flow channel  408  that is surrounded by the end facets of excitation waveguides  604  and collection waveguides  606  substantially defines detection zone  414 . 
         [0070]      FIG. 8  depicts sub-operations suitable for use in forming optics plate  404 . Operation  502  begins with sub-operation  801 , wherein core layer  702  is formed on the top surface of substrate  608 . 
         [0071]    Substrate  608  is a planar substrate that is analogous to the channel plate substrates described above and, in the illustrative embodiment, comprises fused silica in order to suppress autofluorescence. In some embodiments, however, substrate  608  can comprise another material, as discussed above and with respect to channel plates  402 - 1  and  402 - 2 . Substrate  608  defines substrate plane  610 , which lies generally in the x-y plane, as indicated. 
         [0072]    Core layer  702  is a conventional planar layer of stoichiometric silicon nitride, deposited on the top surface of substrate  608  using low-pressure chemical vapor deposition (LPCVD). Core layer  702  has a thickness of approximately 25 nanometers (nm) and defines waveguide plane  704 . In some embodiments, core layer  702  has a different thickness. In some embodiments, core layer  702  comprises a material other than silicon nitride. Materials suitable for use in core layer  702  include any material through which excitation signals can propagate. In some embodiments, core layer  702  is formed with a different suitable formation process. 
         [0073]    In some embodiments, substrate  608  includes a surface layer, such as a silicon oxide, that functions as a lower cladding layer for waveguides formed from core layer  702 . 
         [0074]    At operation  802 , core layer  702  is patterned in conventional fashion to define waveguide cores  706 - 1  through  706 - 8  (referred to, collectively, as waveguide cores  706 ). Typically, core layer  702  is patterned via photolithography and RIE. Waveguide cores  706  are patterned such that each has a width within the range of approximately 1 micron to approximately 4 microns, and typically approximately 2 microns As a result, each of waveguide cores  706  defines a stripe waveguide that is suitable for single-mode operation at the wavelengths of light included in excitation light  210 . Each of waveguide cores  706  includes an end facet  708 , the plurality of which is arranged in a substantially circular arrangement about detection zone  414 . 
         [0075]    In some embodiments, at least one of waveguide cores  706  comprises different materials, is of a different waveguide type, and/or has different dimensions (i.e., thickness or width) than another of surface waveguide cores  706 . Different core materials, types, and/or dimensions enable surface waveguides that are preferable for, for example, different wavelengths, diverse functions (e.g., providing light to or collecting light from detection zone  414 , etc.), and the like. In some embodiments, therefore, at least one of operation  801  and  802  is repeated one or more times. 
         [0076]      FIG. 9A  depicts a schematic drawing of a top view of region  602  after the definition of waveguide cores  706 . 
         [0077]    At operation  803 , intermediate cladding  710  is formed in conventional fashion. Intermediate cladding  710  is a layer of silicon dioxide deposited via LPCVD. Typically, after formation, intermediate cladding  710  is planarized via chemical-mechanical polishing, or another suitable planarization technique. Intermediate cladding  710  has a thickness that is typically within the range of approximately 1 microns to approximately 30 microns. Intermediate cladding  710  operates as both an upper cladding for excitation waveguides  604  and a lower cladding for detection waveguides  606 . 
         [0078]    One skilled in the art will recognize that the thickness of intermediate cladding  710  is a matter of design and is based on several factors, such as the acceptable level of cross-talk between excitation network  216  and collection network  218 , acceptable levels of loss in the excitation and collection waveguides, and the like. 
         [0079]    In some embodiments, intermediate cladding  710  is formed via another deposition technique, such as plasma-enhanced chemical vapor deposition (PECVD), sputtering, spin-on glass deposition, and the like. In some embodiments, intermediate cladding  710  comprises a material other than silicon dioxide. One skilled in the art will recognize that the choice of material for intermediate cladding  710  is based on numerous factors, including the wavelength of light, the materials of substrate  608  and core layers  702  and  712 , material compatibility with fluid  224 , etc. 
