Imaging system for the detection of intraoperative contrast agents in tissue

Abstract

To provide a suitable imaging system for detecting an intraoperative contrast agent in a tissue.SOLUTION: Explained are a system and a method for providing a quantitative standard of a distribution of a contrast agent in a tissue sample by using a combination of high resolution optical slicing imaging modalities to identify a cell distribution. In some examples, the system and the method are used in an intraoperative setting, and may induce a biopsy or surgical operation technique. In one embodiment, an image interpretation algorithm uses one or both of first and second optical slicing imaging modalities to process an obtained image, identifies individual cells, determines places of the individual cells, and measures signals at the places of the individual cells in the image obtained by using the first imaging modality, to output the quantitative standard of the signal derived from a cell-associated contrast agent.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] (Related Applications) This application claims the benefit of U.S. Provisional Application No. 62 / 833,553, filed April 12, 2019, which application is incorporated herein by reference. Summary of the Invention [Means for solving the problem]

[0002] Disclosed herein is a system for imaging a tissue sample, the system comprising: (a) a first optical subsystem configured to acquire high-resolution images of a distribution of a cell-bound contrast agent in the tissue sample using a first optical sectioning imaging modality; (b) a second optical subsystem configured to acquire high-resolution images of the tissue sample morphology using a second optical sectioning imaging modality, the first and second optical subsystems configured to image the same optical plane in the tissue sample; and (c) a processor configured to execute an image interpretation algorithm that processes images acquired using one or both of the first and second optical sectioning imaging modalities to identify and determine the locations of individual cells and output a quantitative measure of a signal derived from the cell-bound contrast agent by measuring signals at corresponding locations of individual cells in the images acquired using the first imaging modality. In some embodiments, the first optical sectioning imaging modality comprises two-photon fluorescence microscopy, confocal fluorescence microscopy, light sheet microscopy, or structured illumination microscopy. In some embodiments, the second optical thinning imaging modality comprises stimulated Raman scattering microscopy, coherent anti-Stokes Raman scattering microscopy, confocal reflectance microscopy, second harmonic generation microscopy, or third harmonic generation microscopy. In some embodiments, the first and second optical subsystems are configured to have an axial resolution of less than 10 μm. In some embodiments, the first and second optical subsystems are configured to have a lateral resolution of less than 5 μm. In some embodiments, the first optical subsystem is further configured to obtain high-resolution images within two or more detection wavelength ranges.In some embodiments, a first detection wavelength range of the two or more detection wavelength ranges includes the emission peak of the cell-bound contrast agent, and a second detection wavelength range of the two or more detection wavelength ranges excludes the emission peak of the cell-bound contrast agent, and the image interpretation algorithm further processes the image obtained using the first detection wavelength to identify individual cells, determine their locations, measure signals at the locations of the individual cells, and output a quantitative measure of the signal derived from the cell-bound contrast agent by correcting the signals by background values ​​measured at corresponding locations of the individual cells in the image obtained using the second detection wavelength range. In some embodiments, the second optical thinning imaging modality comprises stimulated Raman scattering microscopy, and the image is obtained using stimulated Raman scattering microscopy at 2,850 cm, which corresponds to the CH vibration of lipid molecules. -1 In some embodiments, the images are also acquired at a wavenumber of 2,930 cm, which corresponds to the CH vibrations of proteins and nucleic acid molecules. -1In some embodiments, the signal derived from the cell-bound contrast agent comprises a fluorescent signal, a phosphorescent signal, or any combination thereof. In some embodiments, the cell-bound contrast agent comprises an antibody conjugated to a fluorophore, a quantum dot, a nanoparticle, or a phosphorescent material. In some embodiments, the cell-bound contrast agent comprises a fluorogenic enzyme substrate. In some embodiments, the cell-bound contrast agent comprises fluorescein, 5-ALA, BLZ-100, or LUM015. In some embodiments, the cell-bound contrast agent comprises 5-aminolevulinic acid (5-ALA), and the first emission wavelength range comprises 640 nm light and the second emission wavelength comprises wavelengths shorter than 600 nm. In some embodiments, the image interpretation algorithm comprises a Canny edge detection algorithm, a Canny-Delicie edge detection algorithm, a first-order gradient edge detection algorithm, a second-order derivative edge detection algorithm, a phase coherence edge detection algorithm, an image segmentation algorithm, an intensity thresholding algorithm, an intensity clustering algorithm, an intensity histogram-based algorithm, a feature recognition algorithm, a pattern recognition algorithm, a generalized Hough transform algorithm, a circular Hough transform algorithm, a Fourier transform algorithm, a fast Fourier transform algorithm, a wavelet analysis algorithm, an autocorrelation algorithm, or any combination thereof. In some embodiments, the image interpretation algorithm detects individual cells based on image feature size, shape, pattern, intensity, or any combination thereof. In some embodiments, the image interpretation algorithm comprises an artificial intelligence or machine learning algorithm. In some embodiments, the image interpretation algorithm comprises a supervised machine learning algorithm, an unsupervised machine learning algorithm, a semi-supervised machine learning algorithm, or any combination thereof.In some embodiments, the machine learning algorithm comprises an artificial neural network algorithm, a deep convolutional neural network algorithm, a deep recurrent neural network, a generative adversarial network, a support vector machine, a hierarchical clustering algorithm, a Gaussian process regression algorithm, a decision tree algorithm, a logistic model tree algorithm, a random forest algorithm, a fuzzy classifier algorithm, a k-means algorithm, an expectation-maximization algorithm, a fuzzy clustering algorithm, or any combination thereof. In some embodiments, the machine learning algorithm is trained using a training dataset comprising imaging data acquired for archived histopathological tissue samples, imaging data acquired for freshly collected histopathological tissue samples, or any combination thereof. In some embodiments, the training dataset is continuously, periodically, or randomly updated with imaging data acquired by two or more systems deployed for use in the same or different facilities. In some embodiments, the two or more systems are deployed in different facilities, and the training dataset is continuously, periodically, or randomly updated via an internet connection. In some embodiments, the image interpretation algorithm determines the total or average intensity of the signal derived from the cell-bound contrast agent. In some embodiments, the image interpretation algorithm determines whether the signal derived from the cell-bound contrast agent for individual cells exceeds a defined threshold level for contrast-positive cells. In some embodiments, the image interpretation algorithm outputs a total number of contrast-positive cells in the image of the tissue sample, a density of contrast-positive cells in the image of the tissue sample, a percentage of contrast-positive cells in the image of the tissue sample, or any combination thereof. In some embodiments, the image interpretation algorithm also outputs a cellularity score based on images obtained using a second optical thinning imaging modality. In some embodiments, the images of the tissue sample are obtained in vivo. In some embodiments, the images of the tissue sample are obtained ex vivo.In some embodiments, the system is used during a surgical procedure to identify a location for performing a biopsy or to determine whether a resection is complete.

