Systems and methods of raman bandpass microbial detection

Abstract

Provided herein are devices, systems, and methods for performing narrow band Raman Spectroscopy on cells. The method can include incubating the cells with a compound of a known Raman spectrum to determine viability of the cells in the presence of other compounds. The system can include one or more of a light source, a compound dispenser, a band pass filter, a light detector, and processors coupled to the detector.

Description

WSGR Docket No. 68783-701.601SYSTEMS AND METHODS OF RAMAN BANDPASS MICROBIAL DETECTIONCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 717,506, filed November 7, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Bacteria and other microbes cause many diseases and illnesses of varying severity, from respiratory infections to sepsis. To properly treat microbial infections, it is crucial to rapidly determine the phenotype and any associated antimicrobial resistance of microbes harboring the infection. Currently, microbial phenotype and antimicrobial resistance are determined through the labor intensive and lengthy process of culturing microbes of a biological sample of the infected subject on various growth mediums. Spectroscopic techniques, such as Raman spectroscopy, have substantially improved the sensitivity to differentiate microbes based on subtle variations in microbial chemical composition without having to culture the microbes. However, Raman spectroscopy requires a lengthy period of time to scan across an entire Raman spectrum, significantly impacting the ability of Raman spectroscopy to rapidly characterize microbes of a sample to timely treat subjects. Thus, there exists an unmet need of further improving upon detection speed and throughput of the highly sensitive and specific Raman spectroscopy to rapidly identify microbes harboring infections in subjects.SUMMARY

[0003] In an aspect, provided herein is a method for determining a characterization of one or more cells, comprising: obtaining or providing a sample comprising the one or more cells; providing a compound to the sample, wherein when the compound is incorporated by the one or more cells, an intensity of one or more scattered Raman photons emitted by the one or more cells is changed compared to an absence of the incorporation of the compound by the one or more cells; providing a light emission of a light source to the sample, wherein the one or more cells absorb the light emission and emit the one or more scattered Raman photons; detecting the one or more scattered Raman photons of the one or more cells with a detector optically coupled to a band-pass filter, thereby generating one or more Raman optical signals; and determining the characterization of the one or more cells of the sample by processing the one or more Raman optical signals.

[0004] In some embodiments, the one or more cells comprise one or more microbes, one or more mammalian cells, one or more cancer cells, one or more fungi, or any combination thereof. For example, the one or more cells may comprise one or more microbes. In certain embodiments, theWSGR Docket No. 68783-701.601 one or more cells may comprise one or more mammalian cells. In some embodiments, the one or more cells may comprise one or more cancer cells. In embodiments, the one or more cells may comprise one or more fungi.

[0005] In some embodiments, the characterization of the one or more cells is determined in up to about 20 minutes.

[0006] In some embodiments, the light source comprises a laser light source.

[0007] In some embodiments, the detector comprises a photomultiplier tube, an avalanche photodiode, a photodiode, a silicon photomultiplier, a multi -pixel photon counter, a single photon avalanche photodiode, or any combination thereof. In some embodiments, the detector comprises a photomultiplier tube. In some embodiments, the detector comprises an avalanche photodiode. In some embodiments, the detector comprises a photodiode. In some embodiments, the detector comprises a silicon photomultiplier. In some embodiments, the detector comprises a multi-pixel photon counter. In some embodiments, the detector comprises a single photon avalanche photodiode. In some embodiments, a sensitivity of the detector comprises at least about 0.5 ampere / watt (AAV). In some embodiments, the one or more scattered Raman photons are detected by the detector with up to about a 100-microsecond signal acquisition time.

[0008] In some embodiments, the band-pass filter comprises an emission band of about 1 nm to about 20 nm. In some embodiments, the band-pass filter comprises a plurality of band-pass filters. In some embodiments, the plurality of band-pass filters comprises a first band-pass filter and a second band-pass filter, wherein the first band -pass filter comprises a first emission band, and wherein the second band-pass filter comprises a second emission band, and wherein the first emission band and the second emission band do not overlap. In some embodiments, the second band-pass filter is optically coupled to the sample and a second detector, wherein the first bandpass filter is optically coupled to the sample and the detector, and wherein the detector and the second detector differ. In some embodiments, the band-pass filter comprises a transmission efficiency of at least about 70%.

[0009] In some embodiments, the method further comprises scanning the light emission of the light source with a scanner across the sample. In some embodiments, the scanner comprises a stage coupled to the sample, and wherein the stage translates the sample while the light emission of the light source is fixed in position. In some embodiments, the scanner comprises one or more mirrors that direct an optical path of the light emission of the light source across a plane of the sample. In some embodiments, the scanner comprises a stage that translates the detector across the sample. In some embodiments, the scanner comprises a galvanic scanning mirror.

[0010] In some embodiments, the output intensity of the light emission of the light source is modulated at a frequency, and wherein a gain of the detector is modulated at the frequency ofWSGR Docket No. 68783-701.601 modulation of the output intensity of the light emission of the light source. In some embodiments, the output intensity of the light emission of the light source is modulated with an optical chopper, acoustic optic modulator, electric optical modulator, or any combination thereof, optically coupled to the light emission of the light source. In some embodiments, the output intensity of the light emission of the light source is modulated with an optical chopper optically coupled to the light emission of the light source. In some embodiments, the output intensity of the light emission of the light source is modulated with an acoustic optic modulator optically coupled to the light emission of the light source. In some embodiments, the output intensity of the light emission of the light source is modulated with an electric optic modulator optically coupled to the light emission of the light source.

[0011] In some embodiments, the sample comprises a biological sample from a human subject, a mammalian subject, a plant, or any combination thereof. In some embodiments, the sample comprises a biological sample from a human subject. In some embodiments, the sample comprises a biological sample from a mammalian subject. In some embodiments, the same comprises a biological sample from a plant. In some embodiments, the biological sample comprises sputum, blood, urine, cerebral spinal fluid, cervical spinal fluid, bronchial alveolar lavage fluid, fluid collected from a prosthesis, fluid collected from an implant, or any combination thereof. In some embodiments, the biological sample comprises sputum. In some embodiments, the biological sample comprises urine. In some embodiments, the biological sample comprises cerebral spinal fluid. In some embodiments, the biological sample comprises cervical spinal fluid. In some embodiments, the biological sample comprises bronchial alveolar lavage fluid. In some embodiments, the biological sample comprises fluid collected from a prosthesis. In some embodiments, the biological sample comprises fluid collected from an implant.

[0012] In some embodiments, the method is not conducted on the human or animal body. For example, in such embodiments, the sample may be an ex vivo and / or in vitro sample.

[0013] In some embodiments, the one or more cells are not filtered or removed from the biological sample.

[0014] In some embodiments, the band-pass filter comprises a first coating and a second coating, wherein the first coating transmits a first band of light, wherein the second coating transmits a second band of light, and wherein the first band of light and the second band of light differ.

[0015] In some embodiments, the method further comprises providing a second compound to the one or more cells in the sample, wherein the second compound alters a metabolic state of the one or more cells. In some embodiments, the second compound comprises an antimicrobial or a pharmaceutical compound.WSGR Docket No. 68783-701.601

[0016] In some embodiments, the characterization of the one or more cells comprises a phenotype, a metabolic state, antimicrobial resistance, response to the compound, response to the second compound, or any combination thereof, of the one or more cells. In some embodiments, the characterization of the one or more cells comprises a phenotype. In some embodiments, the characterization of the one or more cells comprises a metabolic state. In some embodiments, the characterization of the one or more cells comprises an antimicrobial resistance. In some embodiments, the characterization of the one or more cells comprises a response to the compound. In some embodiments, the characterization of the one or more cells comprises a response to the second compound.

[0017] In some embodiments, the method further comprises generating an image of the one or more cells from the one or more Raman optical signals.

[0018] In some embodiments, the compound comprises a fluid. In some embodiments, the fluid comprises deuterium.

[0019] In some embodiments, a first dichroic mirror is optically coupled to the band-pass filter, wherein the first dichroic mirror is optically coupled to the detector. In some embodiments, a second dichroic mirror is optically coupled to the band-pass filter, wherein the second dichroic mirror is optically coupled to a second detector, wherein the second detector differs from the detector.

[0020] In some embodiments, the detector comprises a single pixel detector.

[0021] In some embodiments, the light source comprises an optical intensity of at least about 15 milliwatt (mW).

[0022] In some embodiments, processing comprises averaging the one or more detected Raman optical signals across a surface of the sample with one or more processors to determine the characterization of the one or more cells.

[0023] In some embodiments, the light source comprises a pulsed light source.

[0024] In some embodiments, the compound is provided to the sample by a compound dispenser. In some embodiments, the compound dispenser comprises a fluidic handling system, a nozzle, a pipette, or any combination thereof. In some embodiments, the compound dispenser comprises a fluidic handling system. In some embodiments, the compound dispenser comprises a nozzle. In some embodiments, the compound dispenser comprises a pipette.

[0025] In some embodiments, the one or more Raman optical signals comprise an integrated intensity of the one or more scattered Raman photons detected by the detector through the band - pass filter.

[0026] In another aspect, provided herein is a computer system for determining a characterization of one or more cells, comprising: one or more processors and memory storingWSGR Docket No. 68783-701.601 one or more programs for execution by the one or more processors, the one or more programs comprising instructions to: provide a compound to a sample comprising the one or more cells, wherein when the compound is incorporated by the one or more cells, an intensity of one or more scattered Raman photons emitted by the one or more cells is changed compared to an absence of the incorporation of the compound by the one or more cells; provide a light emission of a light source to the sample, wherein the one or more cells absorb the light emission and emit the one or more scattered Raman photons; detect the one or more scattered Raman photons of the one or more cells with a detector optically coupled to a band-pass filter, thereby generating one or more Raman optical signals; and determine the characterization of the one or more cells of the sample by processing the one or more Raman optical signals.

[0027] In some embodiments, the one or more cells comprise one or more microbes, one or more mammalian cells, one or more cancer cells, one or more fungi, or any combination thereof. For example, the one or more cells may comprise one or more microbes. In certain embodiments, the one or more cells may comprise one or more mammalian cells. In some embodiments, the one or more cells may comprise one or more cancer cells. In embodiments, the one or more cells may comprise one or more fungi.

[0028] In some embodiments, the characterization of the one or more cells is determined in up to about 20 minutes.

[0029] In some embodiments, the light source comprises a laser light source.

[0030] In some embodiments, the detector comprises a photomultiplier tube, an avalanche photodiode, a photodiode, a silicon photomultiplier, a multi -pixel photon counter, a single photon avalanche photodiode, or any combination thereof. In some embodiments, the detector comprises a photomultiplier tube. In some embodiments, the detector comprises an avalanche photodiode. In some embodiments, the detector comprises a photodiode. In some embodiments, the detector comprises a silicon photomultiplier. In some embodiments, the detector comprises a multi -pixel photon counter. In some embodiments, the detector comprises a single photon avalanche photodiode. In some embodiments, a sensitivity of the detector comprises at least about 0.3 AAV. In some embodiments, the one or more scattered Raman photons are detected by the detector with up to about a 100 microsecond signal acquisition time.

[0031] In some embodiments, the band-pass filter comprises an emission band of about 1 nm to about 20 nm. In some embodiments, the band-pass filter comprises a plurality of band-pass filters. In some embodiments, the plurality of band-pass filters comprises a first band-pass filter and a second band-pass filter, wherein the first band -pass filter comprises a first emission band, wherein the second band-pass filter comprises a second emission band, and wherein the first emission band and the second emission band do not overlap. In some embodiments, the secondWSGR Docket No. 68783-701.601 band-pass filter is optically coupled to the sample and a second detector, wherein the first bandpass filter is optically coupled to the sample and the detector, and wherein the detector and the second detector differ. In some embodiments, the band-pass filter comprises a transmission efficiency of at least about 70%.

[0032] In some embodiments, the instructions further comprise scanning the light emission of the light source with a scanner across the sample. In some embodiments, the scanner comprises a stage coupled to the sample, and wherein the stage translates the sample while the light emission of the light source is fixed in position. In some embodiments, the scanner comprises one or more mirrors that direct an optical path of the light emission of the light source across a plane of the sample. In some embodiments, the scanner comprises a stage that translates the detector across the sample. In some embodiments, the scanner comprises a galvanic scanning mirror.

[0033] In some embodiments, an output intensity of the light emission of the light source is modulated at a frequency, and wherein a gain of the detector is modulated at the frequency of modulation of the light emission of the light source. In some embodiments, the output intensity of the light emission of the light source is modulated with an optical chopper, acoustic optic modulator, electric optical modulator, or any combination thereof, optically coupled to the light emission of the light source. In some embodiments, the output intensity of the light emission of the light source is modulated with an optical chopper optically coupled to the light emission of the light source. In some embodiments, the output intensity of the light emission of the light source is modulated with an acoustic optic modulator optically coupled to the light emission of the light source. In some embodiments, the output intensity of the light emission of the light source is modulated with an electric optic modulator optically coupled to the light emission of the light source.

[0034] In some embodiments, the sample comprises a biological sample from a human subject, a mammalian subject, a plant, or any combination thereof. In some embodiments, the sample comprises a biological sample from a human subject. In some embodiments, the sample comprises a biological sample from a mammalian subject. In some embodiments, the same comprises a biological sample from a plant. In some embodiments, the biological sample comprises sputum, blood, urine, cerebral spinal fluid, cervical spinal fluid, bronchial alveolar lavage fluid, fluid collected from a prosthesis, fluid collected from an implant, or any combination thereof. In some embodiments, the biological sample comprises sputum. In some embodiments, the biological sample comprises urine. In some embodiments, the biological sample comprises cerebral spinal fluid. In some embodiments, the biological sample comprises cervical spinal fluid. In some embodiments, the biological sample comprises bronchial alveolar lavage fluid. In some embodiments, the biological sample comprises fluid collected from aWSGR Docket No. 68783-701.601 prosthesis. In some embodiments, the biological sample comprises fluid collected from an implant.

[0035] In some embodiments, the instructions of the computer system are not conducted on the human or animal body. For example, in such embodiments, the sample may be an ex vivo and / or in vitro sample.

[0036] In some embodiments, the one or more cells are not filtered or removed from the biological sample.

[0037] In some embodiments, the band-pass filter comprises a first coating and a second coating, wherein the first coating transmits a first band of light, wherein the second coating transmits a second band of light, and wherein the first band of light and the second band of light differ.

[0038] In some embodiments, the instructions further comprise providing a second compound to the one or more cells in the sample, and wherein the second compound alters a metabolic state of the one or more cells. In some embodiments, the second compound comprises an antimicrobial or a pharmaceutical compound.