         [0080]    At operation  804 , core layer  712  is formed on intermediate cladding  710 . Core layer  712  is analogous to core layer  702  described above; however, core layer  712  is formed such that it has a thickness of approximately 100 nm. Core layer  712  defines waveguide plane  716 . 
         [0081]    At operation  805 , core layer  712  is patterned in conventional fashion to define stripe waveguide cores  714 - 1  through  714 - 8  (referred to, collectively, as waveguide cores  714 ), which collectively define waveguide plane  716 . Waveguide plane  716  is substantially parallel with substrate plane  610 . Typically, core layer  712  is patterned via photolithography and RIE. 
         [0082]    In order to facilitate collection of light from detection zone  414 , waveguide cores  714  are patterned such that they have width within the range of approximately 1 micron to approximately 4 microns, and typically approximately 2 microns. As a result, each of waveguide cores  714  operates as a multimode waveguide core for the wavelengths of light in collected light  220 . Like waveguide cores  706 , waveguide cores  714  have end facets  718 , which are arranged in a substantially circular arrangement about detection zone  414 . In some embodiments, waveguide cores  714  have a different width or height. 
         [0083]    The dimensions for waveguide cores  706  (and/or waveguide cores  714  provided herein are merely exemplary. One skilled in the art will recognize that the specific dimensions of a waveguide depend on system and application considerations, and that any suitable dimensions for these waveguides is within the scope of the present invention. 
         [0084]    Further, as discussed above vis-à-vis waveguide cores  706 , in some embodiments, at least one of waveguide cores  714  comprises different materials, is of a different waveguide type, and/or has different dimensions (i.e., thickness or width) than another of surface waveguide cores  714 . Different core materials, types, and/or dimensions enable surface waveguides that are preferable for, for example, different wavelengths, diverse functions (e.g., providing light to or collecting light from detection zone  414 , etc.), and the like. In some embodiments, therefore, at least one of operation  804  and  805  is repeated one or more times. 
         [0085]      FIG. 9B  depicts a schematic drawing of a top view of region  602  after the definition of waveguide cores  714 . 
         [0086]    At operation  806 , top cladding layer  720  is formed on waveguide cores  714  to complete formation of collection waveguides  606 . Top cladding layer  720  is analogous to intermediate cladding layer  710 . 
         [0087]    It should be noted that, at the end of operation  806 , each of waveguide cores  706  and  714  extends past perimeter  902 , which defines the extent flow channel  408  as it will be formed in operation  807 . This ensures that each of the pluralities of end facets  708  and  718  will be arranged in a circular pattern located at the edge of the flow channel once it is formed, since the end facets are formed by the deep-RIE process used to form the flow channel. In some embodiments, the end facet of at least one surface waveguide is not formed during the operation in which flow channel  408  is formed. In some embodiments, one or more of end facets  708  and  718  is formed when its respective core layer is patterned to define its corresponding waveguide cores. 
         [0088]    Although the illustrative embodiment comprises excitation waveguides that operate as single-mode waveguides and collection waveguides that operate as multimode waveguides, it will be clear to one skilled in the art, after reading this Specification, how to specify, make, and use alternative embodiments of the present invention comprising at least one excitation waveguide that operates as a multimode waveguide and/or at least one collection waveguide that operates as a single-mode waveguide. 
         [0089]    In some embodiments, collection waveguides  606  are routed individually to the edge of substrate  608  and detected independently. Such an arrangement can, for example, enable maintenance of angle-dependent scattering information. 
         [0090]    At operation  807 , flow channel  408  is formed through the thickness of substrate  608  and its surface layers. Flow channel  408  has a diameter within the range of approximately 20 microns to approximately 120 microns, and is typically approximately 40 microns, which restricts cells in fluid  224  to single-file flow through detection zone  414 . It should be noted that the present invention is applicable to applications other than flow cytometry, wherein the size of flow channel  408  does not necessarily restrict the size of particles or cells in fluid  224 . One skilled in the art will recognize, therefore, that the diameter of flow channel  408  is a matter of application-based design considerations. 