[0003] Also disclosed herein are methods for cellular resolution imaging of tissue samples, the methods including: (a) obtaining high-resolution optically thinned images of the distribution of a cell-bound contrast agent in the tissue sample using a first imaging modality; (b) obtaining high-resolution optically thinned images of the tissue sample morphology in the tissue sample within the same optical focal plane as that of (a) using a second imaging modality; and (c) processing the images obtained using one or both of the first and second imaging modalities using an image interpretation algorithm, wherein the image interpretation algorithm identifies individual cells, determines their locations, and measures the signal at corresponding locations of the individual cells in the images obtained using the first imaging modality to output a quantitative measure of the signal derived from the cell-bound contrast agent at cellular resolution. In some embodiments, the first imaging modality comprises two-photon fluorescence microscopy, confocal fluorescence microscopy, light sheet microscopy, or structured illumination microscopy. In some embodiments, the second imaging modality comprises stimulated Raman scattering microscopy, coherent anti-Stokes Raman scattering microscopy, confocal reflectance microscopy, second harmonic generation microscopy, or third harmonic generation microscopy. In some embodiments, the images obtained using the first imaging modality and the second imaging modality have an axial resolution of less than 10 μm. In some embodiments, the images obtained using the first imaging modality and the second imaging modality have a lateral resolution of less than 5 μm. In some embodiments, the method further includes obtaining images in two or more detection wavelength ranges using the first imaging modality.In some embodiments, a first detection wavelength range of the two or more detection wavelength ranges includes the emission peak of the cell-bound contrast agent, and a second detection wavelength range of the two or more detection wavelength ranges excludes the emission peak of the cell-bound contrast agent, and the image interpretation algorithm further processes the image obtained using the first detection wavelength to identify individual cells, determine their locations, measure the signal at the individual cell locations, and output a quantitative measure of the signal derived from the cell-bound contrast agent by correcting the signal by background values ​​measured at corresponding locations of the individual cells in the image obtained using the second emission wavelength range. In some embodiments, the second imaging modality comprises stimulated Raman scattering microscopy, and the image is obtained using stimulated Raman scattering at 2,850 cm, which corresponds to the CH vibration of lipid molecules. -1 In some embodiments, the images are also acquired at a wavenumber of 2,930 cm, which corresponds to the CH vibrations of proteins and nucleic acid molecules. -1In some embodiments, the signal derived from the cell-bound contrast agent comprises a fluorescent signal, a phosphorescent signal, or any combination thereof. In some embodiments, the cell-bound contrast agent comprises an antibody conjugated to a fluorophore, a quantum dot, a nanoparticle, or a phosphorescent material. In some embodiments, the cell-bound contrast agent comprises a fluorogenic enzyme substrate. In some embodiments, the cell-bound contrast agent comprises fluorescein, 5-ALA, BLZ-100, or LUM015. In some embodiments, the cell-bound contrast agent comprises 5-aminolevulinic acid (5-ALA), and the first emission wavelength range comprises 640 nm light and the second emission wavelength comprises wavelengths shorter than 600 nm. In some embodiments, the image interpretation algorithm comprises a Canny edge detection algorithm, a Canny-Delicie edge detection algorithm, a first-order gradient edge detection algorithm, a second-order derivative edge detection algorithm, a phase coherence edge detection algorithm, an image segmentation algorithm, an intensity thresholding algorithm, an intensity clustering algorithm, an intensity histogram-based algorithm, a feature recognition algorithm, a pattern recognition algorithm, a generalized Hough transform algorithm, a circular Hough transform algorithm, a Fourier transform algorithm, a fast Fourier transform algorithm, a wavelet analysis algorithm, an autocorrelation algorithm, or any combination thereof. In some embodiments, the image interpretation algorithm detects individual cells based on image feature size, shape, pattern, intensity, or any combination thereof. In some embodiments, the image interpretation algorithm comprises an artificial intelligence or machine learning algorithm. In some embodiments, the image interpretation algorithm comprises a supervised machine learning algorithm, an unsupervised machine learning algorithm, a semi-supervised machine learning algorithm, or any combination thereof.In some embodiments, the machine learning algorithm comprises an artificial neural network algorithm, a deep convolutional neural network algorithm, a deep recurrent neural network, a generative adversarial network, a support vector machine, a hierarchical clustering algorithm, a Gaussian process regression algorithm, a decision tree algorithm, a logistic model tree algorithm, a random forest algorithm, a fuzzy classifier algorithm, a k-means algorithm, an expectation-maximization algorithm, a fuzzy clustering algorithm, or any combination thereof. In some embodiments, the machine learning algorithm is trained using a training dataset comprising imaging data acquired for archived histopathological tissue samples, imaging data acquired for freshly collected histopathological tissue samples, or any combination thereof. In some embodiments, the training dataset is continuously, periodically, or randomly updated with imaging data acquired by two or more systems deployed for use in the same or different facilities. In some embodiments, the two or more systems are deployed in different facilities, and the training dataset is continuously, periodically, or randomly updated via an internet connection. In some embodiments, the image interpretation algorithm determines the total or average intensity of the signal derived from the cell-bound contrast agent. In some embodiments, the image interpretation algorithm determines whether the signal derived from the cell-bound contrast agent for individual cells exceeds a defined threshold level for contrast-positive cells. In some embodiments, the image interpretation algorithm outputs a total number of contrast-positive cells in the image of the tissue sample, a density of contrast-positive cells in the image of the tissue sample, a percentage of contrast-positive cells in the image of the tissue sample, or any combination thereof. In some embodiments, the image interpretation algorithm also outputs a cellularity score based on images obtained using a second optical thinning imaging modality. In some embodiments, the images of the tissue sample are obtained in vivo. In some embodiments, the images of the tissue sample are obtained ex vivo.In some embodiments, the methods are used during a surgical procedure to identify a location for performing a biopsy or to determine whether a resection is complete.

