Abstract:
A method and apparatus for enabling chemical identification of individual particles, cells of molecules by obtaining a Raman spectrum of a particle, cell or molecule as it flows past a sensing point in a flow cytometer. The particles, which may be cells or molecules, are associated with a suitable noble metal colloid or colloidal aggregate. Cellular particles may be associated with gold or silver colloidal particles by ultra-sonic sonification while in a sample preparation reservoir containing the gold or silver colloidal suspension. The colloid associated particles are then hydrodynamically focused into a single file by a fluid control module. The surface-enhance Raman Spectrum of individual particles are obtained by illuminating the particle with a laser as the particle flows past a sensing point and gathering the light that is non-elastically scattered (Raman scattered) by the particle. The surface-enhanced Raman spectrum is then analyzed to identify the particle.

Description:
FIELD OF THE INVENTION 
   The present invention relates generally to flow cytometers, and more particularly to portable flow cytometers that use surface-enhanced Raman spectroscopy to identify the specimens that are being counted. 
   BACKGROUND OF THE INVENTION 
   Flow cytometry is a well known means of measuring certain physical and chemical characteristics of cells or particles by sensing certain optical properties of the cells or particles as they travel in suspension, one by one past a sensing point. Flow cytometry is widely used in the biological and medical fields. 
   In a typical flow cytometer, the single file flow of particles is achieved using hydrodynamic focusing of the suspended particles within a sheath fluid. The particles are then individually interrogated by a light beam. In most modern cytometers, the light source is a laser which emits coherent light at a specified wavelength. Each particle interacts with the light beam, and typically the scattered and any emitted fluorescent light produced by each particle is collected. By analyzing the scattered light, physical characteristics such as cell size, shape and internal complexity can be determined. Collecting any emitted fluorescent light also allows any cell component or function that can be detected by a fluorescent compound to be examined. 
   Traditional flow cytometers only detect elastically scattered light (also known as “Rayleigh scattered light”) which does not contain any information about the atomic or molecular structure of the particle. Although such flow cytometers can identify the size and shape, they cannot differentiate between similarly sized, but chemically different molecules or cells, unless the molecules or cells have been tagged with fluorescent markers. 
   In principle, detecting the in-elastically scattered light (also known as “Raman scattered light”) would enable the chemical identification of cells or molecules, as each cell or molecule has a unique Raman spectrum based on its chemical structure. The cross-section for Raman scattering is, however, about 15 orders of magnitude lower than the cross-section for Rayleigh scattering. This means that obtaining the Raman spectrum of an individual particle in a flow cytometer is well beyond the realms of practicality because of the lack of a suitably intense light source and the lack of any suitably sensitive detectors. The Raman cross-section, however, can be dramatically increased by a technique known as surface-enhanced Raman scattering, in which the molecule or particle is placed in contact with a suitably roughened noble metal surface, or in contact with a noble metal colloidal aggregate. Under the right conditions, the cross-section of surface enhance Raman scattering approaches the cross-section for fluorescence emission. 
   In order for flow cytometers to be able to identify individual particle based primarily on the chemical or molecular structure of the particle, what is needed is an apparatus and method that allows particles or cells in a cytometer to be in a condition that produces a sufficiently large surface-enhanced Raman scattering cross-section so that Raman spectra can be obtained from each particle or cell as it passes the cytometer sensing point. Such a system will allow the individual identification of all cells or particles, no matter how similar in size or shape they are to each other, without any need for fluorescent tagging. 
   SUMMARY OF THE INVENTION 
   The present invention relates to methods and apparatus for enabling flow cytometers to identify individual particles primarily by means of their chemical or molecular structure. 
   In a preferred embodiment of the invention, the chemical identification is achieved by obtaining a Raman spectrum of the particle, which may be an individual cell or molecule, as it flows past a sensing point in a flow cytometer. The particles are associated with a suitable noble metal colloid or colloidal aggregate so that their Raman scattering cross-section is surface-enhanced. The particles are then arranged in single file by a fluid control module capable of creating a hydrodynamically focused flow stream, and a light source illuminates each particle as it flows past a sensing point. Because the Raman cross-section is surface-enhanced, the light source may be a conventional laser. The light that is non-elastically scattered (Raman scattered) by the particle is gathered and the surface-enhanced Raman spectrum recorded using suitable dispersion optics and a suitable detector. The surface-enhanced Raman spectrum may then be analyzed to identify the particle from its molecular structure. 