         [0091]    Although in the illustrative embodiment, flow channel  408  is formed via conventional deep-RIE, it will be clear to one skilled in the art, after reading this Specification, how to specify, make, and use alternative embodiments wherein flow channel  408  is formed via a different process, such as sand blasting, laser ablation, wet etching, etc., are also suitable for the formation of the flow channel depending on the materials used in system  200 . 
         [0092]    Returning now to method  500 , at operation  503 , channel plates  402 - 1  and  402 - 2  are joined with optics plate  404  such that channel networks  406 - 1  and  406 - 2  are fluidically coupled with flow channel  408 . In the illustrative embodiment, the plates are joined using a wafer bonding technique, such as fusion bonding, thermo-anodic bonding, etc., to fuse the three plates into a single monolithic element. In some embodiments, the plates are joined via another suitable method, such as clamping, etc. In some embodiments, a fluidic seal is formed between the fluidic elements on each plate using intervening elements, such as O-rings, gaskets, deposited material (e.g., polyimide, SU-8, PMMA, etc.) and the like. 
         [0093]    At operation  504 , ports  414  are fluidically coupled with external fluidic systems, such as a reservoir, pumping system, and waste container for fluid  224 . 
         [0094]    At operation  505 , excitation network is optically coupled with light source  202 . 
         [0095]    Light source  202  is a conventional multi-spectral light source that provides excitation light having wavelengths suitable for exciting the full pallet of fluorochromes used during operation of flow cytometer  200 . In some embodiments, light source  202  includes a plurality of light emitting devices and/or spectral filters, such as lasers, light-emitting diodes (LEDs), superluminescent diodes, and the like. 
         [0096]    At operation  506 , collection network is optically coupled with detector  206 . 
         [0097]    Detector  206  is a conventional detection system operative for detecting one or more of the wavelengths included in collected light  220 , which is received from optofluidic system  204 . Detector  206  includes a plurality of detectors and wavelength filters suitable for discriminating fluorescence signals and scattered signals collected by collection network  216 . Detector  206  provides output signal  222  to processor  208 . 
         [0098]    Processor  208  is a conventional processing system operative for receiving output signal  222  and conducting analysis of the output signal to estimate one or more parameters of fluid  224  and/or cells  226 . 
         [0099]    Returning now to method  300 , at operation  302 , fluid  224  is pumped through optofluidic system  212 , from reservoir  228  to waste container  230 , such that its constituent cells  226  flow through detection zone  414  along flow direction  724 . Flow direction  724  is aligned with the z-direction, as depicted in  FIG. 7B , which is orthogonal to each of substrate plane  610 , and waveguide planes  704  and  716 . In some embodiments, flow direction  724  is not orthogonal with waveguide planes  704  and  716 ; however, it should be noted that it is an aspect of the present invention that flow direction  724  is neither parallel nor coplanar with either of the waveguide planes. 
         [0100]    At operation  303 , light source  202  provides light signal  210  to optofluidic system  204 . 
         [0101]    At operation  304 , cells  226  are interrogated with excitation light  210 . 
         [0102]      FIG. 10  depicts a side view of detection zone  414  during interrogation of a cell  226 . Excitation light is provided to cell  226  by excitation waveguides  604 . 
         [0103]    Interrogation of cell  226  with excitation light  210  gives rise to output light  1002 , which includes forward-scattered, side-scattered, and fluorescent light signals as discussed above. 
         [0104]    At operation  305 , collection waveguides  606  capture a portion of output light  1002  as collected light  220 . 
         [0105]    At operation  306 , collection waveguides  606  convey collected light  220  to detector  206 , which converts it into output signal  222 . 
         [0106]    At operation  307 , processor  208  performs analysis of output signal  222  and provides an estimate of the parameters of interest for cells  226 . 
         [0107]      FIG. 11  depicts a schematic drawing of a top view of region  602  in accordance with a first alternative embodiment of the present invention. System  1100  is analogous to system  200  described above; however, system  1100  includes excitation and collection waveguide pairs that are arranged in arrays that collectively form a polygonal arrangement that surrounds flow channel  408 . 
         [0108]    Each waveguide pair  1102  includes one excitation waveguide  604  and one collection waveguide  606 , as described above and with respect to  FIGS. 7A-B . 