[0039] In some embodiments, the characterization of the one or more cells comprises a phenotype, a metabolic state, antimicrobial resistance, response to the compound, response to the second compound, or any combination thereof, of the one or more cells. In some embodiments, the characterization of the one or more cells comprises a phenotype. In some embodiments, the characterization of the one or more cells comprises a metabolic state. In some embodiments, the characterization of the one or more cells comprises an antimicrobial resistance. In some embodiments, the characterization of the one or more cells comprises a response to the compound. In some embodiments, the characterization of the one or more cells comprises a response to the second compound.

[0040] In some embodiments, the instructions further comprise generating an image of the one or more cells from the one or more Raman optical signals.

[0041] In some embodiments, the compound comprises a fluid. In some embodiments, the fluid comprises deuterium.

[0042] In some embodiments, the system comprises a first dichroic mirror optically coupled to the band-pass filter, wherein the first dichroic mirror is optically coupled to the detector. In some embodiments, the system comprises a second dichroic mirror optically coupled to the band-pass filter, wherein the second dichroic mirror is optically coupled to a second detector, wherein the second detector differs from the detector.

[0043] In some embodiments, the detector comprises a single pixel detector.

[0044] In some embodiments, the light source comprises an optical intensity of at least about 15 mW.WSGR Docket No. 68783-701.601

[0045] In some embodiments, the instructions further comprise averaging one or more detected Raman optical signals across a surface of the sample to determine the characterization of the one or more cells.

[0046] In some embodiments, the light source comprises a pulsed light source.

[0047] In some embodiments, the compound is provided to the sample by a compound dispenser. In some embodiments, the compound dispenser comprises a fluidic handling system, a nozzle, a pipette, or any combination thereof. In some embodiments, the compound dispenser comprises a fluidic handling system. In some embodiments, the compound dispenser comprises a nozzle. In some embodiments, the compound dispenser comprises a pipette.

[0048] In some embodiments, the one or more Raman optical signals comprise an integrated intensity of the one or more scattered Raman photons detected by the detector through the band - pass filter.

[0049] In another aspect, provided herein is a system for determining a characterization of one or more cells, comprising: a light source configured to provide a light emission to a sample comprising one or more cells, wherein the one or more cells absorb the light emission and scatter one or more Raman photons; a compound dispenser to provide a compound to the sample, wherein when the compound is incorporated by the one or more cells, an intensity of the one or more scattered Raman photons emitted by the one or more cells is changed compared to an absence of the incorporation of the compound by the one or more cells; a band-pass filter; a detector optically coupled to the band-pass filter, wherein the detector detects the one or more scattered Raman photons through the band-pass filter, thereby generating one or more detected Raman optical signals; and one or more processors electrically coupled to the detector, wherein the one or more processors process the one or more detected Raman optical signals and determine a characterization of the one or more cells.

[0050] In some embodiments, the one or more cells comprise one or more microbes, one or more mammalian cells, one or more cancer cells, one or more fungi, or any combination thereof. For example, the one or more cells may comprise one or more microbes. In certain embodiments, the one or more cells may comprise one or more mammalian cells. In some embodiments, the one or more cells may comprise one or more cancer cells. In embodiments, the one or more cells may comprise one or more fungi.

[0051] In some embodiments, the one or more processors determine the characterization of the one or more cells in up to about 20 minutes.

[0052] In some embodiments, the light source comprises a laser light source.

[0053] In some embodiments, the detector comprises a photomultiplier tube, an avalanche photodiode, a photodiode, a silicon photomultiplier, a multi -pixel photon counter, a single photonWSGR Docket No. 68783-701.601 avalanche photodiode, or any combination thereof. In some embodiments, the detector comprises a photomultiplier tube. In some embodiments, the detector comprises an avalanche photodiode. In some embodiments, the detector comprises a photodiode. In some embodiments, the detector comprises a silicon photomultiplier. In some embodiments, the detector comprises a multi -pixel photon counter. In some embodiments, the detector comprises a single photon avalanche photodiode. In some embodiments, a sensitivity of the detector comprises at least about 0.3 AAV. In some embodiments, the one or more scattered Raman photons are detected by the detector with up to about a 100 microsecond signal acquisition time.

[0054] In some embodiments, the band-pass filter comprises an emission band of about 1 nm to about 20nm. In some embodiments, the band-pass filter comprises a plurality of band-pass filters. In some embodiments, the plurality of band-pass filters comprises a first band-pass filter and a second band-pass filter, wherein the first band-pass filter comprises a first emission band, wherein the second band-pass filter comprises a second emission band, and wherein the first emission band and the second emission band do not overlap. In some embodiments, the second band-pass filter is optically coupled to the sample and a second detector, wherein the first bandpass filter is optically coupled to the sample and the detector, and wherein the detector and the second detector differ. In some embodiments, the band-pass filter comprises a transmission efficiency of at least about 70%.

[0055] In some embodiments, the system further comprises a scanner that scans the light emission of the light source across the sample. In some embodiments, the scanner comprises a stage coupled to the sample, and wherein the stage translates the sample while the light emission of the light source is fixed in position. In some embodiments, the scanner comprises one or more mirrors that direct an optical path of the light emission of the light source across a plane of the sample. In some embodiments, the scanner comprises a stage to translate the detector across the sample. In some embodiments, the scanner comprises a galvanic scanning mirror.

[0056] In some embodiments, an output intensity of the light emission of the light source is modulated at a frequency, and wherein a gain of the detector is modulated at the frequency of modulation of the light emission of the light source. In some embodiments, the output intensity of the light emission of the light source is modulated with an optical chopper, acoustic optic modulator, electric optical modulator, or any combination thereof, optically coupled to the light emission of the light source. In some embodiments, the output intensity of the light emission of the light source is modulated with an optical chopper optically coupled to the light emission of the light source. In some embodiments, the output intensity of the light emission of the light source is modulated with an acoustic optic modulator optically coupled to the light emission of the light source. In some embodiments, the output intensity of the light emission of the lightWSGR Docket No. 68783-701.601 source is modulated with an electric optic modulator optically coupled to the light emission of the light source.

[0057] In some embodiments, the sample comprises a biological sample from a human subject, a mammalian subject, a plant, or any combination thereof. In some embodiments, the sample comprises a biological sample from a human subject. In some embodiments, the sample comprises a biological sample from a mammalian subject. In some embodiments, the same comprises a biological sample from a plant. In some embodiments, the biological sample comprises sputum, blood, urine, cerebral spinal fluid, cervical spinal fluid, bronchial alveolar lavage fluid, fluid collected from a prosthesis, fluid collected from an implant, or any combination thereof. In some embodiments, the biological sample comprises sputum. In some embodiments, the biological sample comprises urine. In some embodiments, the biological sample comprises cerebral spinal fluid. In some embodiments, the biological sample comprises cervical spinal fluid. In some embodiments, the biological sample comprises bronchial alveolar lavage fluid. In some embodiments, the biological sample comprises fluid collected from a prosthesis. In some embodiments, the biological sample comprises fluid collected from an implant.

[0058] In some embodiments, the system is not conducted on the human or animal body. For example, in such embodiments, the sample may be an ex vivo and / or in vitro sample.

[0059] In some embodiments, the one or more cells are not filtered or removed from the biological sample.

[0060] In some embodiments, the band-pass filter comprises a first coating and a second coating, wherein the first coating transmits a first band of light, wherein the second coating transmits a second band of light, and wherein the first band of light and the second band of light differ.

[0061] In some embodiments, the compound dispenser provides a second compound to the one or more cells in the sample, and wherein the second compound alters a metabolic state of the one or more cells. In some embodiments, the second compound comprises an antimicrobial or a pharmaceutical compound.

[0062] In some embodiments, the characterization of the one or more cells comprises a phenotype, a metabolic state, antimicrobial resistance, response to the compound, or any combination thereof, of the one or more cells. In some embodiments, the characterization of the one or more cells comprises a phenotype. In some embodiments, the characterization of the one or more cells comprises a metabolic state. In some embodiments, the characterization of the one or more cells comprises an antimicrobial resistance. In some embodiments, the characterization of the one or more cells comprises a response to the compound. In some embodiments, the characterization of the one or more cells comprises a response to the second compound.WSGR Docket No. 68783-701.601

[0063] In some embodiments, the one or more processors generate an image from the one or more Raman optical signals.

[0064] In some embodiments, the compound comprises a fluid. In some embodiments, the fluid comprises deuterium.

[0065] In some embodiments, the system further comprises a first dichroic mirror optically coupled to the band-pass filter, wherein the first dichroic mirror is optically coupled to the detector. In some embodiments, the system further comprises a second dichroic mirror optically coupled to the band-pass filter, wherein the second dichroic mirror is optically coupled to a second detector, wherein the second detector differs from the detector.

[0066] In some embodiments, the detector comprises a single pixel detector.

[0067] In some embodiments, the light source comprises an optical power of at least about 15 mW.

[0068] In some embodiments, the one or more processors average one or more detected Raman optical signals across a surface of the sample to determine the characterization of the one or more cells.

[0069] In some embodiments, the light source comprises a pulsed light source.

[0070] In some embodiments, the compound dispenser comprises a fluidic handling system, a nozzle, a pipette, or any combination thereof. In some embodiments, the compound dispenser comprises a fluidic handling system. In some embodiments, the compound dispenser comprises a nozzle. In some embodiments, the compound dispenser comprises a pipette.

[0071] In some embodiments, the one or more Raman optical signals comprise an integrated intensity of the one or more scattered Raman photons detected by the detector through the band - pass filter.

[0072] In another aspect, provided herein is a system for determining a characterization of one or more cells, comprising: a light source configured to provide a light emission to a sample comprising one or more cells, wherein the one or more cells absorb the light emission and scatter one or more Raman photons; a focusing system, wherein the focusing system adjusts a position of the light source to provide the light source at a focus position of a surface of the sample; a Raman spectrometer optically coupled to a filter, wherein the Raman spectrometer detects the one or more scattered Raman photons through the filter, thereby generating one or more detected Raman optical signals; and one or more processors electrically coupled to the Raman spectrometer, wherein the one or more processors process the one or more detected Raman optical signals and determine a characterization of the one or more cells.

[0073] In another aspect, provided herein is a system for determining a characterization of one or more cells, comprising: a light source configured to provide a light emission to a sampleWSGR Docket No. 68783-701.601 comprising one or more cells, wherein the one or more cells absorb the light emission and scatter one or more Raman photons; a widefield imaging system, wherein the widefield imaging system detects one or more visible light scattered photons from the sample comprising the one or more cells, and wherein the visible light scattered photons are generated when the sample absorbs the light emission of the light source; a Raman spectrometer optically coupled to a filter, wherein the Raman spectrometer detects the one or more scattered Raman photons through the filter, thereby generating one or more detected Raman optical signals; and one or more processors electrically coupled to the Raman spectrometer, wherein the one or more processors process the one or more detected Raman optical signals and determine a characterization of the one or more cells.

[0074] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure.Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0075] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0077] FIG. 1 shows an example schematic diagram of a band-pass Raman spectroscopy imaging system, as described in some embodiments herein.

[0078] FIG. 2 shows an example schematic diagram of a Raman spectroscopy imaging system and focusing system, as described in some embodiments herein

[0079] FIG. 3 shows an example transmission intensity graph of a bandpass filter of an imaging system, as described in some embodiments herein.WSGR Docket No. 68783-701.601

[0080] FIG. 4 shows a workflow diagram for a method of characterizing a cell with a band-pass Raman spectroscopy imaging system, as described in some embodiments herein.

[0081] FIG. 5 shows a system diagram of a computer system to implement the methods of the disclosure, as described in some embodiments herein.

[0082] FIGS. 6A-6B show an example Raman spectrum of cells incorporating deuterium on a Raman shift (cm4) scale (FIG. 6 A) and the nanometer wavelength scale (FIG. 6B), as described in some embodiments herein.DETAILED DESCRIPTION

[0083] Due to the variation of antimicrobial resistance across microbes of varying phenotype, the current standard of care of treating individuals with chronic or acute life-threatening infection is to first identify the microbe and any antimicrobial resistance the microbe may exhibit. The current standard of care is to culture the microbes of a subject’s biological sample (e.g., sputum, blood, urine, bronchial lavage fluid, etc.) on varying media substrates and in the presence of antimicrobial compounds. The resulting microbes that grow on the varying media substrates with or without the presence of antibiotics are then visually identified via a light microscope and / or identified by their genetic material using gene sequencing platforms. The process of culturing and identifying microbes is arduous and lengthy. For more severe life-threatening microbial infections, the lengthy (e.g., about 2-5 day) identification and / or microbial susceptibility process may lead to a subject’s worsening conditions, permanent disability and / or injury, or even death. As such, there exists a need for a faster method of determining the type and / or phenotype of microbes present in a subject’s sample to be able to treat a subject timely ahead of any further downstream effects of the infection on the subject’s quality of life.

[0084] Recently, Raman spectroscopy has shown the ability to identify and distinguish one or more microbes from one another based on Raman spectroscopic figure prints of the one or more microbes in a shorter time span, e.g., about 30 minutes to about 3 hours, as compared to traditional microbial culturing methodologies spanning several days of processing necessary to provide bacterial identification and / or antimicrobial susceptibility . Raman Spectroscopy probes a chemical composition of a sample by exciting electrons of chemical bonds of the sample with light energy (e.g., a laser) and detecting scattered photons of light emitted as the electrons relax to a ground vibrational state. Although Raman spectroscopy poses many benefits such as an improvement in detection speed over current culturing methodologies, Raman spectroscopy requires sample processing and culturing ahead of spectroscopic analysis to improve detection signal to noise ratios and accuracy. Such Raman spectroscopy required sample processing approaches substantially limit sample processing time and throughput, thereby preventing wideWSGR Docket No. 68783-701.601 scale use and adoption of Raman spectroscopy in virology clinics. Accordingly, the disclosure describes systems and / or methods that modify the traditional structure of Raman spectroscopy imaging systems and sample preparation to further decrease sample processing required to identify e.g., one or more microbes of a subject’s biological sample.