[0004] Disclosed herein is a system for imaging a tissue sample, the system comprising: a) a high-resolution optical sectioning microscope configured to acquire images of the tissue sample; and b) a processor configured to execute an image interpretation algorithm that detects individual cells in images acquired by the high-resolution optical sectioning microscope and outputs a quantitative measure of a signal derived from a cell-bound contrast agent. In some embodiments, the high-resolution optical sectioning microscope comprises a two-photon fluorescence microscope, a confocal fluorescence microscope, a light sheet microscope, or a structured illumination microscope. In some embodiments, the high-resolution optical sectioning microscope has an axial resolution of less than 10 μm. In some embodiments, the high-resolution optical sectioning microscope has a lateral resolution of less than 5 μm. In some embodiments, the high-resolution optical sectioning microscope is configured to acquire images in two or more emission wavelength ranges. In some embodiments, a first emission wavelength range of the two or more detection wavelength ranges includes the emission peak of the cell-bound contrast agent, and a second emission wavelength range of the two or more detection wavelength ranges excludes the emission peak of the cell-bound contrast agent. An image interpretation algorithm processes an image acquired using the first detection wavelength range to identify individual cells, determine their locations, measure signals at the locations of the individual cells, and correct the signals using background values ​​measured at corresponding locations in an image acquired using the second detection wavelength range, thereby outputting a quantitative measure of the signal derived from the cell-bound contrast agent. In some embodiments, the signal derived from the cell-bound contrast agent comprises a fluorescent signal, a phosphorescent signal, or any combination thereof. In some embodiments, the cell-bound contrast agent comprises an antibody conjugated to a fluorophore, a quantum dot, a nanoparticle, or a phosphorescent material. In some embodiments, the cell-bound contrast agent comprises a fluorogenic enzyme substrate. In some embodiments, the cell-bound contrast agent comprises fluorescein, 5-ALA, BLZ-100, or LUM015. In some embodiments, the cell-bound imaging agent comprises 5-aminolevulinic acid (5-ALA), the first emission wavelength range includes 640 nm light, and the second emission wavelength includes wavelengths shorter than 600 nm.In some embodiments, the image interpretation algorithm comprises a Canny edge detection algorithm, a Canny-Delicie edge detection algorithm, a first-order gradient edge detection algorithm, a second-order derivative edge detection algorithm, a phase coherence edge detection algorithm, an image segmentation algorithm, an intensity thresholding algorithm, an intensity clustering algorithm, an intensity histogram-based algorithm, a feature recognition algorithm, a pattern recognition algorithm, a generalized Hough transform algorithm, a circular Hough transform algorithm, a Fourier transform algorithm, a fast Fourier transform algorithm, a wavelet analysis algorithm, an autocorrelation algorithm, or any combination thereof. In some embodiments, the image interpretation algorithm detects individual cells based on image feature size, shape, pattern, intensity, or any combination thereof. In some embodiments, the image interpretation algorithm comprises an artificial intelligence or machine learning algorithm. In some embodiments, the image interpretation algorithm comprises a supervised machine learning algorithm, an unsupervised machine learning algorithm, a semi-supervised machine learning algorithm, or any combination thereof. In some embodiments, the machine learning algorithm comprises an artificial neural network algorithm, a network, a support vector machine, a hierarchical clustering algorithm, a Gaussian process regression algorithm, a decision tree algorithm, a logistic model tree algorithm, a random forest algorithm, a fuzzy classifier algorithm, a k-means algorithm, an expectation maximization algorithm, a fuzzy clustering algorithm, or any combination thereof. In some embodiments, the machine learning algorithm is trained using a training dataset comprising imaging data obtained for archived histopathological tissue samples, imaging data obtained for freshly collected histopathological tissue samples, or any combination thereof. In some embodiments, the training dataset is continuously, periodically, or randomly updated with imaging data obtained by two or more systems deployed for use in the same or different institutions.In some embodiments, two or more systems are deployed at different facilities, and the training dataset is continuously, periodically, or randomly updated via an internet connection. In some embodiments, the quantitative measure of the signal derived from the cell-bound contrast agent comprises a measure of the amount of contrast agent associated with one or more individual cells in the image. In some embodiments, the quantitative measure of the signal derived from the cell-bound contrast agent comprises a measure of the total number of contrast-positive cells in the image, the density of contrast-positive cells in the image, the percentage of contrast-positive cells in the image, or any combination thereof. In some embodiments, the image of the tissue sample is obtained in vivo. In some embodiments, the image of the tissue sample is obtained ex vivo. In some embodiments, the system is used during a surgical procedure to identify a location for performing a biopsy or to determine whether a resection is complete.

[0005] Disclosed herein are methods for imaging a tissue sample, the method comprising: a) obtaining a high-resolution optically thinned image of the tissue sample; and b) processing the image using an image interpretation algorithm that detects individual cells within the image and outputs a quantitative measure of a signal derived from a cell-bound imaging agent at the location of one or more individual cells. In some embodiments, the high-resolution optically thinned image is obtained using two-photon fluorescence microscopy, confocal fluorescence microscopy, light sheet microscopy, or structured illumination microscopy. In some embodiments, the first imaging modality comprises two-photon fluorescence microscopy, confocal fluorescence microscopy, light sheet microscopy, or structured illumination microscopy. In some embodiments, the obtained image has an axial resolution of less than 10 μm. In some embodiments, the obtained image has a lateral resolution of less than 5 μm. In some embodiments, the signal derived from the cell-bound imaging agent comprises a fluorescent signal, a phosphorescent signal, or any combination thereof. In some embodiments, the cell-bound imaging agent comprises an antibody conjugated to a fluorophore, a quantum dot, a nanoparticle, or a phosphorescent material. In some embodiments, the cell-bound contrast agent comprises a fluorogenic enzyme substrate. In some embodiments, the cell-bound contrast agent comprises fluorescein, 5-ALA, BLZ-100, or LUM015. In some embodiments, the image interpretation algorithm comprises a Canny edge detection algorithm, a Canny-Delicie edge detection algorithm, a first-order gradient edge detection algorithm, a second-order derivative edge detection algorithm, a phase coherence edge detection algorithm, an image segmentation algorithm, an intensity thresholding algorithm, an intensity clustering algorithm, an intensity histogram-based algorithm, a feature recognition algorithm, a pattern recognition algorithm, a generalized Hough transform algorithm, a circle Hough transform algorithm, a Fourier transform algorithm, a fast Fourier transform algorithm, a wavelet analysis algorithm, an autocorrelation algorithm, or any combination thereof.In some embodiments, the image interpretation algorithm detects individual cells based on image feature size, shape, pattern, intensity, or any combination thereof. In some embodiments, the image interpretation algorithm comprises an artificial intelligence or machine learning algorithm. In some embodiments, the image interpretation algorithm comprises a supervised machine learning algorithm, an unsupervised machine learning algorithm, a semi-supervised machine learning algorithm, or any combination thereof. In some embodiments, the machine learning algorithm comprises an artificial neural network algorithm, a deep convolutional neural network algorithm, a deep recurrent neural network, a generative adversarial network, a support vector machine, a hierarchical clustering algorithm, a Gaussian process regression algorithm, a decision tree algorithm, a logistic model tree algorithm, a random forest algorithm, a fuzzy classifier algorithm, a k-means algorithm, an expectation maximization algorithm, a fuzzy clustering algorithm, or any combination thereof. In some embodiments, the machine learning algorithm is trained using a training dataset comprising imaging data obtained on archived histopathological tissue samples, imaging data obtained on freshly collected histopathological tissue samples, or any combination thereof. In some embodiments, the training dataset is continuously, periodically, or randomly updated with imaging data acquired by two or more systems deployed for use in the same or different facilities. In some embodiments, the two or more systems are deployed at different facilities, and the training dataset is continuously, periodically, or randomly updated via an internet connection. In some embodiments, the image interpretation algorithm determines the average intensity of the signal derived from the cell-bound contrast agent. In some embodiments, the image interpretation algorithm determines whether the signal for each individual cell exceeds a defined threshold level for contrast-positive cells.In some embodiments, the image interpretation algorithm outputs a total number of contrast-positive cells in the image, a density of contrast-positive cells in the image, a percentage of contrast-positive cells in the image, or any combination thereof. In some embodiments, the image interpretation algorithm outputs a cellularity score. In some embodiments, the image of the tissue sample is obtained in vivo. In some embodiments, the image of the tissue sample is obtained ex vivo. In some embodiments, the system is used during a surgical procedure to identify a location for performing a biopsy or to determine whether a resection is complete.