         [0109]    Waveguide pairs  1102  are arranged in waveguide arrays  1104 - 1  through  1104 - 8  (referred to, collectively, as waveguide arrays  1104 ), each of which includes eight waveguide pairs arranged in linear fashion. Waveguide arrays  1104  form an octagonal arrangement that is concentric with flow channel  408 . Although in the example shown, waveguide arrays  1104  form a polygon having eight sides, it will be clear to one skilled in the art, after reading this Specification, how to specify, make, and use alternative embodiments wherein waveguide arrays  1104  form a polygon having any practical number of sides. Further one skilled in the art will recognize that waveguide arrays  1104  can include any practical number of waveguide pairs. 
         [0110]    The arrangement of waveguide arrays  1104  about flow channel  408  gives rise to detection zone  1106  having a substantially circular cross-section. In some embodiments, detection zone  1106  has a cross-sectional shape other than circular. 
         [0111]      FIGS. 12A-C  depict simulated illumination patterns across a diameter of a detection zone for different wavelengths of excitation light. 
         [0112]    Patterns  1200 ,  1202 , and  1204  show the illumination pattern across a 120-micron diameter flow channel for TM-polarized light at wavelengths of 404, 532, and 632 nm, respectively. 
         [0113]      FIGS. 13A-C  depict plots of random phase field distribution across detection zone  1106  for different wavelengths of excitation light. 
         [0114]    Plots  1300 ,  1302 , and  1304  depict the distribution of optical power across a 160-micron diameter flow channel for TE-polarized light at wavelengths of 404, 532, and 632 nm, respectively. 
         [0115]      FIGS. 12 and 13  evince that substantially uniform illumination can be realized by providing excitation light from an octagonal pattern of waveguide arrays in accordance with the first illustrative embodiment. 
         [0116]    Although the waveguide arrangements described above enable significant improvement in illumination of a flow channel region, the illumination pattern for any wavelength is determined purely by the arrangement of the facets about the region and are not controllable during operation. It would be desirable to enable control over the shape of the illumination pattern during use, however. 
         [0117]    It is another aspect of the present invention that control over the illumination pattern in the detection zone can be gained by controlling the phase and/or amplitude of the light launched by one or more excitation waveguides into detection zone  414 . In some embodiments, this enables beam shaping capable of providing specific illumination patterns having local intensity maxima at discrete positions within detection zone  414 . 
         [0118]    Further, identification of the light signals captured by an individual collection waveguide can also be improved by controlling the phase of the light signal in that waveguide. 
         [0119]    In some embodiments, at least one of collection waveguides  606  is a single-mode waveguide that includes a polarization filter. Further, in some of these embodiments, excitation light  210  is provided is polarized (e.g., as TM light). In such a configuration, the present invention enables detection of light that is partially converted to another polarization mode (e.g., TE), which provides an indication as to particle shape (e.g., ratio of diameter versus length, etc.), as described by N. G. Khlebtsov, et al., in “Can the Light Scattering Depolarization Ratio of Small Particles Be Greater Than 1/3 ?” J. Phys. Chem. B , Vol. 2005, No. 109, pp. 13578-13584 (2005), which is incorporated herein by reference. 
         [0120]      FIG. 14  depicts an optics plate in accordance with a second alternative embodiment of the present invention. Optics plate  1400  is analogous to optics plate  404  and includes all of the same structure; however, optics plate  1400  also includes phase-control elements  1402 -E- 1  through  1402 -E- 8  and phase-control elements  1402 -C- 1  through  1402 -C- 8  (referred to, collectively, as PCE  1402 -E and PCE  1402 -C, respectively). Each of phase-control elements  1402 -E and  1402 -C is operatively coupled with an excitation waveguide or collection waveguide such that it can control the phase of a light signal propagating through the waveguide. 
         [0121]      FIGS. 15A and 15B  depict cross-section views of phase-control elements  1402 -E-i and  1402 -C-i, respectively, in accordance with the second alternative embodiment of the present invention. Each of phase-control elements  1402 -E-i and  1402 -C-i comprises strain element  1502  that is operatively coupled with its respective waveguide core. Strain element  1502  includes lower electrode  1504 , piezoelectric layer  1506 , and upper electrode  1508 . 