[0085] Provided herein are systems and methods for identifying and / or characterizing one or more cells (e.g., microbes, mammalian, cancer, fungi, etc.) of a biological sample using Raman spectroscopy. For example, in some cases, the systems and / or and methods described herein decrease the amount of time required for the analysis and identification of microbes compared to the time required to identify microbes through culturing and microscope visualization or genomic testing. In some cases, the amount of time required for the analysis may be reduced by limiting and / or targeting the Raman spectroscopic analysis to a one or more bands and / or spectra of the one or more cell’s entire Raman spectra. In some cases, the one or more bands and / or spectra may be representative of the distinguishing aspects between the cells and / or between the presence or absence of cells in a sample. In some cases, the one or more bands and / or spectra of the one or more cells entire Raman spectra may be chosen to target the presence of one or more compounds that may interact with, become absorbed by, and / or incorporated in the one or more cells, described elsewhere herein. For example, the one or more compounds may comprise deuterium, where deuterium has a known and well understood Raman spectral signature. In some cases, deuterium may be provided to a subject’s sample prior to obtaining and / or receiving the Raman spectra of the sample such that the compound may be taken up and / or absorbed by the one or more cells and adjust and / or change the Raman spectra of the one or more cells. In some cases, a presence, identity, and / or characterization of the one or more cells may be determined by detecting and / or determining the presence of the characteristic Raman signature(s) of the one or more compounds. In some cases, the one or more compounds may comprise an antimicrobial compound, a pharmaceutical compound, deuterium, or a combination thereof.

[0086] In some cases, the imaging systems, described elsewhere herein, may comprise one or more filters provided along an optical path coupled to one or more detectors. In some instances, the one or more filters may transmit and / or reflect one or more spectral bands of one or more Raman scattered photons emitted by a sample to target, refine, and / or narrow the analysis of the one or more Raman spectral bands of the sample, as described elsewhere herein. In some cases, by targeting and / or narrowing the analysis of the one or more Raman spectral bands, time otherwise necessary to scan over a sample’s entire Raman spectral band may be reduced. In some cases, the one or more filters may comprise one or more dichroic mirrors, one or more long wavelength pass filters, one or more short wavelength pass filters, one or more band pass filters, and / or one or more notch filters. In some cases, the one or more filters may comprise one or moreWSGR Docket No. 68783-701.601 dichroic mirrors. In some cases, the one or more filters may comprise one or more long wavelength pass filters. In some cases, the one or more filters may comprise one or more notch filters.

[0087] In some cases, a Raman spectrum can be acquired and / or detected through one or more band pass filters to limit the analyzed Raman spectrum to a subset range of emitted Raman scattered photon bands e.g., one or more Raman spectral bands of the one or more cells and / or the one or more compounds taken up, absorbed, and / or that react by and / or with the one or more cells. In some cases, a band pass filter may transmit photons of light of a wavelength band with high efficiency, e.g., at least about 90%, as shown in FIG. 3. Optical band pass filters can be utilized for the detection of various specific bands, e.g., a corresponding Raman spectra of a carbon-deuterium (D2O) (C-D) bond, described elsewhere herein.Imaging Systems

[0088] Described herein are imaging systems (100, 200) for determining a characteristic, phenotype, and / or identity of one or more cells (e.g., microbes, mammalian cells, cancer cells, fungi, etc.) of a sample. In some cases, the imaging system (100, 200), e.g., as shown in FIGS. 1 and 2, may comprise a Raman spectroscopy system. In some cases, the imaging system may comprise a light source 102 (e.g., a laser) that provides a light emission to a sample 118 comprising one or more cells, where the one or more cells may absorb the light emission and scatter one or more Raman photons that are detected by a spectrometer (144) and / or one or more detectors (130, 138) coupled to one or more filters (131, 134) (e.g., one or more band pass filters).

[0089] In some cases, the light source 102 may be optically coupled to one or more polarizers and / or wave plates 104, e.g., a quarter wave plate to control for a polarization of an emitted beam of the light source.

[0090] In some instances, the light source 102 may be optically coupled to laser alignment optical element 106. In some cases, the laser alignment optical element 106 may comprise a kinematic mounted glass element. In some instances, the kinematic mounted glass element 106 may comprise a gimbal mounted glass element. In some cases, the glass element may comprise a glass window. In some cases, the kinematic mounted glass element may be provided within the kinematic mount at an angle with respect to an optical axis of the light source 102. In some cases, an emitted beam of the light source may transmit and / or refract through the mounted glass element to adjust and / or align a beam path of the emission of the light source coupled to one or more imaging system components. By aligning the emitted beam of the light source, coupling efficiency of the one or more optical components of the imaging system are optimized.WSGR Docket No. 68783-701.601

[0091] In some instances, the emitted beam of the light source from the kinematic mounted glass element may be reflected, directed, and / or steered by a first mirror 108 to a beam expanding element 110. In some cases, the first mirror 108 may be adjustable, e.g., by a tip-tilt mechanism to tip and / or tilt the first mirror to control a beam path of the reflected beam. In some instances, the beam expanding element 110 may expand a beam of the light source 102 to achieve a focused beam spot of up to about 1000 nanometer (nm) diameter on the sample 118.

[0092] In some cases, the focused beam spot may comprise a diameter of about 700 nm to about 1,200 nm. In some cases, the focused beam spot may comprise a diameter of about 700 nm to about 750 nm, about 700 nmto about 800 nm, about 700 nm to about 850 nm, about 700 nm to about 900 nm, about 700 nmto about 950 nm, about 700 nm to about 1,000 nm, about 700 nm to about 1,200 nm, about 750 nm to about 800 nm, about 750 nm to about 850 nm, about 750 nm to about 900 nm, about 750 nmto about 950 nm, about 750 nm to about 1,000 nm, about 750 nm to about 1,200 nm, about 800 nm to about 850 nm, about 800 nm to about 900 nm, about 800 nm to about 950 nm, about 800 nmto about 1,000 nm, about 800 nm to about 1,200 nm, about 850 nm to about 900 nm, about 850 nm to about 950 nm, about 850 nm to about 1,000 nm, about 850 nm to about 1,200 nm, about 900 nm to about 950 nm, about 900 nm to about 1,000 nm, about 900 nm to about 1,200 nm, about 950 nm to about 1,000 nm, about 950 nm to about 1,200 nm, or about 1,000 nm to about 1,200 nm. In some cases, the focused beam spot may comprise a diameter of about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, about 1,000 nm, or about 1,200 nm. In some cases, the focused beam spot may comprise a diameter of at least about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, or about 1,000 nm. In some cases, the focused beam spot may comprise a diameter of at most about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, about 1,000 nm, or about 1,200 nm.

[0093] In some cases, an output beam of the light source 102 may be expanded by the beam expanding element 110 by a factor of at least about 5 times expansion.

[0094] In some cases, an output beam of the light source 102 may be expanded by the beam expanding element 110 by a factor of about 5 to about 25. In some cases, an output beam of the light source 102 may be expanded by the beam expanding element 110 by a factor of about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 5 to about 12, about 5 to about 14, about 5 to about 16, about 5 to about 18, about 5 to about 20, about 5 to about 25, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 6 to about 12, about 6 to about 14, about 6 to about 16, about 6 to about 18, about 6 to about 20, about 6 to about 25, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 7 to about 12, about 7 to about 14, about 7 to about 16, about 7 to about 18, about 7 to about 20,WSGR Docket No. 68783-701.601 about 7 to about 25, about 8 to about 9, about 8 to about 10, about 8 to about 12, about 8 to about 14, about 8 to about 16, about 8 to about 18, about 8 to about 20, about 8 to about 25, about 9 to about 10, about 9 to about 12, about 9 to about 14, about 9 to about 16, about 9 to about 18, about 9 to about 20, about 9 to about25, about 10 to about 12, about lOto about 14, about 10 to about 16, about 10 to about 18, about 10 to about20, about 10 to about25, about 12 to about 14, about 12 to about 16, about 12 to about 18, about 12 to about 20, about 12 to about 25, about 14 to about 16, about 14 to about 18, about 14to about 20, about 14 to about25, about 16 to about 18, about 16 to about20, about 16 to about25, about 18 to about 20, about 18 to about 25, or about 20 to about 25. In some cases, an output beam of the light source 102 may be expanded by the beam expanding element 110 by a factor of about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, or about 25. In some cases, an output beam of the light source 102 may be expandedby the beam expanding element llOby a factor of at least about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, or about 20. In some cases, an output beam of the light source 102 may be expanded by the beam expanding element 110 by a factor of at most about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, or about 25.

[0095] In some instances, the expanded output of the beam 111 may be optically coupled to a second mirror 112. In some cases, the second mirror 112 may be adjustable, e.g., by a tip-tilt mechanism to tip and / or tilt the second mirror to control a beam path of the reflected beam.

[0096] In some cases, the second mirror 112 may reflect the expanded beam 111 to a first filter 116. In some instances, the first filter 116 may comprise a first dichroic mirror. In some cases, the first filter may transmit the emitted beam 111 of the light source 102 and reflect one or more scattered photons 113 from the sample 118 to a widefield imaging system 114.

[0097] In some cases, the widefield imaging system 114 may comprise a visible light imaging system to detect one or more visible light photons 113, e.g., one or more visible light photons emitted and / or scattered from the sample as a result of the sample 118 absorbing an emitted beam of the light source. In some cases, the one or more visible light photons may comprise one or more blue-shifted photons. In some cases, the widefield imaging system 114 may detect the one or more scattered visible light photons and convert and / or produce an image from the detected one or more visible light photons of the sample. In some cases, the widefield imaging system may detect the one or more visible light photons scattered from the sample and / or the one or more cells of the sample without moving one or more filters into or out of an optical path of detecting the one or more visible light photons. The widefield imaging system may reduce error in reproducing the location of the one or more scattered visible light photons of the sample and / or the one or more cells of the sample by not moving the one or more filters into or out of theWSGR Docket No. 68783-701.601 optical path of detecting the one or more visible light photons. For example, the one or more filters may induce a spherical and / or chromatic aberration that may shift the position of the one or more visible light scattered photons from the origin of the scattered photons on the sample and / or the one or more cells of the sample. In some cases, the image of the sample detected and / or processed by the widefield imaging system may provide a method for aligning the emitted beam of the light source with the one or more cells of the sample. By aligning the emitted beam of the light source with the one or more cells of the sample, a quantum efficiency of converting the excitation emitted beam of the light source to one or more scattered Raman photons 123 detected by the Raman spectrometer (144) and / or the one or more detectors (138, 130) coupled to one or more filters (131, 134) may be increased compared to a quantum efficiency of converting the excitation emitted beam of the light source to one or more scattered Raman photons 123 without aligning the emitted beam of the light source.

[0098] In some cases, the emitted beam of the light source may be coupled to a second filter 122. In some cases, the second filter 122 may comprise a second dichroic mirror. In some instances, the second filter 122 may reflect the emitted beam of the light source and transmit one or more Raman scattered photons 123 of the sample 118, where the one or more Raman scattered photons are generated, emitted, and / or produced when the sample and / or the one or more cells of the sample absorb the emitted beam of the light source and fall from an excited state to a ground state.

[0099] In some cases, the second dichroic mirror may comprise a reflection -transmittance transition wavelength of about 594 nm.

[0100] In some cases, the second filter 122 may reflect one or more visible light photons, described elsewhere herein, towards the widefield imaging system 114 to be visualized for aligning the emitted beam of the light source and the one or more cells of the sample . In some cases, the sample may comprise a sample and / or one or more cells of the sample provided on and / or within one or more glass cover slips.

[0101] In some instances, the reflected emitted beam of the light source from the second filter 122 may be coupled to an objective lens 120 that focuses the emitted beam of the light source onto the sample and / or the one or more cells of the sample.

[0102] In some cases, the objective lens may comprise a numerical aperture of about 0.72 to about 0.95. In some cases, the objective may comprise a numerical aperture of about 0.72 to about 0.75, about 0.72to about 0.8, about 0.72 to about 0.85, about 0.72to about 0.9, about 0.72 to about 0.95, about 0.75 to about 0.8, about 0.75 to about 0.85, about 0.75 to about 0.9, about 0.75 to about 0.95, about 0.8 to about 0.85, about 0.8 to about 0.9, about 0.8 to about 0.95, about 0.85 to about 0.9, about 0.85 to about 0.95, or about 0.9 to about 0.95. In some cases, theWSGR Docket No. 68783-701.601 objective lens may comprise a numerical aperture of about 0.72, about 0.75, about 0.8, about 0.85, about 0.9, or about 0.95. In some cases, the objective lens may comprise a numerical aperture of at least about 0.72, about0.75, about0.8, about0.85, orabout0.9. In some cases, the objective lens may comprise a numerical aperture of at most about 0.75, about 0.8, about 0.85, about 0.9, or about 0.95.

[0103] In some cases, the objective lens may comprise a magnification of about 40X to about 100X. In some cases, the objective lens may comprise a magnification of about 40X to about 50X, about 40X to about 60X, about 40X to about 70X, about 40X to about 80X, about 40X to about 90X, about 40X to about 100X, about 50X to about 60X, about 50X to about 70X, about 50Xto about 80X, about 50Xto about 90X, about 50X to about 100X, about 60X to about 70X, about 60X to about 80X, about 60X to about 90X, about 60X to about 100X, about 70X to about 80X, about 70X to about 90X, about 70X to about 100X, about 80Xto about 90X, about 80X to about 100X, or about 90X to about 100X. In some cases, the objective lens may comprise a magnification of about 40X, about 50X, about 60X, about 70X, about 80X, about 90X, or about 100X. In some cases, the objective lens may comprise a magnification of at least about 40X, about 50X, about 60X, about 70X, about 80X, or about 90X. In some cases, the objective lens may comprise a magnification of at most about 50X, about 60X, about 70X, about 80X, about 90X, or about 100X.

[0104] In some cases, the imaging system 200 may comprise a focusing system and / or focusing apparatus 148, that may adjust a position of the imaging system objective lens 120 with respect to the sample 118 to focus the emitted beam of the light source onto a surface of the sample and / orthe one ormore cells of the sample. In some cases, the focusing system and / or focusing apparatus may increase the coupling efficiency of collecting the one or more scattered Raman photons 123 from the sample 118. In some cases, the focusing system and / or focusing apparatus may comprise a near infrared light source that may emit an emitted beam of near infrared light 115 towards the sample to determine a focal position of the objective lens 120 with respect to the sample 118. In some cases, the focusing system and / or apparatus 148 may determine a focus of the imaging system 200 by measuring a reflected and / or refracted angle of the near infrared light 115 reflected and / or refracted from a surface of the sample with respect to an optical axis of the objective lens 120. In some cases, the focusing system and / or focusing apparatus 148 may comprise a line sensor to detect the reflected and / or refracted angle of the near infrared light.

[0105] In some cases, the imaging system may comprise a third filter 117 optically coupled to the focusing system and / or focusing apparatus 148, where the third filter may reflect the focusing system and / or focusing apparatus emitted near infrared light 115 to the objective lens 120. InWSGR Docket No. 68783-701.601 some cases, the third filter 117 may comprise a dichroic mirror that reflects near-infrared light and transmits the one or more scattered Raman photons 123.