[0006] Disclosed herein is a method for detecting a cell-bound optical imaging agent in a tissue sample, the method comprising: (a) acquiring a first high-resolution optically thinned image of the tissue sample in a first emission wavelength range that includes the emission peak of the cell-bound contrast agent; (b) acquiring a second high-resolution optically thinned image of the tissue sample in a second emission wavelength range that excludes the emission peak of the cell-bound contrast agent; and (c) applying a pseudocolor algorithm to the first and second images to generate a multicolor image of the tissue sample that facilitates human interpretation. In some embodiments, the first and second high-resolution optically thinned images are acquired using two-photon fluorescence microscopy, confocal fluorescence microscopy, light sheet microscopy, or structured illumination microscopy. In some embodiments, the image acquired using the first emission wavelength range is further processed by an image interpretation algorithm to identify individual cells and their locations and output a total number of contrast-positive cells in the image, a density of contrast-positive cells in the image, a percentage of contrast-positive cells in the image, or any combination thereof. In some embodiments, the image of the tissue sample is obtained in vivo. In some embodiments, the image of the tissue sample is obtained ex vivo. In some embodiments, the method is used during a surgical procedure to identify a location for performing a biopsy or to determine whether a resection is complete.

[0007] Also disclosed herein is a method for guiding surgical resection, the method comprising: a) obtaining high-resolution optically thinned images of the distribution of a cell-bound contrast agent in a tissue sample using a first imaging modality; b) obtaining high-resolution optically thinned images of the tissue sample morphology within the same optical focal plane in the tissue sample using a second imaging modality; and c) processing the images obtained using one or both of the first and second imaging modalities using an image interpretation algorithm, which identifies individual cells, determines their locations, and outputs a quantitative measure of the signal derived from the cell-bound contrast agent at cellular resolution by measuring the signal at corresponding locations of the individual cells in the image obtained using the first imaging modality.

[0008] Disclosed herein is a method for guiding surgical resection, the method comprising: a) obtaining a high-resolution optically sliced ​​image of a tissue sample; and b) processing the image using an image interpretation algorithm that detects individual cells within the image and outputs a quantitative measure of the signal derived from a cell-bound contrast agent at the location of one or more individual cells.

[0009] Disclosed herein is a method for guiding surgical resection, the method including: a) obtaining a first high-resolution optically thinned image of a tissue sample in a first emission wavelength range that includes the emission peak of a cell-bound contrast agent; b) obtaining a second high-resolution optically thinned image of the tissue sample in a second emission wavelength range that excludes the emission peak of the cell-bound contrast agent; and c) applying a pseudocolor algorithm to the first and second images to generate a multicolor image of the tissue sample that facilitates human interpretation.

[0010] In some embodiments, for any of the methods disclosed herein, the images for at least one imaging modality are obtained using two-photon fluorescence microscopy, confocal fluorescence microscopy, light sheet microscopy, or structured illumination microscopy. In some embodiments, the images obtained using at least a second imaging modality are obtained using stimulated Raman scattering microscopy, coherent anti-Stokes Raman scattering microscopy, confocal reflectance microscopy, second harmonic generation microscopy, or third harmonic generation microscopy. In some embodiments, the tissue sample is a brain tissue sample, a breast tissue sample, a lung tissue sample, a pancreatic tissue sample, or a prostate tissue sample. In some embodiments, the images of the tissue sample are obtained in vivo. In some embodiments, the images of the tissue sample are obtained ex vivo. In some embodiments, the image interpretation algorithm used to process the images obtained using the first imaging modality or the second imaging modality outputs a total number of contrast-positive cells in the image, a density of contrast-positive cells in the image, a percentage of contrast-positive cells in the image, or any combination thereof. In some embodiments, the image interpretation algorithm used to process images obtained using the first imaging modality or the second imaging modality comprises a machine learning algorithm. In some embodiments, the machine learning algorithm comprises a supervised machine learning algorithm, an unsupervised machine learning algorithm, a semi-supervised machine learning algorithm, or any combination thereof. In some embodiments, the machine learning algorithm comprises an artificial neural network algorithm, a deep convolutional neural network algorithm, a deep recurrent neural network, a generative adversarial network, a support vector machine, a hierarchical clustering algorithm, a Gaussian process regression algorithm, a decision tree algorithm, a logistic model tree algorithm, a random forest algorithm, a fuzzy classifier algorithm, a k-means algorithm, an expectation maximization algorithm, a fuzzy clustering algorithm, or any combination thereof.In some embodiments, the machine learning algorithm is trained using a training dataset comprising imaging data acquired for archived histopathological tissue samples, imaging data acquired for freshly collected histopathological tissue samples, or any combination thereof. In some embodiments, the training dataset is continuously, periodically, or randomly updated with imaging data acquired by two or more systems deployed for use in the same or different facilities. In some embodiments, the two or more systems are deployed in different facilities, and the training dataset is continuously, periodically, or randomly updated via an internet connection.

[0011] Disclosed herein is a method for guiding surgical resection, the method comprising: a) obtaining high-resolution optically thinned images of the distribution of a cell-bound contrast agent in a tissue sample using two-photon fluorescence microscopy; b) obtaining high-resolution optically thinned images of the tissue sample morphology in the same optical focal plane of the tissue sample using stimulated Raman scattering microscopy; and c) processing the images obtained using stimulated Raman scattering microscopy using an image interpretation algorithm that identifies individual cells, determines their locations, and outputs a quantitative measure of the signal derived from the cell-bound contrast agent at cellular resolution by measuring the signal at locations corresponding to those of the individual cells in the images obtained using two-photon fluorescence microscopy.