         [0122]    Each of lower electrode  1504  and upper electrode  1508  is a layer of electrically conductive material, such as platinum, gold, aluminum, etc. The thickness of lower electrode  1504  and upper electrode  1508  is a matter of design choice. 
         [0123]    Piezoelectric layer  1506  is a layer of piezoelectric material, such as lead zirconate titanate (PZT), having thickness, t. Piezoelectric layer  1506  is patterned to form a substantially rectangular region on which upper electrode  1508  is formed. One skilled in the art will recognize that the width, w, and length, L, of upper electrode  1508  (where w is the dimension of the layer along the direction transverse to the axial direction of its underlying waveguide and L is the dimension of the layer along the axial direction of its underlying waveguide) effectively define the operative dimensions of strain element  1502 . As discussed below and with respect to  FIG. 16 , the operational characteristics of PCE  1402 -E and PCE  1402 -C are based on the values of t, w, and L. 
         [0124]    In some embodiments, one or both of piezoelectric layer  1506  and lower electrode  1504  are not patterned and, therefore, remain over the entire surface of the substrate. In such embodiments, vias are formed through the piezoelectric material to enable electrical contact to be established to the underlying lower electrode. 
         [0125]    In PCE  1402 -E-i, strain element  1502  is disposed on intermediate cladding  710  in a region where core layer  712  has been removed during patterning of collection waveguide cores  714 . 
         [0126]    In PCE  1402 -C-i, strain element  1502  is disposed upper cladding  722  in a region where core layer  702  has been removed during patterning of excitation waveguide cores  706 . 
         [0127]    Processor  208  provides control signals  1510 -E-i and  1510 -C-i to each of PCE  1402 -E-i and PCE  1402 -C-i, respectively. These control signals apply a voltage differential between lower electrode  1504  and upper electrode  1508 , which gives rise to strain in piezoelectric layer  1506 . This strain is transmitted into the underlying waveguide core, resulting in a change in its effective refractive index. 
         [0128]    In similar fashion, the phase of light propagating through collection waveguide  606 - i  is controlled by control signal  1508 -C-i, provided by processor  208 . Control signal  1508 -C-i is a voltage differential applied to lower electrode  1504  and upper electrode  1508 , disposed on upper cladding  722 , as shown. Application of a voltage differential to the electrodes of PCE  1402 -C-i give rise to strain in piezoelectric layer  1506 , which is transmitted into waveguide guide core  714 - i , resulting in a change in its refractive index. 
         [0129]      FIG. 16  depicts simulation results for the change in effective refractive index for excitation waveguide core  706 - i  as a function of the thickness of piezoelectric layer  1506  and the length, L, and width, w, of upper electrode  1508 . 
         [0130]    Plot  1600  shows the change in refractive index for excitation waveguide core  706 - i , as a function of upper electrode width, w, for a piezoelectric layer  1506  having a thickness of 0.5 micron and a upper electrode length of 13.99 mm. 
         [0131]    Plot  1602  shows the change in refractive index for excitation waveguide core  706 - i , as a function of upper electrode width, w, for a piezoelectric layer  1506  having a thickness of 1.0 micron and a upper electrode length of 7.66 mm. 
         [0132]    Plot  1604  shows the change in refractive index for excitation waveguide core  706 - i , as a function of upper electrode width, w, for a piezoelectric layer  1506  having a thickness of 1.5 micron and a upper electrode length of 5.51 mm. 
         [0133]    Plot  1606  shows the change in refractive index for excitation waveguide core  706 - i , as a function of upper electrode width, w, for a piezoelectric layer  1506  having a thickness of 2.0 micron and a upper electrode length of 4.45 mm. 
         [0134]    Plots  1600  through  1606  that a significant change in refractive index can be achieved in a waveguide core operatively coupled with strain element  1502 , which will give rise to a commensurate change in phase for a light signal propagating through the waveguide. 