[0106] In some instances, the near infrared light 115 reflected off of a surface of the sample 118 may be collected by the objective lens 120 and detected by the focusing system and / or focusing apparatus to determine if the surface of the sample 118 is within a focus of the objective lens 120. In some cases, the focusing system and / or focusing apparatus emitted near infrared light 115 may be provided at an offset with respect to the optical axis of the objective lens 120 to avoid potential cross-talk between reflected near infrared light 115 of the focusing system and / or focusing apparatus collected from the surface of the sample and the one or more scattered Raman photons 123.

[0107] In some cases, the one or more scattered Raman photons 123 may be optically coupled from the sample 118 through the objective lens 120 to a fourth filter 124. In some cases, the fourth filter 124 may comprise a long wavelength pass filter.

[0108] In some cases, the one or more scattered Raman photons 123 passing through the fourth filter 124 may be coupled to a confocal spatial filter 126. In some instances, the confocal spatial filter may comprise a pin hole. In some cases, an output of the confocal spatial filter 126 may be coupled to a fifth filter 150.

[0109] In some cases, the fifth filter may comprise a filter that reflects near infrared and / or infrared photons and transmits the one or more scattered Raman photons 123. In some cases, the fifth filter 150 may prevent the scattered photons of the near infrared emitted beam 115 of the focusing system and / or focusing apparatus 148 from being collected and / or detected by a detector thereby reducing noise that would otherwise be detected with the detected signal of the one or more scattered Raman photons.

[0110] In some cases, the one or more scattered Raman photons passing through the confocal spatial filter may be optically coupled to an optical relay of one or more lenses (142, 132, 136). In some cases, the one or more lenses may comprise a periscope 142 and / or one or more achromatic doublets (132, 136). In some instances, the periscope 142 may be optically coupled to one or more lenses (132, 136). In some cases, the one or more lenses may direct and / or guide the one or more Raman scattered photons to a Raman spectrometer 144, where the one or more Raman scattered photons can be detected, processed, and / or further analyzed e.g., by processing and / or analyzing one or more Raman spectral signatures determined and / or identified from the one or more Raman scattered photons.

[0111] In some cases, the output of the confocal spatial filter 126 may be coupled to a third mirror 128, where the third mirror 128 may direct, reflect, and / or steer the one or more scattered Raman photons to a sixth filter 140, as shown in FIG. 1. In some cases, the sixth filter 140 mayWSGR Docket No. 68783-701.601 comprise a dichroic mirror. In some cases, the dichroic mirror may reflect a first one or more wavelengths and / or first one or more bands of the one or more scattered Raman photons and transmit a second one or more wavelengths and / or a second one or more bands of the one or more scattered Raman photons.

[0112] In some instances, the reflected first one or more wavelengths and / or first one or more bands of the one or more scattered Raman photons may be coupled to a seventh filter 131. In some cases, the seventh filter 131 may comprise a band-pass filter. In some cases, the seventh filter may be optically coupled to a lens 132 where the lens may focus the first one or more wavelengths and / or first one or more bands of the one or more scattered Raman photons onto a first detector 130. In some cases, the transmitted second one or more wavelengths and / or second one or more bands of the one or more scattered Raman photons may transmit through the seventh filter and may be optically coupled to an eighth filter 134.

[0113] In some cases, the eighth filter 134 may comprise a band pass filter. In some cases, the eighth filter may be coupled to a lens 136. The lens 136 may focus the second one or more wavelengths and / or second one or more bands of the one or more scattered Raman photons onto a detector 138 to detect the second one or more wavelengths and / or second one or more bands of the one or more scattered Raman photons. In some cases, the seventh filter 131 may comprise a first transmission wavelength band and where the eighth filter 134 may comprise a second transmission wavelength band. In some cases, the first transmission wavelength band and the second transmission wavelength band differ and / or do not overlap. In some cases, the first transmission wavelength band of the seventh filter may comprise a wavelength band with a characteristic Raman spectral band of one or more cells and / or one or more compounds alone or of one or more cells after interacting with the one or more compounds. In some instances, the second transmission wavelength band of the eighth filter may comprise a calibration wavelength band that may be used to calibrate the imaging system.

[0114] In some cases, the light source 102 may comprise a laser light source. In some instances, the light source may comprise a continuous wave (CW) output. In some cases, the light source may comprise a pulsed output. In some cases, the pulsed light source may comprise a pulse frequency of about 1 kilohertz (kHz) to about 40 kHz. In some cases, the light source intensity may be modulated at one or more frequencies. In some cases, the light source emitted beam may be modulated in intensity by an optical chopper, acoustic optic modulator (AOM), electric optical modulator (EOM), or any combination thereof, optically coupled to the light source. In some cases, the light source emitted beam may be modulated in intensity by an optical chopper optically coupled to the light source. In some cases, the light source emitted beam may be modulated in intensity by an acoustic optic modulator (AOM) optically coupled to the lightWSGR Docket No. 68783-701.601 source. In some cases, the light source emitted beam may be modulated by an electric optic modulator optically coupled to the light source.

[0115] FIG. 3 shows an example wavelength transmission graph 300 of a band pass filter of the imaging system, described elsewhere herein. In some cases, the band pass filter may comprise a transmission band with a range of wavelengths to transmit the one or more scattered Raman photons emitted from one or more electrons of a C-D bond when relaxing from an excited to a ground state.

[0116] In some cases, the C-D bond wavelength band may comprise a wavelength band of about 595 nm to about 605nm. In some cases, the band pass filter can reflect and / or block other wavelength and transmit light from about 595 nm to about 605 nm with transmission efficiency greater than about 70%. Such a transmission wavelength band covers the central band of C-D vibration (601-602 nm). In some cases, the transmission efficiency can be greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, or from about 95% to about 100%.

[0117] In some cases, the imaging system, described elsewhere herein, may detect an intensity of one or more Raman spectral bands. Each detected spectral band of the one or more Raman spectral bands may be analyzed with one or more band pass filters. In some cases, for example, a first band-pass filter may be utilized by the imaging system to detect a Raman spectral band for calibration and where a second band-pass filter may be utilized by the imaging system to detect signal of one or more Raman scattered photons of a known compound or one or more cells.

[0118] In some cases, the imaging system may comprise a first hand pass filter and a second band pass filter, where the first band pass filter comprises a transmission wavelength band that differs from the second band pass filter transmission wavelength band. The time needed to acquire signal of the sample and / or the one or more cells of the sample may be reduced by using one or more band pass filters to analyze one or more portions of Raman spectra of the sample and / or the one or more cells of the sample.

[0119] In some cases, the bandwidth of the transmitted wavelength band of the band pass filter can be from about a 1 nm wavelength band to about a 100 nm wavelength band. The bandwidth of the transmitted wavelength band of the filter can be from about 1 nm to about 2 nm, about 1 nm to about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 15 nm, about 1 nm to about 20 nm, about 1 nm to about 50 nm, about 1 nm to about 75 nm, about 1 nm to about 100 nm, about 2 nm to about 5 nm, about 2 nm to about 10 nm, about 2 nm to about 15 nm, about 2 nm to about 20 nm, about 2 nm to about 50 nm, about 2 nm to about 75 nm, about 2 nm to about 100 nm, about 5 nm to about 10 nm, about 5 nm to about 15 nm, about 5 nm to about 20 nm, about 5 nm to about 50 nm, about 5 nm to about 75 nm, about 5 nm to about 100 nm, about 10 nm toWSGR Docket No. 68783-701.601 about 15 nm, about 10 nmto about 20 nm, about 10 nm to about 50 nm, about 10 nm to about 75 nm, about 10 nm to about 100 nm, about 15 nm to about 20 nm, about 15 nm to about 50 nm, about 15 nm to about 75 nm, about 15 nm to about 100 nm, about 20 nm to ab out 50 nm, about 20 nm to about 75 nm, about 20 nm to about lOO nm, about 50 nm to about 75 nm, about 50 nm to about 100 nm, or about 75 nm to about 100 nm. The bandwidth of the transmitted wavelength band of the band pass filter can be at least about 1 nm, about 2 nm, about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 50 nm, or about 75 nm. The bandwidth of the transmitted wavelength band of the band pass filter can be at most about 2 nm, about 5 nm, about 10 nm, about 15 nm, about20 nm, about 50 nm, about 75 nm, or about 100 nm. In some cases, the bandpass filter comprises an emission band of about 1 nm to about 20 nm.

[0120] In some cases, the transmission wavelength band width, the starting transmission wavelength, and / or the ending transmission wavelength of the band pass filter may be selected with respect to, e.g., one or more silent Raman wavelength bands of the one or more cells, described elsewhere herein. In some cases, the transmission wavelength band width of the band pass filter may be selected with respect to e.g., one or more silent Raman wavelength bands of the one or more cells, described elsewhere herein. In some cases, the starting transmission wavelength of the band pass filter may be selected with respect to e.g., one or more silent Raman wavelength bands of the one or more cells, described elsewhere herein. In some cases, the ending transmission wavelength of the band pass filter may be selected with respect to e.g., one or more silent Raman wavelength bands of the one or more cells, described elsewhere herein. For example, the band pass filter may comprise a transmittance band from about 590 nm to about 606 nm. In some cases, a silent Raman wavelength band of the one or more cells may comprise a wavelength band from about 590nm to about 591.5 nm.

[0121] In some cases, the imaging system may comprise a scanner that scans an emitted beam of the light source 102 (e.g., a laser) across the sample and / or the one or more cells. In some cases, the scanner may be optically coupled with the objective lens 120 to scan the emitted beam of the light source (105, 111) across the aperture of the objective lens.

[0122] In some cases, the scanner may comprise a galvanic scanning mirror. In some cases, the scanner may comprise a first scanning mirror and a second scanning mirror, where the first scanning mirror scans the emitted beam of the light source across a first axis of the sample and / or the one or more cells of the sample, and where the second scanning mirror scans the emitted beam of the light source across a second axis at an angle with the first axis across the sample and / or the one or more cells of the sample.

[0123] In some cases, the scanner may comprise a stage. In some cases, the stage may be coupled to the sample 118, where the stage may move the sample in at least about one-WSGR Docket No. 68783-701.601 dimension, at least about two-dimensions, or in up to about three-dimensions. In some cases, the stage may move the sample in at least about one-dimension. In some cases, the stage may move the sample in at least about two-dimensions. In some cases, the stage may move the sample in at least about three-dimensions.

[0124] In some instances, the stage may be coupled to the imaging system where the stage may translate the imaging system and the imaging system objective lens across the sample while the sample may be stationary.

[0125] In some cases, the scanner may scan across an entire surface of the sample and / or the one or more cells of sample. In some cases, the scanner may scan across one or more portions of the sample.

[0126] In some cases, the scanning time of scanning the emitted beam of the light source across the entire sample may vary based at least upon an optical intensity of the emitted beam of the light source and the optical saturation threshold of the sample and / or the one or more cells of the sample. In some cases, the scanning time of the scanner may provide a time that the sample at a location and / or region of the sample is exposed to the emitted beam of the light source. For example, a higher optical intensity emitted beam of the light source may require a shorter exposure time of the high optical intensity of the emitted beam of the light source to the sample compared with a longer exposure time of a lower optical intensity emitted beam of the light source. Accordingly, the imaging systems described herein may adjust and / or tune the output optical intensity of the light source and the scanning time to further decrease the time required to characterize and / or identify the one or more cells of the sample.

[0127] In some cases, the light exposure time of the emitted beam of the light source provided to the sample (e.g., one or more regions of the sample) may be from about 100 microseconds (ps) to about 60 minutes. In some cases the light exposure time of the emitted beam of the light source provided to the sample may be from about 100 ps to about 500 ps, about 100 ps to about 1 millisecond (ms), about 100 ps to about 1 s, about 100 ps to about 4 s, about 100 ps to about 30 s, about 100 ps to about 1 min, about 100 ps to about 20 mins, about 100 ps to about 40 mins, about 100 ps to about 60 mins, about 500 ps to about 1 ms, about 500 ps to about 1 s, about 500 ps to about 4 s, about 500 ps to about 30 s, about 500 ps to about 1 min, about 500 ps to about 20 mins, about 500 ps to about 40 mins, about 500 ps to about 60 mins, about 1 ms to about 1 s, about 1 ms to about 4 s, about 1 ms to about 30 s, about 1 ms to about 1 min, about 1 ms to about 20 mins, about 1 ms to about 40 mins, about 1 ms to about 60 mins, about 1 s to about 4 s, about 1 s to about 30 s, about 1 s to about 1 min, about 1 s to about 20 mins, about 1 s to about 40 mins, about 1 s to about 60 mins, about 4 s to about 30 s, about 4 s to about 1 min, about 4 s to about 20 mins, about 4 s to about 40 mins, about 4 s to about 60 mins, about 30 s to about 1 min, about 30WSGR Docket No. 68783-701.601 s to about 20 mins, about 30 s to about 40 mins, about 30 s to about 60 mins, about 1 min to about 20 mins, about 1 min to about 40 mins, about 1 min to about 60 mins, about 20 mins to about 40 mins, about 20 mins to about 60 mins, or about 40 mins to about 60 mins. In some cases, the light exposure time of the emitted beam of the light source provided to the sample may be at least about 100 ps, about 500 ps, about 1 min, about 1 min, about 4 s, about 30 s, about 1 min, about 20 mins, or about 40 mins. In some cases, the light exposure time of the emitted beam of the light source provided to the sample may be at most about 500 ps, about 1 min, about 1 min, about 4 s, about 30 s, about 1 min, about 20 mins, about 40 mins, or about 60 mins.

[0128] In some cases, a collected signal and / or spectra of one or more scattered Raman photons of the sample and / or the one or more cells of the sample may be averaged across one or more regions of the sample to reduce noise of the signal collected.