[0012] Disclosed herein is a method for guiding surgical resection, the method comprising: a) obtaining high-resolution optically thinned images of a tissue sample using two-photon fluorescence microscopy; and b) processing the images using an image interpretation algorithm that detects individual cells within the images and outputs a quantitative measure of the signal derived from the cell-bound contrast agent with cellular resolution at the location of one or more individual cells.

[0013] Disclosed herein is a method for guiding surgical resection, the method including: a) obtaining a first high-resolution, optically thinned, two-photon fluorescence image of a tissue sample in a first emission wavelength range that includes the emission peak of a cell-bound contrast agent; b) obtaining a second high-resolution, optically thinned, two-photon fluorescence image of the tissue sample in a second emission wavelength range that excludes the emission peak of the cell-bound contrast agent; and c) applying a pseudocolor algorithm to the first and second two-photon fluorescence images to generate a multicolor image of the tissue sample that facilitates human interpretation.

[0014] In any of the methods disclosed herein, in some embodiments, images for at least one imaging modality are obtained using confocal fluorescence microscopy, light sheet microscopy, or structured illumination microscopy instead of two-photon fluorescence microscopy. In some embodiments, images obtained using at least a second imaging modality are obtained using coherent anti-Stokes Raman scattering microscopy, confocal reflectance microscopy, second harmonic generation microscopy, or third harmonic generation microscopy instead of stimulated Raman scattering microscopy. In some embodiments, the tissue sample is a brain tissue sample, a breast tissue sample, a lung tissue sample, a pancreatic tissue sample, or a prostate tissue sample. In some embodiments, the images of the tissue sample are obtained in vivo. In some embodiments, the images of the tissue sample are obtained ex vivo. In some embodiments, the image interpretation algorithm used to process the images obtained using two-photon fluorescence microscopy outputs a total number of contrast-positive cells in the image, a density of contrast-positive cells in the image, a percentage of contrast-positive cells in the image, or any combination thereof. In some embodiments, the image interpretation algorithm used to process images obtained using two-photon fluorescence or stimulated Raman scattering microscopy comprises a machine learning algorithm. In some embodiments, the machine learning algorithm comprises a supervised machine learning algorithm, an unsupervised machine learning algorithm, a semi-supervised machine learning algorithm, or any combination thereof. In some embodiments, the machine learning algorithm comprises an artificial neural network algorithm, a deep convolutional neural network algorithm, a deep recurrent neural network, a generative adversarial network, a support vector machine, a hierarchical clustering algorithm, a Gaussian process regression algorithm, a decision tree algorithm, a logistic model tree algorithm, a random forest algorithm, a fuzzy classifier algorithm, a k-means algorithm, an expectation maximization algorithm, a fuzzy clustering algorithm, or any combination thereof.In some embodiments, the machine learning algorithm is trained using a training dataset comprising imaging data acquired for archived histopathological tissue samples, imaging data acquired for freshly collected histopathological tissue samples, or any combination thereof. In some embodiments, the training dataset is continuously, periodically, or randomly updated with imaging data acquired by two or more systems deployed for use in the same or different facilities. In some embodiments, the two or more systems are deployed in different facilities, and the training dataset is continuously, periodically, or randomly updated via an internet connection. The present invention provides, for example, the following. (Item 1) 1. A system for imaging a tissue sample, the system comprising: a) a first optical subsystem configured to obtain high-resolution images of the distribution of a cell-bound contrast agent in the tissue sample using a first optical slice imaging modality; b) a second optical subsystem configured to obtain high-resolution images of the tissue sample morphology using a second optical thinning imaging modality, wherein the first and second optical subsystems are configured to image the same optical plane within the tissue sample; and c) a processor configured to invoke an image interpretation algorithm; and Equipped with The image interpretation algorithm processes images obtained using one or both of the first and second optical thinning imaging modalities to identify individual cells, determine their locations, and output a quantitative measure of the signal derived from the cell-bound contrast agent by measuring signals at the locations corresponding to those of the individual cells in the images obtained using the first imaging modality. (Item 2) Item 10. The system of item 1, wherein the first optical slice imaging modality comprises two-photon fluorescence microscopy, confocal fluorescence microscopy, light sheet microscopy, or structured illumination microscopy. (Item 3) Item 4. The system of item 1 or 2, wherein the second optical thinning imaging modality comprises stimulated Raman scattering microscopy, coherent anti-Stokes Raman scattering microscopy, confocal reflectance microscopy, second harmonic generation microscopy, or third harmonic generation microscopy. 4. The system of any one of items 1-3, wherein the first and second optical subsystems are configured to have an axial resolution of less than 10 μm. (Item 5) 5. The system of any one of items 1-4, wherein the first and second optical subsystems are configured to have a lateral resolution of less than 5 μm. (Item 6) 6. The system of any one of items 1-5, wherein the first optical subsystem is further configured to obtain high-resolution images in two or more detection wavelength ranges. (Item 7) a first detection wavelength range of the two or more detection wavelength ranges includes an emission peak of the cell-bound contrast agent; a second detection wavelength range of the two or more detection wavelength ranges excludes an emission peak of the cell-bound contrast agent; 7. The system of claim 6, wherein the image interpretation algorithm further processes the image obtained using the first detection wavelength to identify individual cells, determine their locations, measure the signal at the locations of the individual cells, and correct the signal by background values ​​measured at corresponding locations of the individual cells in the image obtained using the second detection wavelength range, thereby outputting a quantitative measure of the signal derived from the cell-bound contrast agent. (Item 8) The second optical thinning imaging modality comprises stimulated Raman scattering microscopy, and the images are taken at 2,850 cm corresponding to CH vibrations of lipid molecules. -18. The system of any one of items 3-7, wherein the wavelength is obtained at a wave number of . (Item 9) 9. The system of claim 8, wherein the image is also obtained at a wavenumber of 2,930 cm, which corresponds to CH vibrations of protein and nucleic acid molecules. (Item 10) 10. The system of any one of items 1-9, wherein the cell-bound contrast agent comprises fluorescein, 5-ALA, BLZ-100, or LUM015. (Item 11) 11. The system of any one of items 6-10, wherein the cell-bound imaging agent comprises 5-aminolevulinic acid (5-ALA), the first emission wavelength range includes 640 nm light, and the second emission wavelength includes wavelengths shorter than 600 nm. (Item 12) 12. The system of any one of items 1-11, wherein the image interpretation algorithm detects individual cells based on image feature size, shape, pattern, intensity, or any combination thereof. (Item 13) Item 13. The system of any one of items 1-12, wherein the image interpretation algorithm comprises a supervised machine learning algorithm, an unsupervised machine learning algorithm, a semi-supervised machine learning algorithm, or any combination thereof. (Item 14) 14. The system of claim 13, wherein the machine learning algorithm is trained using a training dataset comprising imaging data obtained for archived histopathological tissue samples, imaging data obtained for freshly collected histopathological tissue samples, or any combination thereof. (Item 15) Item 15. The system of item 14, wherein the training dataset is continuously, periodically, or randomly updated with imaging data acquired by two or more systems deployed for use in the same or different facilities. (Item 16) 16. The system of any one of items 1-15, wherein the image interpretation algorithm determines a total or average intensity of the signal derived from the cell-bound contrast agent. (Item 17) 17. The system of any one of items 1-16, wherein the image interpretation algorithm determines whether the signal derived from the cell-bound contrast agent for an individual cell exceeds a defined threshold level for a contrast agent-positive cell. (Item 18) Item 19. The system of item 17, wherein the image interpretation algorithm outputs a total number of contrast-positive cells in the image of the tissue sample, a density of contrast-positive cells in the image of the tissue sample, a percentage of contrast-positive cells in the image of the tissue sample, or any combination thereof. 