Instrument and method for optical particle sensing

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 the known cell, microbe or virus,
• b) incubating a first concentration of the therapeutic agent with one or more of 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-d with varying amounts of the therapeutic agent;
• and
• f) comparing the first and second spectra obtained using each varying amount to determine the minimum amount of therapeutic agent required to effectively bind the known cell, microbe or virus, wherein said minimal amount is an effective dosage,
• wherein the first and second spectra are obtained using a device comprising:
• i) two or more lasers which produce two or more beams of laser light,
• ii) a first beam splitter that combines beams of laser light from the two or more lasers,
• iii) a first focusing lens for focusing the combined beams of laser light from the beam splitter,
• iv) a flow cell positioned in the path of the focused beams of laser light from the first focusing lens, wherein the flow cell is adapted to receive and pass the sample medium comprising a dispersion of the biological particles, wherein the focused beams of laser light from the first focusing lens are scattered when they interact with one or more of the particles, thereby forming scattered beams of laser light,
• v) a mirror positioned between the flow cell and a spatial filter, to reflect a portion of the scattered beams of laser light,
• vi) an extinction detector positioned in the path of the reflected portion of the scattered beams of laser light,
• vii) the 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,
• viii) a first 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, thereby forming a first collimated beam of laser light,
• ix) a second beam splitter positioned between the first collimating lens and a second focusing lens, to split the first collimated beam of laser light into a first beam of laser light and a second beam of laser light, wherein the first beam of laser light is not diverted from an original path that is toward the second focusing lens, and the second beam of laser light is diverted from the original path onto a second path, thereby forming a diverted beam of laser light,
• x) a second collimating lens positioned along the second path, for collimating the diverted beam of laser light, thereby forming a second collimated beam of laser light,
• xi) the second focusing lens positioned in the path of the first beam of laser light, for focusing the first beam of laser light, thereby forming a second focused beam of laser light,
• xii) a first scatter detector positioned in the path of the second focused beam of laser light, and
• xiii) a second scatter detector positioned in the path of the second collimated beam of laser light,
• wherein the first scatter detector detects light at a first frequency corresponding to one of the beams of laser light originating from a first laser of the two or more lasers and the second scatter detector detects light at a second frequency corresponding to a second beam of laser light originating from a second laser of the two or more lasers,
• wherein the focused light scattering techniques used to obtain the first and second spectra comprise:
• f) having at least one of the two or more lasers produce one or more beams of laser light,
• g) passing each beam of laser light through the first focusing lens, which focuses the beams of laser light such that the effective width of the beams of laser light in a direction transverse to the path of the beams of laser light is between about 0.05 and 0.5 μm, thereby forming the focused beams of laser light,
• h) passing the focused beams of laser light through the flow cell, and, in doing so, through the sample medium as it passes through the flow cell, so that the focused beam of laser light is scattered when it interacts with the one or more of the known cell, microbe, or virus in the sample medium, and the focused beam of laser light is not scattered if it does not interact with the one or more of the known cell, microbe, or virus in the sample medium, depending on whether the one or more of the known cell, microbe, or virus are in the path of the focused beam of laser light, and
• i) using the extinction detector and the scatter detectors to generate data for the first and second spectra.

A method of determining the efficacy of a putative 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, or a plurality of these particles, each with a receptor to which the putative therapeutic agent will bind, wherein the sample medium is, or k derived from, a fluid selected from the group consisting of blood, blood products, water, cerebrospinal fluid, ascites, pleural fluid, and synovial fluid,
• b) incubating the sample medium with the 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(s) wherein the first and second spectra are obtained using a device comprising:
• i) two or more lasers which produce two or more beams of laser light,
• ii) a first beam splitter that combines beams of laser light from the two or more lasers,
• iii) a first focusing lens for focusing the combined beams of laser light from the beam splitter,
• iv) a flow cell positioned in the path of the focused beams of laser light from the first focusing lens, wherein the flow cell is adapted to receive and pass the sample medium comprising a dispersion of the biological particles, wherein the focused beams of laser light from the first focusing lens are scattered when they interact with one or more of the particles, thereby forming scattered beams of laser light,
• v) a mirror positioned between the flow cell and a spatial filter, to reflect a portion of the scattered beams of laser light,
• vi) an extinction detector positioned in the path of the reflected portion of the scattered beams of laser light,
• vii) the 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,
• viii) a first 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, thereby forming a first collimated beam of laser light,
• ix) a second beam splitter positioned between the first collimating lens and a second focusing lens, to split the first collimated beam of laser light into a first beam of laser light and a second beam of laser light, wherein the first beam of laser light is not diverted from an original path that is toward the second focusing lens, and the second beam of laser light is diverted from the original path onto a second path, thereby forming a diverted beam of laser light,
• x) a second collimating lens positioned along the second path, for collimating the diverted beam of laser light, thereby forming a second collimated beam of laser light,
• xi) the second focusing lens positioned in the path of the first beam of laser light, for focusing the first beam of laser light, thereby forming a second focused beam of laser light,
• xii) a first scatter detector positioned in the path of the second focused beam of laser light, and
• xiii) a second scatter detector positioned in the path of the second collimated beam of laser light,
• wherein the first scatter detector detects light at a first frequency corresponding to one of the beams of laser light originating from a first laser of the two or more lasers and the second scatter detector detects light at a second frequency corresponding to a second beam of laser light originating from a second laser of the two or more lasers,
• wherein the focused light scattering techniques used to obtain the first and second spectra comprise:
• e) having at least one of the two or more lasers produce one or more beams of laser light,
• f) passing each beam of laser light through the first focusing lens, which focuses the beams of laser light such that the effective width of the beams of laser light in a direction transverse to the path of the beams of laser light is between about 0.05 and 0.5 μm, thereby forming the focused beams of laser light,
• g) passing the focused beams of laser light through the flow cell, and, in doing so, through the sample medium as it passes through the flow cell, so that the focused beam of laser light is scattered when it interacts with one or more of the biological particles in the sample medium, and the focused beam of laser light is not scattered if it does not interact with the one or more of the biological particles in the sample medium, depending on whether the one or more of the biological particles are in the path of the focused beam of laser light, and
• using the extinction detector and the scatter detectors to generate data for the first and second spectra.