Instrument and Method for Optical Particle Sensing

Abstract

Devices for detecting particle sizes and distributions using focused light scattering techniques, by passing a sample through a focused beam of light, are disclosed. In one embodiment, the devices include one or more lasers, whose light is focused into a narrow beam and into a flow cell, and dispersions are passed through the flow cell using hydrodynamic sample injection. In another embodiment, a plurality of lasers is used, optionally with hydrodynamic sample injection. Particles pass through and scatter the light. The scattered light is then detected using scatter and extinction detectors, and, optionally, fluorescence detectors, and the number and size of the particles is determined. Particles in the size range of 0.1 to 10 μιη can be measured. Using the device, significantly smaller particles can be detected than if techniques such as EQELS, flow cytometry, and other conventional devices for measuring biological particles.

1 - 62 . (canceled) 
     
     
         63 . A method of identifying a biological particle of interest in a sample medium, said method comprising:
 a) introducing a sample medium, which may or may not include a biological particle of interest, into a device for measuring the size and/or number of particles in a sample medium,   wherein the sample medium is, or is derived from, one or more fluids selected from the group consisting of blood, blood products, water, cerebrospinal fluid, ascites, pleural fluid, and synovial fluid,   wherein the device for measuring the size and/or number of particles in a sample medium comprises:
 i) one or more lasers which produce one or more beams of laser light, each of which beams of laser light following a path, 
 ii) a first focusing lens positioned in the path of one of the beams of laser light, 
   which first focusing lens focuses the beams of laser light such that the effective width in a direction transverse to the path of the beams of laser light is between about 0.05 and 0.5 μm, thereby forming a focused beam of laser light,
 iii) a flow cell positioned in the path of the focused beam of laser light, wherein the flow cell is adapted to receive and pass a sample medium comprising a dispersion of particles, 
 iv) a spatial filter positioned in the path of the scattered beams of laser light, which allows the scattered beams of laser light to pass through, and does not allow the beams of laser light that are not scattered to pass through, 
 v) a collimating lens positioned in the path of the scattered beams of laser light that passed through the spatial filter, to collimate the scattered beams of laser light that passed through the spatial filter, 
 vi) a second focusing lens positioned in the path of the collimated beams of laser light, for focusing the collimated beams of laser light that passed through the collimating lens, 
 vii) a scatter detector positioned in the path of the focused beams of laser light that passed through the second focusing lens, 
 viii) a mirror positioned between the flow cell and the spatial filter, to reflect a portion of the scattered beams of laser light, and 
 ix) an extinction detector positioned in the path of the reflected portion of the scattered beams of laser light, 
   b) passing the sample medium through one or more focused beams of laser light in the device, so that the focused beam of laser light is scattered when it interacts with one or more of the particles, and the focused beam of laser light is not scattered if it does not interact with the one or more particles, depending on whether the one or more particles are in the path of the focused beam of laser light,   c) using focused light scattering techniques to prepare a spectrum showing particle size distribution from within the sample medium,   wherein focused light scattering techniques comprise passing a sample media through a particular path, where a focused beam of light passes through the sample media,   wherein the focused beam is of a size such that a particle in the size range of 0.1 to 10 μm is sufficient to block all of the beam, or a significant enough part of the beam, so that the particle size can be measured,   
       and
 c) identifying the presence or absence of the biological particle of interest by comparing the sizes of the particles in the sample medium with the known size of the particle of interest. 
 
     
     
         64 . The method of  claim 63 , wherein the size of the particles in the sample medium is compared with a reference database comprising a library of particle sizes obtained using focused light scattering techniques, wherein the library comprises ten or more spectra. 
     
     
         65 . The method of  claim 63 , wherein, after an initial determination is made that a particle of interest is present in the sample medium, a confirmatory assay is performed, wherein the confirmatory assay involves taking an EQELS spectra of the sample medium, and verifying the existence of a biological particle by identifying a feature unique to the biological particle. 
     
     
         66 . The method of  claim 63 , wherein the biological particle of interest is a lymphocyte, erythrocyte, B-cell, T-cell, neutrophil, monocyte, bacteria, fungi, viruses, protozoa, tumor cell, red blood cell, white blood cell, granulocyte, platelet, cancer cell, or stem cell. 
     
     
         67 . The method of  claim 63 , wherein the biological microparticle has a size ranging from about 0 μm to about 20 μm. 
     