20. The system of claim 18, wherein the image interpretation algorithm also outputs a cellularity score based on images obtained using the second optical sectioning imaging modality. (Item 20) 20. The system of any one of items 1-19, wherein the image of the tissue sample is obtained in vivo. (Item 21) 20. The system of any one of items 1-19, wherein the image of the tissue sample is obtained ex vivo. (Item 22) 22. The system of any one of items 1-21, wherein the system is used during a surgical procedure to identify a location for performing a biopsy or to determine whether a resection is complete. (Item 23) 1. A method for cellular resolution imaging of a tissue sample, the method comprising: a) obtaining high-resolution optical slice images of the distribution of a cell-associated contrast agent in said tissue sample using a first imaging modality; b) obtaining high-resolution optically sliced ​​images of tissue sample morphology in the tissue sample within the same optical focal plane as that of (a) using a second imaging modality; c) using an image interpretation algorithm to process images obtained using one or both of said first and second imaging modalities; Including, The method, wherein the image interpretation algorithm identifies individual cells, determines their locations, and outputs a quantitative measure of the signal derived from the cell-bound contrast agent at cellular resolution by measuring the signal at corresponding locations of the individual cells in the image obtained using the first imaging modality. (Item 24) 24. The method of claim 23, wherein the first imaging modality comprises two-photon fluorescence microscopy, confocal fluorescence microscopy, light sheet microscopy, or structured illumination microscopy. (Item 25) 25. The method of claim 23 or 24, wherein the second imaging modality comprises stimulated Raman scattering microscopy, coherent anti-Stokes Raman scattering microscopy, confocal reflectance microscopy, second harmonic generation microscopy, or third harmonic generation microscopy. (Item 26) 26. The method of any one of items 23-25, wherein the images obtained using the first imaging modality and the second imaging modality have an axial resolution of less than 10 μm. (Item 27) 27. The method of any one of items 23-26, wherein the images obtained using the first imaging modality and the second imaging modality have a lateral resolution of less than 5 μm. (Item 28) 28. The method of any one of items 23-27, further comprising obtaining images in two or more detection wavelength ranges using the first imaging modality. (Item 29) a first detection wavelength range of the two or more detection wavelength ranges includes an emission peak of the cell-bound contrast agent; a second detection wavelength range of the two or more detection wavelength ranges excludes an emission peak of the cell-bound contrast agent; 29. The method of claim 28, wherein the image interpretation algorithm further processes images obtained using the first detection wavelength to identify individual cells, determine their locations, measure signals at the locations of the individual cells, and correct the signals by background values ​​measured at corresponding locations of the individual cells in the images obtained using the second emission wavelength range, thereby outputting a quantitative measure of the signals derived from the cell-bound contrast agent. (Item 30) The second imaging modality comprises stimulated Raman scattering microscopy, and the images are taken at 2,850 cm corresponding to CH vibrations of lipid molecules. -1 30. The method according to any one of items 25-29, wherein the wavelength is obtained at a wave number of (Item 31) 31. The method of claim 30, wherein the image is also obtained at a wavenumber of 2,930 cm, which corresponds to CH vibrations of protein and nucleic acid molecules. (Item 32) 32. The method of any one of items 23-31, wherein the cell-bound imaging agent comprises fluorescein, 5-ALA, BLZ-100, or LUM015. (Item 33) 33. The method of any one of items 28-32, wherein the cell-bound imaging agent comprises 5-aminolevulinic acid (5-ALA), the first emission wavelength range comprises 640 nm light, and the second emission wavelength comprises wavelengths shorter than 600 nm. (Item 34) 34. The method of any one of items 23-33, wherein the image interpretation algorithm detects individual cells based on image feature size, shape, pattern, intensity, or any combination thereof. (Item 35) 35. The method of any one of items 23-34, wherein the image interpretation algorithm comprises a supervised machine learning algorithm, an unsupervised machine learning algorithm, a semi-supervised machine learning algorithm, or any combination thereof. (Item 36) 36. The method of claim 35, wherein the machine learning algorithm is trained using a training dataset comprising imaging data obtained on archived histopathological tissue samples, imaging data obtained on freshly collected histopathological tissue samples, or any combination thereof. (Item 37) 37. The method of claim 36, wherein the training dataset is continuously, periodically, or randomly updated with imaging data acquired by two or more systems deployed for use in the same or different facilities. (Item 38) 38. The method of any one of items 23-37, wherein the image interpretation algorithm determines the total intensity or the average intensity of the signal derived from the cell-bound contrast agent. (Item 39) 39. The method of any one of items 23-38, wherein the image interpretation algorithm determines whether the signal derived from the cell-bound contrast agent for an individual cell is above a defined threshold level for a contrast agent-positive cell. (Item 40) 40. The method of claim 39, wherein the image interpretation algorithm outputs a total number of contrast agent-positive cells in the image of the tissue sample, a density of contrast agent-positive cells in the image of the tissue sample, a percentage of contrast agent-positive cells in the image of the tissue sample, or any combination thereof. (Item 41) 41. The method of claim 40, wherein the image interpretation algorithm also outputs a cellularity score based on images obtained using the second optical sectioning imaging modality. (Item 42) 42. The method of any one of items 23-41, wherein the image of the tissue sample is obtained in vivo. (Item 43) 42. The method of any one of items 23-41, wherein the image of the tissue sample is obtained ex vivo. (Item 44) 44. The method of any one of items 23-43, wherein the method is used during a surgical procedure to identify a location for performing a biopsy or to determine whether a resection is complete. (Item 45) 1. A system for imaging a tissue sample, the system comprising: a) a high-resolution optical sectioning microscope configured to obtain images of the tissue sample; and b) a processor configured to invoke an image interpretation algorithm; Equipped with The system, wherein the image interpretation algorithm detects individual cells in the images obtained by the high-resolution optical sectioning microscope and outputs a quantitative measure of the signal derived from the cell-bound contrast agent. (Item 46) 46. ​​The system of claim 45, wherein the high-resolution optical sectioning microscope comprises a two-photon fluorescence microscope, a confocal fluorescence microscope, a light sheet microscope, or a structured illumination microscope. (Item 47) 47. The system of any one of items 45-46, wherein the high-resolution optical sectioning microscope is configured to obtain images in two or more emission wavelength ranges. (Item 48) a first emission wavelength range of the two or more detection wavelength ranges includes an emission peak of the cell-bound contrast agent; a second emission wavelength range of the two or more detection wavelength ranges excludes an emission peak of the cell-bound contrast agent; Item 48. The system of item 47, wherein the image interpretation algorithm processes images obtained using the first detection wavelength range to identify individual cells, determine their locations, measure the signal at the locations of the individual cells, and correct the signal using a background value measured at a corresponding location in an image obtained using the second detection wavelength range, thereby outputting a quantitative measure of the signal derived from the cell-bound contrast agent. (Item 49) 49. The system of any one of items 45-48, wherein the cell-bound contrast agent comprises fluorescein, 5-ALA, BLZ-100, or LUM015. (Item 50) 50. The system of any one of items 47-49, wherein the cell-bound imaging agent comprises 5-aminolevulinic acid (5-ALA), the first emission wavelength range includes 640 nm light, and the second emission wavelength includes wavelengths shorter than 600 nm. (Item 51) 51. The system of any one of items 45-50, wherein the image interpretation algorithm detects individual cells based on image feature size, shape, pattern, intensity, or any combination thereof. (Item 52) 52. The system of any one of items 45-51, wherein the image interpretation algorithm comprises a supervised machine learning algorithm, an unsupervised machine learning algorithm, a semi-supervised machine learning algorithm, or any combination thereof. (Item 53) 53. The system of claim 52, wherein the machine learning algorithm is trained using a training dataset comprising imaging data obtained for archived histopathological tissue samples, imaging data obtained for freshly collected histopathological tissue samples, or any combination thereof. (Item 54) 54. The system of any one of items 45-53, wherein the quantitative measure of the signal derived from the cell-bound contrast agent comprises a measure of the amount of contrast agent associated with one or more individual cells in the image. (Item 55) 55. The system of any one of items 45-54, wherein the quantitative measure of the signal derived from the cell-bound contrast agent comprises a measure of the total number of contrast-positive cells in the image, the density of contrast-positive cells in the image, the percentage of contrast-positive cells in the image, or any combination thereof. (Item 56) 56. The system of any one of items 45-55, wherein the image of the tissue sample is obtained in vivo. (Item 57) 56. The system of any one of items 45-55, wherein the image of the tissue sample is obtained ex vivo. (Item 58) 58. The system of any one of items 45-57, wherein the system is used during a surgical procedure to identify a location for performing a biopsy or to determine whether a resection is complete. (Item 59) 1. A method of imaging a tissue sample, the method comprising: a) obtaining a high-resolution optical section image of the tissue sample; b) processing said image using an image interpretation algorithm; Including, The image interpretation algorithm detects individual cells within the image and outputs a quantitative measure of the signal derived from the cell-bound contrast agent at the location of one or more individual cells. 