[0129] In some cases, the optical intensity of the light source (e.g., an optical intensity of the emitted beam of the light source) may be about 1 mW to about 60 mW. In some cases, the optical intensity of the light source may be about 1 mW to about 2 mW, about 1 mW to about 3 mW, about 1 mW to about 4 mW, about 1 mW to about 5 mW, about 1 mW to about 8 mW, about 1 mW to about 10 mW, about 1 mW to about 15 mW, about 1 mW to about 20 mW, about 1 mW to about 30 mW, about 1 mW to about 40 mW, about 1 mW to about 60 mW, about 2 mW to about 3 mW, about 2 mW to about 4 mW, about 2 mW to about 5 mW, about 2 mW to about 8 mW, about 2 mW to about 10 mW, about 2 mW to about 15 mW, about 2 mW to about 20 mW, about 2 mW to about 30 mW, about 2 mW to about 40 mW, about 2 mW to about 60 mW, about3 mW to about 4 mW, about 3 mW to about 5 mW, about 3 mW to about 8 mW, about 3 mW to about 10 mW, about 3 mW to about 15 mW, about 3 mW to about 20 mW, about 3 mW to about 30 mW, about 3 mW to about 40 mW, about 3 mW to about 60 mW, about 4 mW to about 5 mW, about 4 mW to about 8 mW, about 4 mW to about 10 mW, about 4 mW to about 15 mW, about 4 mW to about 20 mW, about 4 mW to about 30 mW, about 4 mW to about 40 mW, about4 mW to about 60 mW, about 5 mW to about 8 mW, about 5 mW to about 10 mW, about 5 mW to about 15 mW, about 5 mW to about 20 mW, about 5 mW to about 30 mW, about 5 mW to about 40 mW, about 5 mW to about 60 mW, about 8 mW to about 10 mW, about 8 mW to about 15 mW, about 8 mW to about 20 mW, about 8 mW to about 30 mW, about 8 mW to about 40 mW, about 8 mW to about 60 mW, about 10 mW to about 15 mW, about 10 mW to about 20 mW, about 10 mW to about 30 mW, about 10 mW to about 40 mW, about 10 mW to about 60 mW, about 15 mW to about 20 mW, about 15 mW to about 30 mW, about 15 mW to about 40 mW, about 15 mW to about 60 mW, about 20 mW to about 30 mW, about 20 mW to about 40 mW, about 20 mW to about 60 mW, about 30 mW to about 40 mW, about 30 mW to about 60 mW, or about 40 mW to about 60 mW. In some cases, the optical intensity of the light sourceWSGR Docket No. 68783-701.601 may be about 1 mW, about 2 mW, about 3 mW, about 4 mW, about 5 mW, about 8 mW, about 10 mW, about 15 mW, about 20 mW, about 30 mW, about 40 mW, or about 60 mW. In some cases, the optical intensity of the light source may be at least about 1 mW, about 2 mW, about 3 mW, about 4 mW, about 5 mW, about 8 mW, about 10 mW, about 15 mW, about 20 mW, about 30 mW, or about 40 mW. In some cases, the optical intensity of the light source may be at most about 2 mW, about 3 mW, about 4 mW, about 5 mW, about 8 mW, about 10 mW, about 15 mW, about 20 mW, about 30 mW, about 40 mW, or about 60 mW.

[0130] The sample, as described herein, may comprise a biological sample from a subject. In some embodiments, the biological sample is not part of the subject’s body. For example, the biological sample may be obtained or collected from the patient’s body. In some embodiments, the biological sample may be ex vivo and / or in vitro.

[0131] In some cases, the subject may comprise a human subject, a mammalian subject, a plant, or any combination thereof. In some cases, the subject may comprise a human. In some cases, the subject may comprise a mammalian subject. In some cases, the subject may comprise a plant.

[0132] The sample can comprise sputum, blood, urine, cerebral spinal fluid, cervical spinal fluid, bronchial alveolar lavage fluid, fluid collected from a prosthesis, fluid collected from an implant, or any combination thereof. In some cases, the sample may comprise sputum. In some cases, the sample may comprise blood. In some cases, the sample may comprise cerebral spinal fluid. In some cases, the sample may comprise cervical spinal fluid. In some cases, the sample may comprise bronchial alveolar lavage fluid. In some cases, the sample may comprise fluid collected from a prosthesis. In some cases, the sample may comprise fluid collected from an implant.

[0133] In some cases, the sample may comprise one or more cells, as described elsewhere herein. The one or more cells of sample can be removed and / or filtered from the sample prior to imaging the sample with the imaging systems described elsewhere herein. In some cases, the one or more cells of the sample can remain within and / or in the sample further reducing time required for sample preparation to remove the cells.

[0134] In some cases, the entire sample may be used, for example when the entire sample is expected to have the same or similar emission properties. In some cases, the one or more cells may comprise microbes, mammalian cells, cancer cells, fungi, any other cells with a known spectra or silent Raman spectral zone that overlaps with one or more compounds Raman spectra, or any combination thereof. In some cases, the one or more cells may comprise microbes. In some cases, the one or more cells may comprise mammalian cells. In some cases, the one or more cells may comprise cancer cells. In some cases, the one or more cells may comprise fungi. In some cases, the one or more cells may comprise any other cells with a known spectra or silent Raman spectral zone that overlaps with one or more compounds Raman spectra. A silent zoneWSGR Docket No. 68783-701.601 can be a wavelength band range of a Raman spectra for a cell where there is low signal variation in emitted signal of the one or more scattered Raman photons, such that the signal is constant and additions to the signal in that zone (e.g., by taking up a compound) are visible.

[0135] In some cases, the light source may comprise a center wavelength of about 532 nm, about 638 nm, about 785 nm, and / or about 1064 nm. In some cases, the imaging system may comprise a notch filter to block reflected and / or scattered light of the emitted beam of the light source, e.g., reflected light of the emitted beam of the light source from the sample from being detected and adding noise to the detected signal of the one or more scattered Raman photons.

[0136] In some cases, the one or more detectors (138, 130, 144) may comprise a constant or a varying sensitivity. In some cases, the one or more detectors may comprise a sensitivity to detect a single scattered Raman photon. In some cases, the one or more detectors may comprise a detector that counts the number of scattered Raman photons e.g., of one or more wavelength bands of one or more scattered Raman photons.

[0137] In some cases, the one or more detectors (138, 130), described elsewhere herein, may comprise a photomultiplier tube. In some cases, the one or more detectors may comprise an avalanche photodiode. In some cases, the one or more detectors may comprise one or more photodiodes. In some cases, the one or more detectors may comprise one or more silicon photomultiplier detectors. In some cases, the one or more detectors may comprise a multi-pixel photon counter. In some cases, the one or more detectors may comprise a single photon avalanche photodiode. In some cases, the one or more detectors may comprise an adjustable gain. The adjustable gain of the detector may be modulated at one or more light source intensity modulation frequencies , described elsewhere herein.

[0138] The sensitivity of the detector may be about 0.3 ampere / Watt (A / W) to about 5 A / W. The sensitivity of the detector may be about 0.3 A / W to about 0.4 A / W, about 0.3 A / W to about 0.5 A / W, about 0.3 A / W to about 0.6 A / W, about 0.3 A / W to about 0.8 A / W, about 0.3 A / W to about 1 A / W, about 0.3 A / W to about 2 A / W, about 0.3 A / W to about 3 A / W, about 0.3 A / W to about 4 A / W, about 0.3 A / W to about 5 A / W, about 0.4 A / W to about 0.5 A / W, about 0.4 A / W to about 0.6 A / W, about 0.4 A / W to about 0.8 A / W, about 0.4 A / W to about 1 A / W, about 0.4 A / W to about 2 A / W, about 0.4 A / W to about 3 A / W, about 0.4 A / W to about 4 A / W, about 0.4 A / W to about 5 A / W, about 0.5 A / W to about 0.6 A / W, about 0.5 A / W to about 0.8 A / W, about 0.5 A / W to about 1 A / W, about 0.5 A / W to about2 A / W, about 0.5 A / W to about 3 A / W, about 0.5 A / W to about 4 A / W, about 0.5 A / W to about 5 A / W, about 0.6 A / W to about 0.8 A / W, about 0.6 A / W to about 1 A / W, about 0.6 A / W to about 2 A / W, about 0.6 A / W to about 3 A / W, about 0.6 A / W to about 4 A / W, about 0.6 A / W to about 5 A / W, about 0.8 A / W to about 1 A / W, about 0.8 A / W to about 2 A / W, about 0.8 A / W to about 3 A / W, about 0.8 A / W to about 4 A / W, about 0.8 A / WWSGR Docket No. 68783-701.601 to about 5 AAV, about 1 AAV to about 2 AAV, about 1 AAV to about 3 AAV, about 1 AAV to about 4 AAV, about 1 AAV to about 5 AAV, about 2 AAV to about 3 AAV, about 2 AAV to about 4 AAV, about 2 AAV to about 5 AAV, about 3 AAV to about 4 AAV, about 3 AAV to about 5 AAV, or about 4 AAV to about 5 AAV. The sensitivity of the detector may be about 0.3 AAV, about 0.4 AAV, about 0.5 AAV, about 0.6 AAV, about 0.8 AAV, about 1 AAV, about 2 AAV, about 3 AAV, about 4 AAV, or about 5 AAV. The sensitivity of the detector may be at least about 0.3 AAV, about 0.4 AAV, about 0.5 AAV, about 0.6 AAV, about 0.8 AAV, about 1 AAV, about 2 AAV, about 3 AAV, or about 4 AAV. The sensitivity of the detector may be at most about 0.4 AAV, about 0.5 AAV, about 0.6 AAV, about 0.8 AAV, about 1 AAV, about 2 AAV, about 3 AAV, about 4 AAV, or about 5 AAV.

[0139] In some cases, the imaging system may comprise a compound dispenser to provide one or more compounds to a sample such that the sample and / or the one or more cells of the sample may absorb and / or interact with the compound. The compound may be a fluid. The compound can be deuterium. The compound may comprise an antimicrobial compound, a pharmaceutical compound, or any combination thereof. In some cases, the compound may comprise an antimicrobial compound. In some cases, the compound may comprise a pharmaceutical compound. In some cases, the compound dispenser may comprise a fluidic handling system, a nozzle, a pipette, or any combination thereof. In some cases, the compound dispenser may comprise a fluidic handling system. In some cases, the compound dispenser may comprise a nozzle. In some cases, the compound dispenser may comprise a pipette.Methods

[0140] Provided herein is a method for determining a characteristic of one or more cells of a sample 400, as shown in FIG. 4. In some cases, the method for determining the characteristic of the one or more cells of sample may be conducted with and / or implemented on the imaging system(s) and / or the computer system(s), described elsewhere herein. In embodiments, the method is not carried out on the subject’ s body. For example, in such embodiments, the method is carried out on a sample obtained from or collected from the subject, e.g., the sample maybe ex vivo and / or in vitro.

[0141] The method 400 for determining a characteristic of one or more cells of the sample may comprise: obtaining or providing a sample comprising one or more cells 402; providing a compound to the sample, where when the compound is incorporated by the one or more cells, an intensity of one or more scattered Raman photons emitted by the one or more cells is changed compared to an absence of the incorporation of the compound by the one or more cells 404; providing a light emission of a light source to the sample, where the one or more cells absorb theWSGR Docket No. 68783-701.601 light emission and emit the one or more scattered Raman photons 406; detecting the one or more scattered Raman photons of the one or more cells with a detector optically coupled to a band -pass filter, thereby generating one or more Raman optical signals 408; and determining a characterization of the one or more cells of the sample by processing the one or more Raman optical signals 410.

[0142] In some cases, the compound can be deuterium. In some instances, the compound may comprise one or more compounds. In some instances, the one or more compounds may comprise an antimicrobial compound and / or a pharmaceutical compound. In some cases, a compound dispenser may provide the compound to the sample. In some cases, the compound dispenser may comprise a fluidic handling system, a nozzle, a pipette, or any combination thereof. In some cases, the compound may be a fluid. In some cases, the compound dispenser may comprise a fluidic handling system. In some cases, the compound dispenser may comprise a nozzle. In some cases, the compound dispenser may comprise a pipette.

[0143] In some cases, the sample may comprise a sample from a human subject, a mammalian subject, a plant, or any combination thereof. In some cases, the sample may comprise a sample from a human subject. In some cases, the sample may comprise a sample from a mammalian subject. In some cases, the sample may comprise a sample from a plant.

[0144] In some cases, the sample may comprise a biological sample. In some cases, the sample may comprise sputum, blood, urine, cerebral spinal fluid, cervical spinal fluid, bronchial alveolar lavage fluid, fluid collected from a prosthesis, fluid collected from an implant, or any combination thereof. In some cases, the sample may comprise sputum. In some cases, the sample may comprise blood. In some cases, the sample may comprise cerebral spinal fluid. In some cases, the sample may comprise cervical spinal fluid. In some cases, the sample may comprise bronchial alveolar lavage fluid. In some cases, the sample may comprise fluid collected from a prosthesis. In some cases, the sample may comprise fluid collected from an implant.

[0145] In some cases, the one or more cells may comprise one or more microbes, one or more mammalian cells, one or more cancer cells, one or more fungi, or any combination thereof. In some cases, the one or more cells may comprise microbes. In some cases, the one or more cells may comprise mammalian cells. In some cases, the one or more cells may comprise cancer cells. In some cases, the one or more cells may comprise fungi. In some cases, the one or more cells may comprise any other cells with a known spectra or silent Raman spectral zone that overlaps with one or more compounds Raman spectra.

[0146] In some cases, the characterization of the one or more cells may be determined in about 2 minutes to about 40 minutes. In some cases, the characterization of the one or more cells may be determined in about 2 minutes to about 5 minutes, about 2 minutes to about 10 minutes, about 2WSGR Docket No. 68783-701.601 minutes to about 15 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 25 minutes, about 2 minutes to about 30 minutes, about 2 minutes to about 35 minutes, about 2 minutes to about 40 minutes, about 5 minutes to about 10 minutes, about 5 minutes to about 15 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 25 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 35 minutes, about 5 minutes to about 40 minutes, about 10 minutes to about 15 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 25 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 35 minutes, about 10 minutes to about 40 minutes, about 15 minutes to about 20 minutes, about 15 minutes to about 25 minutes, about 15 minutes to about 30 minutes, about 15 minutes to about 35 minutes, about 15 minutes to about 40 minutes, about 20 minutes to about 25 minutes, about 20 minutes to about 30 minutes, about20 minutes to about 35 minutes, about20 minutes to about 40 minutes, about 25 minutes to about 30 minutes, about 25 minutes to about 35 minutes, about 25 minutes to about 40 minutes, about 30 minutes to about 35 minutes, about 30 minutes to about 40 minutes, or about 35 minutes to about 40 minutes. In some cases, the characterization of the one or more cells is determined in about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes. In some cases, the characterization of the one or more cells may be determined in at least about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes. In some cases, the characterization of the one or more cells may be determinedin at most about 5 minutes, about 10 minutes, about 15 minutes, about20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes.

[0147] In some cases, the light source, described elsewhere herein, may comprise a laser light source. In some cases, the light source may comprise an optical intensity, describe elsewhere herein. For example, the light source may comprise an optical intensity of at least about 15mW.

[0148] In some cases, the light source may comprise a pulsed light source or a continuous wave output light source. In some cases, the one or more Raman optical signals may comprise an integrated intensity of the one or more scattered Raman photons detected by the detector through one or more filters, as described elsewhere herein.

[0149] In some instances, the detector may comprise a photomultiplier tube, an avalanche photodiode, a photodiode, a silicon photomultiplier, a multi -pixel photon counter, a single photon avalanche photodiode, or any combination thereof. In some embodiments, the detector comprises a photomultiplier tube. In some embodiments, the detector comprises an avalanche photodiode. In some embodiments, the detector comprises a photodiode. In some embodiments, the detector comprises a silicon photomultiplier. In some embodiments, the detector comprises a multi -pixelWSGR Docket No. 68783-701.601 photon counter. In some embodiments, the detector comprises a single photon avalanche photodiode.