     
         68 . A method of determining the efficacy of a putative therapeutic agent, comprising:
 a) introducing a sample medium, which sample medium comprises a biological particle with a receptor to which a putative therapeutic agent will bind, into a device for measuring the size and/or number of particles in a sample medium,   wherein the device for measuring the size and/or number of particles in a sample medium comprises:
 i) one or more lasers which produce one or more beams of laser light, each of which beams of laser light following a path, 
 ii) a first focusing lens positioned in the path of one of the beams of laser light, 
   which first focusing lens focuses the beams of laser light such that the effective width in a direction transverse to the path of the beams of laser light is between about 0.05 and 0.5 μm, thereby forming a focused beam of laser light,
 iii) a flow cell positioned in the path of the focused beam of laser light, wherein the flow cell is adapted to receive and pass a sample medium comprising a dispersion of particles, 
 iv) a spatial filter positioned in the path of the scattered beams of laser light, which allows the scattered beams of laser light to pass through, and does not allow the beams of laser light that are not scattered to pass through, 
 v) a collimating lens positioned in the path of the scattered beams of laser light that passed through the spatial filter, to collimate the scattered beams of laser light that passed through the spatial filter, 
 vi) a second focusing lens positioned in the path of the collimated beams of laser light, for focusing the collimated beams of laser light that passed through the collimating lens, 
 vii) a scatter detector positioned in the path of the focused beams of laser light that passed through the second focusing lens, 
 viii) a mirror positioned between the flow cell and the spatial filter, to reflect a portion of the scattered beams of laser light, and 
 ix) an extinction detector positioned in the path of the reflected portion of the scattered beams of laser light, 
   b) passing the sample medium through one or more focused beams of laser light in the device, so that the focused beam of laser light is scattered when it interacts with one or more of the particles, and the focused beam of laser light is not scattered if it does not interact with the one or more particles, depending on whether the one or more particles are in the path of the focused beam of laser light,   c) using focused light scattering techniques to prepare a spectrum showing particle size and distribution from within the sample medium,   wherein focused light scattering techniques comprise passing a sample media through a particular path, where a focused beam of light passes through the sample media,   wherein the focused beam is of a size such that a particle in the size range of 0.1 to 10 μm is sufficient to block all of the beam, or a significant enough part of the beam, so that the particle size can be measured,   b) incubating the sample medium with a putative therapeutic agent,   c) obtaining a second spectra showing particle size and distribution on the incubated sample medium using focused light scattering techniques, and   d) determining whether the particle size and distribution has been altered by the incubation of the putative therapeutic agent, a change in the particle size and/or distribution is indicative of a complex formation of the putative therapeutic agent and the biological particle.   
     
     
         69 . The method of  claim 68 , wherein the biological microparticle is selected from tumor cells, red blood cells, white blood cells, granulocytes, platelets, monocytes, neutrophils, lymphocytes, cancer cells, bacteria, viruses, and fungi. 
     
     
         70 . A method for determining whether a biological particle will form a complex with a known therapeutic agent, comprising:
 a) obtaining a spectra showing particle size and distribution using focused light scattering techniques on a sample medium comprising a biological particle with a receptor to which a known therapeutic agent may or may not bind, using a device for measuring the size and/or number of particles in a sample medium,   wherein the device for measuring the size and/or number of particles in a sample medium comprises:
 i) one or more lasers which produce one or more beams of laser light, each of which beams of laser light following a path, 
 ii) a first focusing lens positioned in the path of one of the beams of laser light, 
   which first focusing lens focuses the beams of laser light such that the effective width in a direction transverse to the path of the beams of laser light is between about 0.05 and 0.5 μm, thereby forming a focused beam of laser light,
 iii) a flow cell positioned in the path of the focused beam of laser light, wherein the flow cell is adapted to receive and pass a sample medium comprising a dispersion of particles, 
 iv) a spatial filter positioned in the path of the scattered beams of laser light, which allows the scattered beams of laser light to pass through, and does not allow the beams of laser light that are not scattered to pass through, 
 v) a collimating lens positioned in the path of the scattered beams of laser light that passed through the spatial filter, to collimate the scattered beams of laser light that passed through the spatial filter, 
 vi) a second focusing lens positioned in the path of the collimated beams of laser light, for focusing the collimated beams of laser light that passed through the collimating lens, 
 vii) a scatter detector positioned in the path of the focused beams of laser light that passed through the second focusing lens, 
 viii) a mirror positioned between the flow cell and the spatial filter, to reflect a portion of the scattered beams of laser light, and 
 ix) an extinction detector positioned in the path of the reflected portion of the scattered beams of laser light, 
   wherein the sample medium is, or is derived from, a fluid selected from the group consisting of blood, blood products, water, cerebrospinal fluid, ascites, pleural fluid, and synovial fluid,   wherein focused light scattering techniques comprise passing a sample media through a particular path, where a focused beam of light passes through the sample media, and   wherein the focused beam is of a size such that a particle in the size range of 0.1 to 10 μm is sufficient to block all of the beam, or a significant enough part of the beam, so that the particle size can be measured,   b) incubating the sample medium with a known therapeutic agent,   b) obtaining a second spectra showing particle size and distribution on the incubated sample medium using focused light scattering techniques, and   c) determining whether the particle size and distribution has been altered by the incubation of the known therapeutic agent, a change in the particle size and/or distribution is indicative of a complex formation of the known therapeutic agent and the biological particle.   
     