   In a preferred embodiment of the present invention, cellular particles are associated with gold or silver colloidal particles by ultra-sonic sonification while in a sample preparation reservoir containing a gold or silver colloidal suspension. 
   These and other aspects of the invention will be described below in greater detail, and by reference to the attached drawings. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIG. 1  is a cross-sectional view of an exemplary embodiment of a Raman detection based cytometer. 
       FIG. 2  is a schematic view of an exemplary embodiment of a Raman detection based cytometer. 
       FIG. 3  is a schematic view of an exemplary embodiment of a removable cartridge of the present invention. 
       FIG. 4  is a cross-sectional view of the cytometer flow channel of an exemplary embodiment of a Raman detection based cytometer. 
       FIG. 5  is a schematic view of the illumination and detection optics of an exemplary embodiment of a Raman detection based cytometer. 
   

   DETAILED DESCRIPTION 
   The Raman based flow cytometry of the present invention counts individually identified particles within a sample by combining non-destructive, molecule specific Raman spectroscopy with a flow cytometer&#39;s ability to sequentially observe individual molecules. 
   One difficulty in implementing Raman based flow cytometry is that Raman scattering has a very low cross-section, i.e., only a very small percentage of the photons in a light beam focused on a sample are Raman (in-elastically) scattered. The effective Raman cross-section of a sample may, however, be increased by the technique of surface enhanced Raman Scattering, in which each molecule is associated with a noble metal colloid or colloidal aggregate. This association can be done by, for instance, sonification of cells in a noble metal colloidal solution. 
   The cytometric technique of hydrodynamic focusing may then be used to arrange the prepared molecules in a single file, flowing as a core stream within a fluid sheath, ready for individual interrogation by a light source. Hydrodynamic focusing requires precision pumping and control of the sample and sheath fluids within appropriately designed channels, as described in detail below. 
   An intense light source, preferably a laser source, may be focused onto the molecules flowing in the cytometer core-stream, and the in-elastically scattered light collected, detected and analyzed by Raman spectroscopy. For the identification of the molecules, the detection system needs appropriate wavelength selecting elements that provide sufficient detail of the molecule&#39;s characteristic Raman spectrum. 
   An exemplary embodiment of the present invention, having the elements for performing Raman detection based cytometer on individual molecules will now be discussed in detail by reference to the accompanying drawings in which, as far as possible, like numbers represent like elements. 
     FIG. 1  is a cross-sectional view of an exemplary embodiment of a Raman detection based flow cytometer, comprising an instrument housing  10 , a removable cartridge  12 , a pressurization module  14 , control electronics  16 , an illumination source  18 , source steering optics  20 , source focusing optics  22 , Raman scattered light collecting optics  24 , a light dispersing optical element  26  and a light detecting element  28 . 
     FIG. 2  is a schematic view of an exemplary, portable embodiment of a Raman detection based flow cytometer, comprising a lower section  32  having batteries  34 , illumination source  18 , illumination steering and collimating optics module  38 , control electronics  16 ; an upper section  30 , comprising a scattered light detection and dispersion module  40 , a light detecting element  28 , a manual pressurizing unit  44 , first pressure chambers  72   a ,  72   b  and  72   c , second pressure chambers  74   a ,  74   b  and  74   c , first valves  76   a ,  76   b  and  76   c  and second set of valves  78   a ,  78   b  and  78   c ; and a removable cartridge  12 , comprising a sample supply lumen  48 , a fluid control unit  50 , a flow stream  52 , a Raman chamber  61  and a waste reservoir  54 . The removable cartridge  12  may be made primarily of materials such as molded plastic that allow the cartridge to be mass produced and disposable. 
     FIG. 3  is a schematic view of an exemplary embodiment of a removable cartridge  12  of the present invention, in which the fluid control unit  50  comprises an ultrasonic source  55 , a sample conditioning reservoir  56 , a sample storage reservoir  58 , a sheath fluid reservoir  60 , flow sensors  62   a ,  62   b  and  62   c , a hydrodynamic focusing module  64 , a flow channel  52  in which a sheath fluid  51  surrounds a core stream  51 . 