[0150] In some cases, the detector may comprise a sensitivity, as described elsewhere herein, e.g., a sensitivity of at least about 0.3 A / W.

[0151] In some cases, the one or more scattered Raman photons may be detected by the detector with a signal acquisition time, as described elsewhere herein, e.g., a signal acquisition time of up to about 100 ps.

[0152] In some cases, the one or more scattered Raman photons emitted and / or scattered by the one or more cells may be detected by one or more detectors of an imaging system, described elsewhere herein, where one or more filters may be provided in an optical path between the one or more cells and one or more detectors. In some cases, the one or more filters may comprise a band-pass filter.

[0153] In some instances, the band-pass filter may comprise an emission band, described elsewhere herein, e.g., an emission band of about 1 nm to about 20 nm. In some cases, the one or more filters may comprise a plurality of band-pass filters. In some cases, the plurality of bandpass filters may comprise a first band-pass filter and a second band-pass filter, where the first band-pass filter comprises a first emission band, and where the second band -pass filter comprises a second emission band, andwhere the first emission band and the second emission band do not overlap. In some cases, the second band -pass filter may be optically coupled to the sample and a second detector, where the first band -pass filter may be optically coupled to the sample and the detector, and where the detector and the second detector differ.

[0154] In some cases, the band-pass filter may comprise a transmission efficiency of at least about 70%.

[0155] In some cases, the band-pass filter may comprise a first coating and a second coating, where the first coating may transmit a firsthand of light, where the second coating may transmit a second band of light, and where the first band of light and the second band may differ.

[0156] In some cases, the band-pass filter may be optically coupled to a first dichroic mirror, where the first dichroic mirror may be optically coupled to the detector. In some cases, the bandpass filter may be optically coupled to a second dichroic mirror, where the second dichroic mirror is optically coupled to a second detector. In some cases, the detector and the second detector may be different detectors. In some cases, the detector may be a single pixel detector.

[0157] In some cases, the method 400 may comprise scanning the light emission of the light source across one or more portions and / or the entirety of the sample and / or the one or more cells of the sample with a scanner, described elsewhere herein.WSGR Docket No. 68783-701.601

[0158] In some cases, the scanner may comprise a stage coupled to the sample, where the stage may translate the sample while the light emission of the light source is fixed in position.

[0159] In some cases, the scanner may comprise one or more mirrors that direct an optical path of the light emission of the light source across a plane of the sample. In some instances, the scanner may comprise a stage that translates a detector across the sample.

[0160] In some cases, the scanner may comprise a first rotating and / or oscillating mirror and / or a second rotating and / or oscillating mirror. In some cases, the first rotating and / or oscillating mirror and / or the second rotating and / or oscillating mirror may comprise a galvanic scanning mirror.

[0161] In some cases, an output intensity of the light emission of the light source may be modulated at a frequency, as described elsewhere herein. In some instances, a gain of the detector may be modulated at the frequency of modulation of the output intensity of the light emission of the light source.

[0162] In some cases, the output intensity of the light emission of the light source may be modulated with an optical chopper, acoustic optic modulator, electric optical modulation, or any combination thereof, optically coupled to the light emission of the light source. In some cases, the light source emitted beam may be modulated in intensity by an optical chopper optically coupled to the light source. In some cases, the light source emitted beam may be modulated in intensity by an acoustic optic modulator (AOM) optically coupled to the light source. In some cases, the light source emitted beam may be modulated by an electric optic modular optically coupled to the light source.

[0163] In some cases, the method 400 may comprise, generating an image of the sample and / or the one or more cells of the sample from one or more scattered photons, e.g., the one or more visible light photons and / or the one or more Raman scattered photons, described elsewhere herein.

[0164] In some cases, the method 400 may comprise processing one or more detected Raman optical signals across a surface of the sample with one or more processors, to average one or more detected Raman optical signals.

[0165] In some cases, the characterization of the one or more cells may comprise determining and / or identifying a phenotype, a metabolic state, antimicrobial resistance, response to the compound, or any combination thereof, of the one or more cells. In some embodiments, the characterization of the one or more cells comprises a phenotype. In some embodiments, the characterization of the one or more cells comprises a metabolic state. In some embodiments, the characterization of the one or more cells comprises an antimicrobial resistance. In someWSGR Docket No. 68783-701.601 embodiments, the characterization of the one or more cells comprises a response to the compound.

[0166] In some cases, a second compound can be provided to the one or more cells in the sample, where the second compound may differ from the compound provided to the sample in method 400. For example, the compound and / or the second compound can alter a metabolic state of the one or more cells.

[0167] In some cases, the compound and / or the second compound may comprise an antimicrobial or a pharmaceutical compound to treat a disease or infection associated with the one or more cells of the sample. Such a method of providing an antimicrobial compound and / or a pharmaceutical compound can be used to test for an antimicrobial resistance and / or the response of the one or more cells to the pharmaceutical compound. For example, one can add an antimicrobial compound to a sample comprising one or more microbial cells to assess whether the antimicrobial compound may lyse and / or destroy the one or more microbes present in the sample. After the addition of the antimicrobial compound, a second compound e.g., deuterium may be provided to the sample comprising the one or more cells such that the deuterium may incorporate and / or be absorbed into living cells after the exposure of the one or more microbes to the antimicrobial compound. If a deuterium band is detected during imaging, those cells that took up the deuterium (e.g., those cells are alive) may indicate an antimicrobial resistance of the cells to the antimicrobial compound provided to the one or more cells of the samples .

[0168] In some cases, the method may comprise separating, filtering, and / or removing the one or more cells from the biological sample ahead of providing the compound to the one or more cells, providing a light emission of the light source, and / or detecting the one or more scattered Raman photons of the one or more cells.

[0169] In some cases, one or more processors, described elsewhere herein, may process the one or more detected Raman optical signals. In some cases, the one or more processors may process the one or more detected Raman optical signals into Raman intensity based spectroscopic information, such as the Raman intensity spectra as shown in FIGS. 6A-6B. In some cases, the one or more processors may process the one or more scattered Raman photons and / or one or more visible light photons, described elsewhere herein, to produce an image of the sample and / or the one or more cells of the sample.Computer Systems

[0170] A block diagram is shown depicting an exemplary machine that includes a computer system 500 (e.g., a processing or computing system), as shown in FIG. 5, within which a set of instructions can execute for causing a device to perform or execute any one or more of theWSGR Docket No. 68783-701.601 aspects and / or methodologies for static code scheduling of the present disclosure. The components in FIG. 5 are examples only and do not limit the scope of use or functionality of any hardware, software, embedded logic component, or a combination of two or more such components implementing particular embodiments.

[0171] Computer system 500 may include one or more processors 501, a memory 503, and a storage 508 that communicate with each other, and with other components of the imaging system, via a bus 540. In some cases, the one or more processors may communicate, actuate, and / or operate the light source, opto -mechanical components of the imaging system (e.g., kinematic mounts and tip-tilt stages), the one or more detectors, the Raman spectrometer, the scanner, compound dispenser, widefield imaging system, autofocusing system and / or autofocusing device, or any combination thereof. In some cases, the one or more processors may communicate, actuate, and / or operate the light source. In some cases, the one or more processors may communicate, actuate, and / or operate the opto-mechanical components of the imaging system (e.g., kinematic mounts and tip-tilt stages). In some cases, the one or more processors may communicate, actuate, and / or operate the one or more detectors. In some cases, the one or more processors may communicate, actuate, and / or operate the Raman spectrometer. In some cases, the one or more processors may communicate, actuate, and / or operate the scanner. In some cases, the one or more processors may communicate, actuate, and / or operate the compound dispenser. In some cases, the one or more processors may communicate, actuate, and / or operate the widefield imaging system. In some cases, the one or more processors may communicate, actuate, and / or operate the autofocusing system. In some cases, the one or more processors may communicate, actuate, and / or operate the autofocusing device.

[0172] In some cases, the one or more processors may process one or more scattered photons (e.g., visible light and / or Raman scattered photons) to generate one or more graphs of Raman spectra intensity and / or one or more images of the sample and / or the one or more cells of the sample, e.g., as shown in FIGS. 6A-6B.

[0173] The programs, described elsewhere herein, may comprise instructions to provide a compound to a sample comprising the cells. When the compound is incorporated by the cells, an intensity of scattered Raman photons emitted by the cells can be changed compared to an absence of the incorporation of the compound by the cells. The programs may comprise instructions to provide a light emission of a light source to the sample comprising one or more cells. The one or more cells may absorb the light emission and emit the one or more scattered Raman photons. The programs may comprise instructions to detect the scattered Raman photons of the cells with one or more detectors optically coupled to a band -pass filter, thereby generating one or more Raman optical signals. The programs may comprise instructions to determine theWSGR Docket No. 68783-701.601 characterization of the cells of the sample by processing the one or more Raman optical signals, as described elsewhere herein.

[0174] The bus 540 may also link a display 532, one or more input devices 533 (which may, for example, include a keypad, a keyboard, a mouse, a stylus, etc.), one or more output devices 534, one or more storage devices 535, and various tangible storage media 536. All of these elements may interface directly or via one or more interfaces or adaptors to the bus 540. For instance, the various tangible storage media 536 can interface with the bus 540 via storage medium interface 526

[0175] Computer system 500 may have any suitable physical form, including but not limited to one or more integrated circuits (ICs), printed circuit boards (PCBs), mobile handheld devices (such as mobile telephones or PDAs), laptop or notebook computers, distributed computer systems, computing grids, or servers.

[0176] Computer system 500 may comprise one or more processor(s) 501 (e.g., central processing units (CPUs), general purpose graphics processing units (GPGPUs), or quantum processing units (QPUs) that carry out functions.

[0177] Processor(s) 501 may comprise a cache memory unit 502 for temporary local storage of instructions, data (e.g., one or more Raman spectra or intensity of one or more Raman spectra of the sample and / or the one or more cells of the sample), or computer addresses.

[0178] Processor(s) 501 may be configured to assist in execution of computer readable instructions. Computer system 500 may provide functionality for the components depicted in FIG. 5 as a result of the processor(s) 501 executing non-transitory, processor-executable instructions embodied in one or more tangible computer-readable storage media, such as memory 503, storage 508, storage devices 535, and / or storage medium 536.

[0179] The computer-readable media may store software that implements particular embodiments, and processor(s) 501 may execute the software. Memory 503 may read the software from one or more other computer-readable media (such as mass storage device(s) 535, 536) or from one or more other sources through a suitable interface, such as network interface 520.

[0180] The software may cause processor(s) 501 to carry out one or more processes or one or more steps of one or more processes described or illustrated herein. Carrying out such processes or steps may include defining data structures stored in memory 503 and modifying the data structures as directed by the software.

[0181] The memory 503 may include various components (e.g., machine readable media) including, but not limited to, a random access memory component (e.g., RAM 504) (e.g., static RAM (SRAM), dynamic RAM (DRAM), ferroelectric random access memory (FRAM), phase-WSGR Docket No. 68783-701.601 change random access memory (PRAM), etc.), a read-only memory component (e.g., ROM 505), and any combinations thereof. ROM 505 may act to communicate data and instructions unidirectionally to processor(s) 501, and RAM 504 may act to communicate data and instructions bidirectionally with processor(s) 501. ROM 505 and RAM 504 may include any suitable tangible computer-readable media described below.

[0182] In one example, a basic input / output system 506 (BIOS), including basic routines that help to transfer information between elements within computer system 500, such as during startup, may be stored in the memory 503.

[0183] Fixed storage 508 may be connected bidirectionally to processor(s) 501, optionally through storage control unit 507. Fixed storage 508 provides additional data storage capacity and may also include any suitable tangible computer-readable media described herein. Storage 508 may be used to store operating system 509, executable(s) 510, data 511 (e.g., the one or more Raman spectra and / or corresponding intensity recorded for the one or more Raman spectra of the sample and / or the one or more cells of the sample), applications 512 (application programs), and the like. Storage 508 can also include an optical disk drive, a solid-state memory device (e.g., flash-based systems), or a combination of any of the above. Information in storage 508 may be incorporated as virtual memory in memory 503.

[0184] In one example, storage device(s) 535 may be removably interfaced with computer system 500 (e.g., via an external port connector (not shown)) via a storage device interface 525. Particularly, storage device(s) 535 and an associated machine-readable medium may provide non-volatile and / or volatile storage of machine-readable instructions, data structures, program modules, and / or other data for the computer system 500. In one example, software may reside, completely or partially, within a machine-readable medium on storage device(s) 535. In another example, software may reside, completely or partially, within processor(s) 501.

[0185] Computer system 500 may also include an input device 533. In one example, a user of computer system 500 may enter commands and / or other information into computer system 500 via input device(s) 533. Examples of an input device(s) 533 include, but are not limited to, an alpha-numeric input device (e.g., a keyboard), a pointing device (e.g., a mouse or touchpad), a touchpad, a touch screen, a multi-touch screen, a joystick, a stylus, a gamepad, an audio input device (e.g., a microphone, a voice response system, etc.), an optical scanner, a video or still image capture device (e.g., a camera), or any combinations thereof. In some embodiments, the input device may be a Kinect, Leap Motion, or the like. Input device(s) 533 may be interfaced to bus 540 via any of a variety of input interfaces 523 including, but not limited to, serial, parallel, game port, USB, FIREWIRE, THUNDERBOLT, or any combination of the above.WSGR Docket No. 68783-701.601

[0186] In particular embodiments, when computer system 500 is connected to network 530, computer system 500 may communicate with other devices, specifically mobile devices and enterprise systems, distributed computing systems, cloud storage systems, cloud computing systems, and the like, connected to network 530. Communications to and from computer system 500 may be sentthrough network interface 520. For example, network interface 520 may receive incoming communications (such as requests or responses from other devices) in the form of one or more packets (such as Internet Protocol (IP) packets) from network 530, and computer system 500 may store the incoming communications in memory 503 for processing. Computer system 500 may similarly store outgoing communications (such as requests or responses to other devices) in the form of one or more packets in memory 503 and communicated to network 530 from network interface 520. Processor(s) 501 may access these communication packets stored in memory 503 for processing.

[0187] Examples of the network interface 520 include, but are not limited to, a network interface card, a modem, and any combination thereof. Examples of a network 530 or network segment 530 include, but are not limited to, a distributed computing system, a cloud computing system, a wide area network (WAN) (e.g., the Internet, an enterprise network), a local area network (LAN) (e.g., a network associated with an office, a building, a campus or other relatively small geographic space), a telephone network, a direct connection between two computing devices, a peer-to-peer network, and any combinations thereof. A network, such as network 530, may employ a wired and / or a wireless mode of communication. In general, any network topology may be used.