         [0135]    One skilled in the art will recognize that piezoelectric-layer-based PCE  1042  is merely one example of a phase control element within the scope of the present invention. For example, phase can be controlled via thermo-optic modulation (i.e., via a heater disposed on a waveguide), birefringence modulation using a magnetostrictive element, etc. Further, in some embodiments, control over the illumination pattern in the detection zone is provided by controlling amplitude of the light launched by one or more excitation waveguides using an amplitude modulator, such as a Mach-Zehnder interferometer structure. 
         [0136]    In some embodiments, at least some of excitation waveguides  604  include phase and/or amplitude controllers such that the excitation waveguides are operative for steering an illumination pattern around detection zone  414 . As a result, a single fixed-location collection waveguide can be used to collect scattered/fluorescent light from the detection zone. In some cases, this affords a simpler detection scheme and/or enables the use of a single large and sensitive detector (e.g., an avalanche photodiode, photomultiplier tube, etc.) APD to detect output optical signals that are too weak to collect with a conventional detector array. By correlating the detected light with the direction of the steered illumination pattern, angular information is retained. 
         [0137]      FIG. 17  depicts a picture of a conventional flow cytometer flow cell in accordance with the prior art. Flow cell  1700  includes cell body  1702 , channel  1704 , fluid port  1706 , and lens  1708 . 
         [0138]    Channel  1704  is formed through the length of cell body  1702  such that it defines a long conduit for conveying fluid through detection zone  414 . Detection zone  414  is defined by the position of lens  1708 , which is integrated into the flow cell such that it focuses free-space excitation light into the detection zone and collects light (e.g., forward- and side-scattered light and fluorescence signals) from the detection zone. 
         [0139]      FIGS. 18A-B  depict a flow cytometry flow cell in accordance with a third alternative embodiment of the present invention. Flow cell  1800  represents embodiments of the present invention that have substantially the same form factor as prior-art flow cells, but afford improved optical system performance and simpler operation. Flow cell  1800  comprises cell body  1802 , channel  1804 , fluid port  1706 , and optofluidic system  1806 . 
         [0140]    Cell body  1802  is analogous to cell body  1702 ; however cell body includes two conventional cell body portions  1702 - 1  and  1702 - 2 , which are attached to either side of optofluidic system  1806 . Typically, cell body portions  1702 - 1  and  1702 - 2  are joined with optofluidic system  1806  via a conventional joining technology, such as fusion bonding, glue, etc. Cell body portions  1702 - 1  and  1702 - 2  include sections of channel  1704 , which bookend and fluidically couple flow channel  408  to collectively define channel  1804 . 
         [0141]    Optofluidic system  1806  is analogous to optics plate  404  and comprises plate  1808  and detection system  1812 , which includes excitation network  1814 , collection network  1816 , and flow channel  408 . Excitation network  1814  and collection network  1816  are analogous to excitation network  216  and collection network  218  and are formed in waveguide plane  1810 , which is defined by the top surface of plate  1808 . 
         [0142]    It should be noted that the waveguides of excitation network  1814  and collection network  1816  are formed in the same waveguide plane. As a result, all of the excitation waveguides are arranged about one side of flow channel  408 , while all of the collection waveguides are arranged about the other side of the flow channel. While the illumination pattern provided by such an arrangement is not as uniform as in some other embodiments of the present invention, it still typically represents a significant improvement over illumination patterns provided by prior-art flow cytometer arrangements (e.g., that shown of system  1700 ). In some embodiments, the waveguides of excitation network  1814  and collection network  1816  are disposed in two or more waveguide planes, as described above. Further, in some embodiments, the waveguides of excitation network  1814  and collection network  1816  are arranged in another arrangement about flow channel  408 , such as those arrangements described above. 
         [0143]    One skilled in the art will recognize that there are several ways to optically couple to and from excitation network  1814  and collection network  1816 , such as butt coupling optical fibers to the waveguide networks, focusing free-space optical signals into the end facets of the networks, etc. 
         [0144]    It is to be understood that the disclosure teaches just one example of the illustrative embodiment and that many variations of the invention can easily be devised by those skilled in the art after reading this disclosure and that the scope of the present invention is to be determined by the following claims.