60. The method of claim 59, wherein the high-resolution optical slice image is obtained using two-photon fluorescence microscopy, confocal fluorescence microscopy, light sheet microscopy, or structured illumination microscopy. (Item 61) 61. The method of any one of items 59-60, wherein the first imaging modality comprises two-photon fluorescence microscopy, confocal fluorescence microscopy, light sheet microscopy, or structured illumination microscopy. (Item 62) 62. The method of any one of items 59-61, wherein the cell-bound imaging agent comprises fluorescein, 5-ALA, BLZ-100, or LUM015. (Item 63) 63. The method of any one of items 59-62, wherein the image interpretation algorithm detects individual cells based on image feature size, shape, pattern, intensity, or any combination thereof. (Item 64) 64. The method of any one of items 59-63, wherein the image interpretation algorithm comprises an artificial intelligence or machine learning algorithm. (Item 65) Item 65. The method of item 64, wherein the machine learning algorithm comprises a supervised machine learning algorithm, an unsupervised machine learning algorithm, a semi-supervised machine learning algorithm, or any combination thereof. (Item 66) 66. The method of any one of items 64-65, wherein the machine learning algorithm is trained using a training dataset comprising imaging data obtained on archived histopathological tissue samples, imaging data obtained on freshly collected histopathological tissue samples, or any combination thereof. (Item 67) 67. The method of any one of items 59-66, wherein the image interpretation algorithm determines the average intensity of the signal derived from the cell-bound contrast agent. (Item 68) 68. The method of any one of items 59-67, wherein the image interpretation algorithm determines whether the signal for each individual cell is above a defined threshold level for a contrast agent-positive cell. (Item 69) 69. The method of claim 68, wherein the image interpretation algorithm outputs the total number of contrast-positive cells in the image, the density of contrast-positive cells in the image, the percentage of contrast-positive cells in the image, or any combination thereof. (Item 70) 70. The method of any one of items 59-69, wherein the image interpretation algorithm outputs a cellularity score. (Item 71) 71. The method of any one of items 59-70, wherein the image of the tissue sample is obtained in vivo. (Item 72) 72. The method of any one of items 59-71, wherein the image of the tissue sample is obtained ex vivo. (Item 73) 73. The method of any one of items 59-72, wherein the method is used during a surgical procedure to identify a location for performing a biopsy or to determine whether a resection is complete. (Item 74) 1. A method for the detection of a cell-bound optical imaging agent in a tissue sample, said method comprising: a) obtaining a first high-resolution optically sliced ​​image of the tissue sample in a first emission wavelength range that includes an emission peak of the cell-bound contrast agent; b) obtaining a second high-resolution optically sliced ​​image of the tissue sample in a second emission wavelength range that excludes the emission peak of the cell-bound contrast agent; c) applying a pseudocolor algorithm to the first and second images to generate a multicolor image of the tissue sample that facilitates human interpretation; A method comprising: (Item 75) 75. The method of claim 74, wherein the first and second high-resolution optically sliced ​​images are obtained using two-photon fluorescence microscopy, confocal fluorescence microscopy, light sheet microscopy, or structured illumination microscopy. (Item 76) 76. The method of claim 74 or 75, wherein the image obtained using the first emission wavelength range is further processed by an image interpretation algorithm to identify individual cells and their locations and output a total number of contrast agent-positive cells in the image, a density of contrast agent-positive cells in the image, a percentage of contrast agent-positive cells in the image, or any combination thereof. (Item 77) 77. The method of any one of items 74-76, wherein the image of the tissue sample is obtained in vivo. (Item 78) 78. The method of any one of items 74-77, wherein the image of the tissue sample is obtained ex vivo. (Item 79) 79. The method of any one of items 74-78, wherein the system is used during a surgical procedure to identify a location for performing a biopsy or to determine whether a resection is complete. (Item 80) 1. A method of inducing surgical resection, said method comprising: a) obtaining high-resolution optically thinned images of the distribution of a cell-bound contrast agent in a tissue sample using two-photon fluorescence microscopy; b) obtaining high-resolution optically thinned images of tissue sample morphology within the same optical focal plane of said tissue sample using stimulated Raman scattering microscopy; c) using an image interpretation algorithm to process images obtained using stimulated Raman scattering microscopy; Including, The method wherein the image interpretation algorithm identifies individual cells, determines their locations, and outputs a quantitative measure of the signal derived from the cell-bound contrast agent at cellular resolution by measuring the signal at corresponding locations of the individual cells in the image obtained using two-photon fluorescence microscopy. (Item 81) 1. A method of inducing surgical resection, said method comprising: a) obtaining high-resolution optically thinned images of a tissue sample using two-photon fluorescence microscopy; b) processing said image using an image interpretation algorithm; Including, The method, wherein the image interpretation algorithm detects individual cells within the image and outputs a quantitative measure of the signal derived from the cell-bound contrast agent with cellular resolution at one or more individual cell locations. (Item 82) 1. A method of inducing surgical resection, said method comprising: a) obtaining a first high-resolution optically thinned two-photon fluorescence image of the tissue sample in a first emission wavelength range that includes an emission peak of a cell-bound contrast agent; b) obtaining a second high-resolution optically thinned two-photon fluorescence image of the tissue sample in a second emission wavelength range that excludes the emission peak of the cell-bound contrast agent; c) applying a pseudocolor algorithm to the first and second two-photon fluorescence images to generate a multicolor image of the tissue sample that facilitates human interpretation; A method comprising: (Item 83) 83. The method of any one of items 80-82, wherein the images for at least one imaging modality are obtained using confocal fluorescence microscopy, light sheet microscopy, or structured illumination microscopy instead of two-photon fluorescence microscopy. (Item 84) 84. The method of any one of items 80-83, wherein the images obtained using at least a second imaging modality are obtained using coherent anti-Stokes Raman scattering microscopy, confocal reflectance microscopy, second harmonic generation microscopy, or third harmonic generation microscopy instead of stimulated Raman scattering microscopy. (Item 85) 85. The method of any one of items 80-84, wherein the tissue sample is a brain tissue sample, a breast tissue sample, a lung tissue sample, a pancreatic tissue sample, or a prostate tissue sample. (Item 86) 86. The method of any one of items 80-85, wherein the image of the tissue sample is obtained in vivo. (Item 87) 87. The method of any one of items 80-86, wherein the image of the tissue sample is obtained ex vivo. (Item 88) 88. The method of any one of items 80-87, wherein the image interpretation algorithm used to process the image obtained using two-photon fluorescence microscopy outputs a total number of contrast agent-positive cells in the image, a density of contrast agent-positive cells in the image, a percentage of contrast agent-positive cells in the image, or any combination thereof. (Item 89) 89. The method of any one of items 80-88, wherein the image interpretation algorithm used to process images obtained using two-photon fluorescence or stimulated Raman scattering microscopy comprises a machine learning algorithm. (Item 90) 90. The method of claim 89, wherein the machine learning algorithm comprises a supervised machine learning algorithm, an unsupervised machine learning algorithm, a semi-supervised machine learning algorithm, or any combination thereof. (Item 91) Item 91. The method of item 89 or item 90, wherein the machine learning algorithm comprises an artificial neural network algorithm, a deep convolutional neural network algorithm, a deep recurrent neural network, a generative adversarial network, a support vector machine, a hierarchical clustering algorithm, a Gaussian process regression algorithm, a decision tree algorithm, a logistics model tree algorithm, a random forest algorithm, a fuzzy classifier algorithm, a k-means algorithm, an expectation maximization algorithm, a fuzzy clustering algorithm, or any combination thereof. (Item 92) 92. The method of any one of items 89-91, wherein the machine learning algorithm is trained using a training dataset comprising imaging data obtained on archived histopathological tissue samples, imaging data obtained on freshly collected histopathological tissue samples, or any combination thereof. (Item 93) Item 93. The method of item 92, wherein the training dataset is continuously, periodically, or randomly updated with imaging data acquired by two or more systems deployed for use in the same or different facilities. (Item 94) Item 94. The method of item 93, wherein the two or more systems are deployed in different facilities and the training dataset is continuously, periodically, or randomly updated via an internet connection. (Incorporated by reference)