   The sample may be prepared off-line or in the sample conditioning reservoir  56 . Examples of sample preparation techniques that enhance the Raman cross-section of molecules include loading cells with colloidal gold by fluid-phase uptake or sonification, as described in detail in, for instance, in the article entitled “Surface-Enhanced Raman Spectroscopy in Single Living Cells Using Gold Nanoparticles”, by Kneipp et al, Applied Spectroscopy, Volume 56, number 2, 2002, pp 150-154, the contents of which are hereby incorporated by reference. The molecules may also have enhanced Raman cross-sections by attaching them to micro-beads filled with noble-metal (copper, gold, silver, platinum, palladium and iridium) colloids, as described in, for instance, in the article entitled “Flow Analysis-based Surface-Enhanced Raman Spectroscopy Employing Exchangable Microbeads as SERS-active Surfaces” by Lendl et al. in Applied Spectroscopy, Volume 54, number 7, 2000, pp 1012-1018, the contents of which are hereby incorporated by reference. Noble metal colloids can also be made to aggregate into clusters suitable for individual molecules to attach to, as described in detail in, for instance, the article, “Single Molecule Detection Using Surface Enhanced-Raman Scattering (SERS)” by Kneip et al. in Physical Review Letters, volume 78, number 9, 1997, pp 1667-1670, the contents of which are hereby incorporated by reference. Molecules may also be attached to microstructures, such as liposomes, that are filled or coated with noble metal colloids. Molecules themselves may also be coated with noble metal colloids. Other possible methods for associating the cellular particles with the nobel metal colloidal particles include, but are not limited to, the manual injection of the metal colloids into individual cells and treating the metal colloidal particles as projectiles and firing them into the cellular particles. 
   In a preferred embodiment, intended for Raman based cytometry of individual cells, the sample conditioning reservoir  56  is adjacent to an ultrasonic source  55 , which may be controlled by control electronics  16 . The sample conditioning reservoir  56  comprises a cell supporting, buffer solution containing aggregated clusters of 60 nm gold nano-particles. Once sample cells have been fed into the buffer solution, a short burst of ultrasound ruptures the cell membrane, permitting gold colloid uptake by the cell. On cessation of the ultrasound, the cell membrane typically self-anneals within seconds. 
   The prepared sample fluid is then fed from the sample conditioning reservoir  56  into the sample storage reservoir  58 . The sample fluid is then fed into the hydrodynamic focusing module  64 , which may be a fluidic circuit engineered to perform hydrodynamic focusing, as described in detail in, for instance, U.S. Pat. No. 6,597,438, titled “Portable Flow Cytometery” issued to Cabuz et al. on Jul. 22, 2003, the contents of which are hereby incorporated by reference. The hydrodynamic focusing causes molecules of the sample to fall into single file a long a core stream  53  surrounded by a sheath fluid  51  within the flow channel  52 . The velocity of the sheath fluid  51  is preferably about 9 times that of the core stream  53 . Additionally, the velocity of the sheath fluid  51  and the core stream  53  remain sufficiently low to maintain laminar flow in the flow channel  52 . 
   In a preferred embodiment, the required velocity of the sheath fluid and the sample fluid are provided by a combination of a manual pressurizing unit  44 , coupled via pressure chambers  46   b  and  46   c  to fluid reservoirs  58  and  60  and fluid flow sensors  62   b  and  62   c , all acting in a closed feed-back loop under control electronics  16 . 
   The manual pressurizing unit  44  may, for instance, be manually powered plungers or bulbs with a check valve. In either case, the manually generated pressure is preferably provided to first pressure chambers  72   a ,  72   b  and  72   c . First valves  76   a ,  76   b  and  76   b  are then provided for controllably releasing the pressure in the first chambers  72   a ,  72   b  and  72   c  to the second pressure chambers  74   a ,  74   b  and  74   c . Second valves  78   a ,  78   b  and  78   c  may be provided in the second pressure chambers  74   a ,  74   b  and  74   c  for controllably venting the pressure in the second pressure chambers  74   a ,  74   b  and  74   c . The control electronics  16 , which typically comprises a programmable micro-processor, opens the first valves  76   a ,  76   b  or  76   c  when the fluid flow in the corresponding downstream fluid stream drops below a first predetermined value and opens the second, vent value  78   a ,  78   b  or  78   c  when the fluid flow in the downstream fluid increases above a second predetermined value. Each value is preferably an array of electrostatically actuated micro-valves that are individually addressable and controllable. The fluid flow sensors  62   a ,  62   b  and  62   c  are preferably thermal anemometer type flow sensors. 