     
         71 . The method of  claim 70 , wherein the biological microparticle is selected from tumor cells, red blood cells, white blood cells, granulocytes, platelets, monocytes, neutrophils, lymphocytes, cancer cells, bacteria, viruses, and fungi. 
     
     
         72 . A method for determining an effective dosage of a therapeutic agent against a known cell, microbe or virus comprising:
 a) generating a first spectrum showing particle size and distribution using focused light scattering for a known cell, microbe or virus, using a device for measuring the size and/or number of particles in a sample medium,   wherein the device for measuring the size and/or number of particles in a sample medium comprises:
 i) one or more lasers which produce one or more beams of laser light, each of which beams of laser light following a path, 
 ii) a first focusing lens positioned in the path of one of the beams of laser light, 
   which first focusing lens focuses the beams of laser light such that the effective width in a direction transverse to the path of the beams of laser light is between about 0.05 and 0.5 μm, thereby forming a focused beam of laser light,
 iii) a flow cell positioned in the path of the focused beam of laser light, wherein the flow cell is adapted to receive and pass a sample medium comprising a dispersion of particles, 
 iv) a spatial filter positioned in the path of the scattered beams of laser light, which allows the scattered beams of laser light to pass through, and does not allow the beams of laser light that are not scattered to pass through, 
 v) a collimating lens positioned in the path of the scattered beams of laser light that passed through the spatial filter, to collimate the scattered beams of laser light that passed through the spatial filter, 
 vi) a second focusing lens positioned in the path of the collimated beams of laser light, for focusing the collimated beams of laser light that passed through the collimating lens, 
 vii) a scatter detector positioned in the path of the focused beams of laser light that passed through the second focusing lens, 
 viii) a mirror positioned between the flow cell and the spatial filter, to reflect a portion of the scattered beams of laser light, and 
 ix) an extinction detector positioned in the path of the reflected portion of the scattered beams of laser light; 
   wherein focused light scattering techniques comprise passing a sample media through a particular path, where a focused beam of light passes through the sample media, and   wherein the focused beam is of a size such that a particle in the size range of 0.1 to 10 μm is sufficient to block all of the beam, or a significant enough part of the beam, so that the particle size can be measured,   b) incubating a first concentration of a therapeutic agent with the known cell, microbe or virus;   c) generating a second spectrum showing particle size and distribution using focused light scattering of the combination of the therapeutic agent and the known cell, microbe or virus; and   d) comparing the first and second spectra, wherein a change in the particle size and/or distribution is indicative of binding of the therapeutic agent and the cell, microbe or virus, and wherein binding is indicative of inhibition of the known cell, microbe or virus;   e) repeating steps a-e with varying amounts of the therapeutic agent; and   f) comparing the spectra to determine the minimum amount of therapeutic agent required to effectively bind the known cell, microbe or virus.   
     
     
         73 . The method of  claim 72 , wherein the known cell, microbe or virus is a cell selected from the group consisting of tumor cells, red blood cells, white blood cells, granulocytes, platelets, monocytes, neutrophils, lymphocytes, and cancer cells. 
     
     
         74 . The method of  claim 63 , wherein the device further comprises a hydrodynamic flow injector for introducing the sample medium into the flow cell. 
     
     
         75 . The method of  claim 63 , further comprising a detector positioned in the path of the beams of light passing through the second chromatic filter. 
     
     
         76 . The method of  claim 75 , wherein the detector is a fluorescence detector. 
     
     
         77 . The method of  claim 63 , wherein the device further comprises:
 k) a processor adapted to receive information from the various detectors, and to responsively generate an output correlative of the size and/or number of particles in the sample medium.   
     
     
         78 . The method of  claim 77 , further comprising a memory map for storing information on the size and/or number of particles in the sample medium. 
     
     
         79 . The method of  claim 77 , further comprising a video display interface operatively coupled to the processor for outputting information on the size and/or number of particles in the sample medium. 
     
     
         80 . The method of  claim 63 , wherein the first focusing lens focuses the beams of light such that the effective width in a direction transverse to the axis of the light beam is between about 0.05 and 0.25 μm. 
     
     
         81 . The method of  claim 63 , wherein the first focusing lens focuses the beams of light such that the effective width in a direction transverse to the axis of the light beam is between about 0.05 and 0.15 μm.