[0188] Information and data can be displayed through a display 532. Examples of a display 532 include, but are not limited to, a liquid crystal display (LCD), a thin film transistor liquid crystal display (TFT-LCD), an organic liquid crystal display (OLED) such as a passive -matrix OLED (PMOLED), active-matrix OLED (AMOLED) display, a plasma display, and any combinations thereof. The display 532 can interface with the processor(s) 501, memory 503, and fixed storage 508, as well as other devices, such as input device(s) 533, via the bus 540. The display 532 may be linked to the bus 540 via a video interface 522, and transport of data between the display 532 and the bus 540 can be controlled via the graphics control 521.

[0189] In some embodiments, the display may be a video projector. In some embodiments, the display may be a head-mounted display (HMD) such as a VR headset. In further embodiments, suitable VR headsets may comprise, by way of non-limiting examples, HTC Vive, Oculus Rift, Samsung Gear VR, Microsoft HoloLens, Razer OSVR, FOVE VR, Zeiss VR One, Avegant Glyph, Freefly VR headset, and the like. In still further embodiments, the display may be a combination of devices such as those disclosed herein.WSGR Docket No. 68783-701.601

[0190] In addition to a display 532, computer system 500 may include one or more other peripheral output devices 534 including, but not limited to, an audio speaker, a printer, a storage device, and any combinations thereof . Such peripheral output devices may be connected to the bus 540 via an output interface 524. Examples of an output interface 524 include, but are not limited to, a serial port, a parallel connection, a USB port, a FIREWIRE port, a THUNDERBOLT port, and any combinations thereof.

[0191] In some cases, the one or more processors of the system may process the one or more scattered Raman photons and / or a corresponding one or more Raman spectra of the one or more scattered Raman photons and generate a displayed response to a user’s inquiry regarding whether the one or more cells of the sample have a certain characteristic. In some cases, the characteristic can be a phenotype, a metabolic state, antimicrobial resistance, response to the compound, or any combination thereof, of the one or more cells of the sample. In some embodiments, the characterization of the one or more cells comprises a phenotype. In some embodiments, the characterization of the one or more cells comprises a metabolic state. In some embodiments, the characterization of the one or more cells comprises an antimicrobial resistance. In some embodiments, the characterization of the one or more cells comprises a response to the compound.

[0192] In some cases, the one or more processors can send a message (e.g., sending a message to a lab technician for review or directly to a licensed physician or microbiologist). The message may display a text or image result on the display, described elsewhere herein.

[0193] The display may be integrated with the imaging system and / or the display may comprise a standalone display coupled to the imaging system. The display may comprise a display of a personal screen (e.g., smartphone or personal computer). The display may comprise a web-portal that may be accessed by a personal computing device (e.g., smart-phone, tablet, and / or a personal computer) electrically coupled to a network address of the imaging system.

[0194] In some embodiments, computer system 500 may provide functionality as a result of logic hardwired or otherwise embodied in a circuit, which may operate in place of or together with software to execute one or more processes or one or more steps of one or more processes described or illustrated herein. Reference to software in this disclosure may encompass logic, and reference to logic may encompass software. Moreover, reference to a computer -readable medium may encompass a circuit (such as an IC) storing software for execution, a circuit embodying logic for execution, orboth, where appropriate. The present disclosure encompasses any suitable combination of hardware, software, or both.

[0195] Those of skill in the art will appreciate that the various illustrative logical blocks, modules, circuits, and algorithm steps described in connection with the embodiments disclosedWSGR Docket No. 68783-701.601 herein may be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and steps have been described above generally in terms of their functionality.

[0196] The various illustrative logical blocks, modules, and circuits described in connection with the embodiments disclosed herein may be implemented or performed with a general purpose processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general purpose processor may be a microprocessor, but in the alternative, the processor may be any conventional processor, controller, microcontroller, or state machine. A processor may also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.

[0197] The steps of a method or algorithm described in connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by one or more processor(s), or in a combination of the two. A software module may reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium known in the art.

[0198] In some cases, the computer system may execute the method(s), described elsewhere herein. In some cases, the steps and / or the operations embodied in software that the computer system executes may be automatic e.g., based on characteristics of the sample and / or the compound provided to the sample and / or the one or more cells that the computer may receive and / or determine from stored memory. In some cases, the steps and / or operations that the computer system executes may be guided by a user, e.g., as a user places sample into the imaging system and proceeds to input information of the sample and / or information of the one or more cells of the sample as input parameters into the imaging system.

[0199] An exemplary storage medium may be coupled to the processor such the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium may be integral to the processor. The processor and the storage medium may reside in an ASIC. The ASIC may reside in a user terminal. In the alternative, the processor and the storage medium may reside as discrete components in a user terminal.

[0200] In accordance with the description herein, suitable computing devices include, by way of non-limiting examples, cloud computing platforms, distributed computing platforms, server clusters, server computers, desktop computers, laptop computers, notebook computers, sub-WSGR Docket No. 68783-701.601 notebook computers, netbook computers, netpad computers, set -top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, tablet computers, and the like.

[0201] In some embodiments, the computing device includes an operating system configured to perform executable instructions. The operating system is, for example, software, including programs and data, which manages the device’s hardware and provides services for execution of applications. Those of skill in the art will recognize that suitable server operating systems include, by way of non -limiting examples, FreeBSD, OpenBSD, NetBSD®, Linux, Apple® Mac OS X Server®, Oracle® Solaris®, Windows Server®, and Novell® NetWare®. Those of skill in the art will recognize that suitable personal computer operating systems include, by way of non- limiting examples, Microsoft® Windows®, Apple® Mac OS X®, UNIX®, and UNIX-like operating systems such as GNU / Linux®. In some embodiments, the operating system is provided by cloud computing.DEFINITIONS

[0202] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0203] Throughout this application, various embodiments may be presented in a range format. It should be under stood th at the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure.Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0204] The ranges disclosed herein also encompass any and all overlap, sub -ranges, and combinations thereof. Language such as “up to,” “at least,” “greater than,” “less than,” “between,” and the like includes the number recited. Numbers preceded by a term such as “approximately”, “about”, and “substantially” as used herein include the recited numbers, and also represent an amount close to the stated amount that still performs a desired function orWSGR Docket No. 68783-701.601 achieves a desired result. The term “about” or “approximately” may mean within an acceptable error range for the particular value, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, the terms “approximately”, “about”, and “substantially” may refer to an amount that is within less than 10% of, within less than 5% of, within less than 1 % of, within less than 0.1% of, and within less than 0.01% of the stated amount. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value may be assumed.

[0205] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural referencesunless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0206] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0207] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.EXAMPLES

[0208] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.Example 1: Microbial Incorporation of Deuterium to Determine Antimicrobial Compound Resistance

[0209] A microbiologist sets out to determine whether a microbe causing an infection in a subject is resistantto an antimicrobial compound. The microbiologist collects a sample from the subject with the infection. The microbiologist plates the sample comprising the one or more microbes and adds the antimicrobial compound to the sample. The microbiologist allows theWSGR Docket No. 68783-701.601 sample and antimicrobial compound to incubate for 1 hour. The microbiologist then adds deuterium, expecting that any live microbes (and thus resistant to the antimicrobial compound) will take up the deuterium. The microbiologist performs Raman spectroscopy on the sample using an imaging system, described elsewhere herein, by providing an emitted beam of a light source to one or more regions of the sample for up to 20 minutes. No signal is determined at the anticipated Raman spectra of the compound. The microbiologist understands this outcome to indicate that the one or more microbes present in the sample are not resistant to the antimicrobial compound. With this information the doctor prescribes the antimicrobial compound to the subject to treat their infection.Example 2: High Intensity Low Exposure Time Raman Spectroscopy

[0210] A microbiologist sets out to determine whether a microbe causing an infection of a subject is resistantto an antimicrobial compound. The microbiologist collects a sample from the subject with the infection. The microbiologist plates the sample comprising the one or more microbes and adds the antimicrobial compound to the sample. The microbiologist allows the sample and antimicrobial compound to incubate for 1 hour. The microbiologist then adds deuterium, expecting that any live microbes (and thus resistant to the antimicrobial compound) will take up the deuterium. The microbiologist performs Raman spectroscopy using the imaging systems, described elsewhere herein, by providing an emitted beam of a light source at 50mW output optical power to one or more regions of the sample for 2 seconds to avoid photodamage and / or photobleaching of the sample and / or the one or more cells of the sample. The emitted 50mW optical power emitted beam of the light source is rapidly moved across the sample and / or the one or more cells of the sample and one or more scattered Raman photons are detected across the sample and averaged. The microbiologist then observes the recorded, processed, and displayed Raman spectra of the sample for an average measured intensity of photons scattered in the deuterium spectroscopy band. The microbiologist observers a band at 2173 cm , thereby confirming that the one or more microbes of the sample appear to be resistantto the antimicrobial compound.Example 3: Effect of Incubation Time on Cellular Deuterium Incorporation

[0211] E.coli was grown in lithium borate buffer with 40% D2O. A time-course of single cell Raman spectra (SCRS) was taken of the E. coli. FIG. 6A shows a graph of Raman shift by cm4with intensity on the y-axis. FIG. 6B shows a graph of Raman shift in a nanometer scale with intensity on the y-axis. In both, the C-D spectra band 600 began forming at about 20 minutes of incubation of deuterium with E.coli. The bandwas observed around 2173 cm-1(FIG. 6A), whichWSGR Docket No. 68783-701.601 was in the ‘silent zone’ from about 1847 cm-1to about 2668 cm4of single cell Raman spectra in most natural cells including prokaryotic and eukaryotic cells. This reflects a silent zone of from about 590 nm to about 620 nm in FIG. 6B for an incident laser of 532 nm. As microbes fall into these categories, this zone is silent (nothing shown) in the lowest line representing 0 minutes. As shown in FIG. 6B, the central band for C-D vibration is around 601-602 nm.

[0212] Thus, the results shown in FIGS. 6A-6B indicate that the spectral Raman band of deuterium can be identified in the Raman silent zone of microbes and can be utilized as a proxy for detecting the presence of microbes in a sample (e.g., after treating a sample with an antimicrobial compound).

[0213] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicingthe present disclosure. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WSGR Docket No. 68783-701.601CLAIMSWHAT IS CLAIMED IS:

1. A method for determining a characterization of one or more cells, comprising: obtaining or providing a sample comprising the one or more cells; providing a compound to the sample, wherein when the compound is incorporated by the one or more cells, an intensity of one or more scattered Raman photons emitted by the one or more cells is changed compared to an absence of the incorporation of the compound by the one or more cells; providing a light emission of a light source to the sample, wherein the one or more cells absorb the light emission and emit the one or more scattered Raman photons; detecting the one or more scattered Raman photons of the one or more cells with a detector optically coupled to a band-pass filter, thereby generating one or more Raman optical signals; and determining the characterization of the one or more cells of the sample by processing the one or more Raman optical signals.

2. The method of claim 1, wherein the one or more cells comprise one or more microbes, one or more mammalian cells, one or more cancer cells, one or more fungi, or any combination thereof.

3. The method of claims 1 or 2, wherein the characterization of the one or more cells is determined in up to about 20 minutes.

4. The method of any one of claims 1-3, wherein the light source comprises a laser light source.

5. The method of any one of claims 1-4, wherein the detector comprises a photomultiplier tube, an avalanche photodiode, a photodiode, a silicon photomultiplier, a multi - pixel photon counter, a single photon avalanche photodiode, or any combination thereof.

6. The method of any one of claims 1-5, wherein a sensitivity of the detector comprises at least about 0.5 ampere / watt (A / W).

7. The method of any one of claims 1-6, wherein the one or more scattered Raman photons are detected by the detector with up to about a 100 microsecond signal acquisition time.

8. The method of any one of claims 1-7, wherein the band-pass filter comprises an emission band of about 1 nm to about 20 nm.WSGR Docket No. 68783-701.6019. The method of any one of claims 1-8, wherein the band-pass filter comprises a plurality of band-pass filters.

10. The method of claim 9, wherein the plurality ofband-pass filters comprises a first band-pass filter and a second band-pass filter, wherein the first band-pass filter comprises a first emission band, and wherein the second band-pass filter comprises a second emission band, and wherein the first emission band and the second emission band do not overlap.11 . The method of claim 10, wherein the second band-pass filter is optically coupled to the sample and a second detector, wherein the first band -pass filter is optically coupled to the sample and the detector, and wherein the detector and the second detector differ.

12. The method of any one of claims 1-10, wherein the band-pass filter comprises a transmission efficiency of at least about 70%.

13. The method of any one of claims 1-12, further comprising scanning the light emission of the light source with a scanner across the sample.

14. The method of claim 13, wherein the scanner comprises a stage coupled to the sample, and wherein the stage translates the sample while the light emission of the light source is fixed in position.

15. The method of claim 13 , wherein the scanner comprises one or more mirrors that direct an optical path of the light emission of the light source across a plane of the sample.

16. The method of claim 13, wherein the scanner comprises a stage that translates the detector across the sample.

17. The method of claim 13, wherein the scanner comprises a galvanic scanning mirror.

18. The method of any one of claims 1-17, wherein an output intensity of the light emission of the light source is modulated at a frequency, and wherein a gain of the detector is modulated at the frequency of modulation of the output intensity of the light emission of the light source.

19. The method of claim 18, wherein the output intensity of the light emission of the light source is modulated with an optical chopper, acoustic optic modulator, electric optical modulator, or any combination thereof, optically coupled to the light emission of the light source.WSGR Docket No. 68783-701.60120. The method of any one of claims 1-19, wherein the sample comprises a biological sample from a human subject, a mammalian subject, a plant, or any combination thereof.

21. The method of claim 20, wherein the biological sample comprises sputum, blood, urine, cerebral spinal fluid, cervical spinal fluid, bronchial alveolar lavage fluid, fluid collected from a prosthesis, fluid collected from an implant, or any combination thereof.

22. The method of claims 20 or 21, wherein the one or more cells are not filtered or removed from the biological sample.

23. The method of any one of claims 1-22, wherein the band-pass filter comprises a first coating and a second coating, wherein the first coating transmits a first band of light, wherein the second coating transmits a second band of light, and wherein the first band of light and the second band of light differ.

24. The method of any one of claims 1-23, further comprising providing a second compound to the one or more cells in the sample, wherein the second compound alters a metabolic state of the one or more cells.

25. The method of claim 24, wherein the second compound comprises an antimicrobial or a pharmaceutical compound.

26. The method of claims 24 or 25, wherein the characterization of the one or more cells comprises a phenotype, a metabolic state, antimicrobial resistance, response to the compound, response to the second compound, or any combination thereof, of the one or more cells.