   The hydrodynamically focused core stream  53 , containing sample particles arranged in single file, surrounded by a sheath fluid  51 , flows through flow channel  52  into Raman chamber  61 . 
     FIG. 4  is a cross-sectional view of the cytometer flow channel of an exemplary embodiment of a Raman detection based cytometer, comprising the flow stream  52 , an upper and a lower glass substrate  56  and  54 , an illuminating micro-lens  58 , a detecting micro-lens  60 , a source pass filter and source absorbing filters  64  and  66 , a focused core stream  53 , containing sample particles. 
     FIG. 5  is a schematic view of the illumination and detection optics of an exemplary embodiment of a Raman detection based cytometer, comprising an illumination source  18 , source focusing optics  22 , a focused core stream  53  containing sample particles  68 , Raman-scattered light-collecting optics  24 , a light dispersing optical element  26  and a light detecting element  28 . 
   In a preferred embodiment, the Raman chamber  61  comprises glass substrates  56  and  54 , flow channel  52 , micro-lenses  58  and  60 , source-pass filter  62 , and source-absorbing filter  64  and  66 . 
   Light emitted by light source  18  is directed as a collimated beam onto micro-lens  58  by beam steering and collimating optics  38 . Micro-lens  58  focuses the light though source-pass filter  62  onto particles in the core stream  53 . Light that is in-elastically (Raman) scattered from particle in the core stream  42  passes through source-absorbing filter  56  and is directed towards detector  28  by micro-lens  60  and steering and discrimination optics  40 . 
   Light source  18  may be a laser or array of lasers having a wavelength, a power and a polarization suitable for Raman spectroscopy. Prior art light sources include argon-ion pumped Ti:sapphire lasers operating at 830 nm, providing 200 mW of power focused down to about 2×10 5  W/cm 2  at the sample. Other suitable light sources include Nd:Yag laser operating at 1064 nm and helium neon lasers operating at 632.8 nm. A Vertical Cavity Surface Emitting Laser (VCSEL) is particularly suited to portable implementation. VCSEL are available that operate at wavelengths in the range from 850 nm to 670 nm. A 10×10 micron emitting surface VCSEL typically has a power of about 1 mW, producing a power density of about 1×10 3  W/cm 2  when focused to a Gaussian spot of about 10 microns. Any suitable solid state laser may be used as light source  18 . 
   The light source  18  may also be a single light source, such as a solid state laser or a diode laser, movably attached to one or more stepper motors so that either the sources lateral position or distance from the core-stream  53 , or both, may be adjusted to achieve optimal scattering. Adjustment of such a movable source in focus and/or lateral displacement with respect to the core stream  53  may include a feed-back loop responsive to light reflected by a difference in refractive index between the core stream  53  and the sheath fluid  51 . The adjustment feed-back loop may also be accomplished by, for instance, using light reflected or scattered from tracking or calibration micro-beads that are interspersed with the molecules of interest in the core-stream 
   Steering and discrimination optics  40  and detector  28  may be, for instance, a Sentinal Raman Spectrometer having a Charge Couple Device detector (CCD), as supplied by Bruker Optics, Inc. of Bilerica, Mass. Detector  28  may also be any other low noise, high quantum efficiency multichannel detector or CCD array. Steering and discrimination optic module may also comprise any suitable combination of filters, acoustoptic tunable filters (AOTF), gratings, diffraction optics and holographic optics. In a preferred embodiment, the steering and discrimination optic module is a suitable holographic diffraction grating that combines wavelength selection with focusing the collected Raman scattered light onto the detector  28 . 
   Source-pass filter  62  is a coating that band passes only the source wavelength, thereby eliminating collecting Raman scattered light that occurs before the flow channel. Any suitable well-know multi-layer or holographic band pass filter may be used. 
   Source-absorbing filters  64  and  66  are coatings that reject the source wavelength, thereby eliminating Raman scattering after the flow channel. Any suitable well-known multi-layer or holographic band pass filter may be used. 
   Once the core stream  53  and surrounding sheath fluid  51  have passed through Raman chamber  61 , they drain into waste reservoir  54 . 
   Although the invention has been described in language specific to structural features and/or methodological acts, it is to be understood that the invention defined in the appended claims is not necessarily limited to the specific features or acts described. Rather, the specific features and acts are disclosed as exemplary forms of implementing the claimed invention.