27. The method of claims 1-26, further comprising generating an image of the one or more cells from the one or more Raman optical signals.

28. The method of any one of claims 1-27, wherein the compound comprises a fluid.

29. The method of claim 28, wherein the fluid comprises deuterium.

30. The method of any one of claims 1-29, further comprising a first dichroic mirror optically coupled to the band-pass filter, wherein the first dichroic mirror is optically coupled to the detector.WSGR Docket No. 68783-701.60131 . The method of any one of claim 1-30, further comprising a second dichroic mirror optically coupled to the band-pass filter, wherein the second dichroic mirror is optically coupled to a second detector, wherein the second detector differs from the detector.

32. The method of any one of claims 1-31, wherein the detector comprises a single pixel detector.

33. The method of any one of claims 1-32, wherein the light source comprises an optical intensity of at least about 15 mW.

34. The method of any one of claims 1-33, wherein processing comprises averaging the one or more detected Raman optical signals across a surface of the sample with one or more processors to determine the characterization of the one or more cells.

35. The method of any one of claims 1-34, wherein the light source comprises a pulsed light source.

36. The method of any one of claims 1-35, wherein the compound is provided to the sample by a compound dispenser.

37. The method of claim 36, wherein the compound dispenser comprises a fluidic handling system, a nozzle, a pipette, or any combination thereof.

38. The method of any one of claims 1-37, wherein the one or more Raman optical signals comprise an integrated intensity of the one or more scattered Raman photons detected by the detector through the band-pass filter.

39. A computer system for determining a characterization of one or more cells, comprising: one or more processors and memory storing one or more programs for execution by the one or more processors, the one or more programs comprising instructions to: provide a compound to a sample comprising the one or more cells, wherein when the compound is incorporated by the one or more cells, an intensity of one or more scattered Raman photons emitted by the one or more cells is changed compared to an absence of the incorporation of the compound by the one or more cells; provide a light emission of a light source to the sample, wherein the one or more cells absorb the light emission and emit the one or more scattered Raman photons;WSGR Docket No. 68783-701.601 detect the one or more scattered Raman photons of the one or more cells with a detector optically coupled to a band-pass filter, thereby generating one or more Raman optical signals; and determine the characterization of the one or more cells of the sample by processing the one or more Raman optical signals.

40. The computer system of claim 39, wherein the one or more cells comprise one or more microbes, one or more mammalian cells, one or more cancer cells, one or more fungi, or any combination thereof.

41. The computer system of claims 39 or 40, wherein the characterization of the one or more cells is determined in up to about 20 minutes.

42. The computer system of any one of claims 39-41, wherein the light source comprises a laser light source.

43. The computer system of any one of claims 39-42, wherein the detector comprises a photomultiplier tube, an avalanche photodiode, a photodiode, a silicon photomultiplier, a multi - pixel photon counter, a single photon avalanche photodiode, or any combination thereof.

44. The computer system of any one of claims 39-43, where a sensitivity of the detector comprises at least about 0.3 AAV.

45. The computer system of any one of claims 39-43, wherein the one or more scattered Raman photons are detected by the detector with up to about a 100 microsecond signal acquisition time.

46. The computer system of any one of claims 39-45, wherein the band-pass filter comprises an emission band of about 1 nm to about 20 nm.

47. The computer system of any one of claims 39-45, wherein the band-pass filter comprises a plurality of band-pass filters.

48. The computer system of claim 47, wherein the plurality of band-pass filters comprises a first band-pass filter and a second band -pass filter, wherein the first band-pass filter comprises a first emission band, wherein the second band-pass filter comprises a second emission band, and wherein the first emission band and the second emission band do not overlap.WSGR Docket No. 68783-701.60149. The computer system of claim 48, wherein the secondhand-pass filter is optically coupled to the sample and a second detector, wherein the first band-pass filter is optically coupled to the sample and the detector, and wherein the detector and the second detector differ.

50. The computer system of any one of claims 39-49, wherein the band-pass filter comprises a transmission efficiency of at least about 70%.

51. The computer system of any one of claims 39-50, wherein the instructions further comprise scanning the light emission of the light source with a scanner across the sample.

52. The computer system of claim 51, wherein the scanner comprises a stage coupled to the sample, and wherein the stage translates the sample while the light emission of the light source is fixed in position.

53. The computer system of claim 51, wherein the scanner comprises one or more mirrors that direct an optical path of the light emission of the light source across a plane of the sample.

54. The computer system of claim 51, wherein the scanner comprises a stage that translates the detector across the sample.

55. The computer system of claim 51, wherein the scanner comprises a galvanic scanning mirror.

56. The computer system of any one of claims 39-55, wherein an output intensity of the light emission of the light source is modulated at a frequency, and wherein a gain of the detector is modulated at the frequency of modulation of the light emission of the light source.

57. The computer system of claim 56, wherein the output intensity of the light emission of the light source is modulated with an optical chopper, acoustic optic modulator, electric optical modulator, or any combination thereof, optically coupled to the light emission of the light source.

58. The computer system of any one of claims 39-57, wherein the sample comprises a biological sample from a human subject, a mammalian subject, a plant, or any combination thereof.WSGR Docket No. 68783-701.60159. The computer system of claim 58, wherein the biological sample comprises sputum, blood, urine, cerebral spinal fluid, cervical spinal fluid, bronchial alveolar lavage fluid, fluid collected from a prosthesis, fluid collected from an implant, or any combination thereof.

60. The computer system of claims 58 or 59, wherein the one or more cells are not filtered or removed from the biological sample.

61. The computer system of any one of claims 39-60, wherein the band-pass filter comprises a first coating and a second coating, wherein the first coating transmits a first band of light, wherein the second coating transmits a secondhand of light, and wherein the first band of light and the second band of light differ.

62. The computer system of any one of claims 39-61, wherein the instructions further comprise providing a second compound to the one or more cells in the sample, and wherein the second compound alters a metabolic state of the one or more cells.

63. The computer system of claim 62, wherein the second compound comprises an antimicrobial or a pharmaceutical compound.

64. The computer system of claims 62 or 63, wherein the characterization of the one or more cells comprises a phenotype, a metabolic state, antimicrobial resistance, response to the compound, response to the second compound, or any combination thereof, of the one or more cells.

65. The computer system of claims 39-64, wherein the instructions further comprise generating an image of the one or more cells from the one or more Raman optical signals.

66. The computer system of any one of claims 39-65, wherein the compound comprises a fluid.

67. The computer system of claim 66, wherein the fluid comprises deuterium.

68. The computer system of any one of claims 39-67, comprising a first dichroic mirror optically coupled to the band-pass filter, wherein the first dichroic mirror is optically coupled to the detector.

69. The computer system of any one of claims 39-68, further comprising a second dichroic mirror optically coupled to the band -pass filter, wherein the second dichroic mirror is optically coupled to a second detector, wherein the second detector differs from the detector.WSGR Docket No. 68783-701.60170. The computer system of any one of claims 39-69, wherein the detector comprises a single pixel detector.

71. The computer system of any one of claims 39-70, wherein the light source comprises an optical intensity of at least about 15 mW.

72. The computer system of any one of claims 39-71, wherein the instructions further comprise averaging one or more detected Raman optical signals across a surface of the sample to determine the characterization of the one or more cells.

73. The computer system of any one of claims 39-72, wherein the light source comprises a pulsed light source.

74. The computer system of any one of claims 39-73, wherein the compound is provided to the sample by a compound dispenser.

75. The computer system of claim 74, wherein the compound dispenser comprises a fluidic handling system, a nozzle, a pipette, or any combination thereof.

76. The computer system of any one of claims 39-75, wherein the one or more Raman optical signals comprise an integrated intensity of the one or more scattered Raman photons detected by the detector through the band -pass filter.

77. A system for determining a characterization of one or more cells, comprising: a light source configured to provide a light emission to a sample comprising one or more cells, wherein the one or more cells absorb the light emission and scatter one or more Raman photons; a compound dispenser to provide a compound to the sample, wherein when the compound is incorporated by the one or more cells, an intensity of the one or more scattered Raman photons emitted by the one or more cells is changed compared to an absence of the incorporation of the compound by the one or more cells; a band-pass filter; a detector optically coupled to the band -pass filter, wherein the detector detects the one or more scattered Raman photons through the band -pass filter, thereby generating one or more detected Raman optical signals; and one or more processors electrically coupled to the detector, wherein the one or more processors process the one or more detected Raman optical signals and determine a characterization of the one or more cells.WSGR Docket No. 68783-701.60178. The system of claim 77, wherein the one or more cells comprise one or more microbes, one or more mammalian cells, one or more cancer cells, one or more fungi, or any combination thereof.

79. The system of claims 77 or 78, wherein the one or more processors determine the characterization of the one or more cells in up to about 20 minutes.

80. The system of any one of claims 77-79, wherein the light source comprises a laser light source.

81. The system of any one of claims 77-80, wherein the detector comprises a photomultiplier tube, an avalanche photodiode, a photodiode, a silicon photomultiplier, a multi - pixel photon counter, a single photon avalanche photodiode, or any combination thereof.

82. The system of any one of claims 77-81, where a sensitivity of the detector comprises at least about 0.3 AAV.

83. The system of any one of claims 77-82, wherein the one or more scattered Raman photons are detected by the detector with up to about a 100 microsecond signal acquisition time.

84. The system of any one of claims 77-83, wherein the band-pass filter comprises an emission band of about 1 nm to about 20nm.

85. The system of any one of claims 77-84, wherein the band-pass filter comprises a plurality of band-pass filters.

86. The system claim 85, wherein the plurality of band-pass filters comprises a first band-pass filter and a second band-pass filter, wherein the first band-pass filter comprises a first emission band, wherein the second band-pass filter comprises a second emission band, and wherein the first emission band and the second emission band do not overlap.

87. The system of claim 86, wherein the second band-pass filter is optically coupled to the sample and a second detector, wherein the first band-pass filter is optically coupled to the sample and the detector, and wherein the detector and the second detector differ.

88. The system of any one of claims 77-87, wherein the band-pass filter comprises a transmission efficiency of at least about 70%.

89. The system of any one of claims 77-88, further comprising a scanner that scans the light emission of the light source across the sample.WSGR Docket No. 68783-701.60190. The system of claim 89, wherein the scanner comprises a stage coupled to the sample, and wherein the stage translates the sample while the light emission of the light source is fixed in position.91 . The system of claim 89, wherein the scanner comprises one or more mirrors that direct an optical path of the light emission of the light source across a plane of the sample.

92. The system of claim 89, wherein the scanner comprises a stage to translate the detector across the sample.

93. The system of claim 89, wherein the scanner comprises a galvanic scanning mirror.

94. The system of any one of claims 77-93, wherein an output intensity of the light emission of the light source is modulated at a frequency, and wherein a gain of the detector is modulated at the frequency of modulation of the light emission of the light source.

95. The system of claim 94, wherein the output intensity of the light emission of the light source is modulated with an optical chopper, acoustic optic modulator, electric optical modulator, or any combination thereof, optically coupled to the light emission of the light source.

96. The system of any one of claims 77-95, wherein the sample comprises a biological sample from a human subject, a mammalian subject, a plant, or any combination thereof.

97. The system of claim 96, wherein the biological sample comprises sputum, blood, urine, cerebral spinal fluid, cervical spinal fluid, bronchial alveolar lavage fluid, fluid collected from a prosthesis, fluid collected from an implant, or any combination thereof.

98. The system of claims 96 or 97, wherein the one or more cells are not filtered or removed from the biological sample.

99. The system of any one of claims 77-98, wherein the band-pass filter comprises a first coating and a second coating, wherein the first coating transmits a first band of light, wherein the second coating transmits a second band of light, and wherein the first band of light and the second band of light differ.

100. The system of any one of claims 77-99, wherein the characterization of the one or more cells comprises a phenotype, a metabolic state, antimicrobial resistance, response to the compound, or any combination thereof, of the one or more cells.WSGR Docket No. 68783-701.601101. The system of any one of claims 77-100, wherein the one or more processors generate an image from the one or more Raman optical signals.

102. The system of any one of claims 77-101, wherein the compound comprises a fluid.

103. The system of claim 102, wherein the fluid comprises deuterium.

104. The system of any one of claims 77-103, further comprising a first dichroic mirror optically coupled to the band-pass filter, wherein the first dichroic mirror is optically coupled to the detector.

105. The system of claim 104, further comprising a second dichroic mirror optically coupled to the band-pass filter, wherein the second dichroic mirror is optically coupled to a second detector, wherein the second detector differs from the detector.

106. The system of any one of claims 77-105, wherein the detector comprises a single pixel detector.

107. The system of any one of claims 77-106, wherein the light source comprises an optical power of at least about 15 milliwatts (mW).

108. The system of any one of claims 77-107, wherein the one or more processors average one or more detected Raman optical signals across a surface of the sample to determine the characterization of the one or more cells.

109. The system of any one of claims 77-108, wherein the light source comprises a pulsed light source.

110. The system of any one of claims 77-109, wherein the compound dispenser comprises a fluidic handling system, a nozzle, a pipette, or any combination thereof.

111. The system of any one of claims 77-110, wherein the one or more Raman optical signals comprise an integrated intensity of the one or more scattered Raman photons detected by the detector through the band-pass filter.

112. A system for determining a characterization of one or more cells, comprising: a light source configured to provide a light emission to a sample comprising one or more cells, wherein the one or more cells absorb the light emission and scatter one or more Raman photons;WSGR Docket No. 68783-701.601 a focusing system, wherein the focusing system adjusts a position of the light source to provide the light source at a focus position of a surface of the sample; a Raman spectrometer optically coupled to a filter, wherein the Raman spectrometer detects the one or more scattered Raman photons through the filter, thereby generating one or more detected Raman optical signals; and one or more processors electrically coupled to the Raman spectrometer, wherein the one or more processors process the one or more detected Raman optical signals and determine a characterization of the one or more cells.

113. A system for determining a characterization of one or more cells, comprising: a light source configured to provide a light emission to a sample comprising one or more cells, wherein the one or more cells absorb the light emission and scatter one or more Raman photons; a widefield imaging system, wherein the widefield imaging system detects one or more visible light scattered photons from the sample comprising the one or more cells, and wherein the visible light scattered photons are generated when the sample absorbs the light emission of the light source; a Raman spectrometer optically coupled to a filter, wherein the Raman spectrometer detects the one or more scattered Raman photons through the filter, thereby generating one or more detected Raman optical signals; and one or more processors electrically coupled to the Raman spectrometer, wherein the one or more processors process the one or more detected Raman optical signals and determine a characterization of the one or more cells.

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