[0015] All publications, patents, and patent applications mentioned herein are 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. In the event of a conflict between a term in this specification and a term in an incorporated reference, the term in this specification shall control. [Brief explanation of the drawings]

[0016] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.

[0017] [Figure 1A] FIG. 1A provides a non-limiting example of a visible light image of brain tissue during a surgical procedure to remove a glioma.

[0018] [Figure 1B] FIG. 1B provides a non-limiting example of a fluorescent image of the brain tissue illustrated in FIG. 1A, in which 5-aminolevulinic acid (5-ALA) HCl is used as an imaging agent to better visualize and guide the removal of gliomas.

[0019] [Figure 2] Figure 2 provides a schematic diagram of a high-resolution optical microscope that combines stimulated Raman scattering (SRS) microscopy for imaging tissue morphology with two-photon fluorescence (2P) microscopy for imaging the distribution of fluorescent contrast agents.

[0020] [Figure 3A] FIG. 3A provides a non-limiting example of a two-photon fluorescence image of a brain tumor tissue sample dosed with 5-ALA as an imaging agent.

[0021] [Figure 3B] FIG. 3B provides a non-limiting example of a two-photon fluorescence image of the brain tumor tissue sample shown in FIG. 3A after processing the image and identifying individual cells within the image.

[0022] [Figure 4A] FIG. 4A provides a non-limiting example of an ex vivo two-photon fluorescence image of biopsied tissue from a patient medicated with 5-ALA as an imaging agent.

[0023] [Figure 4B]FIG. 4B provides a non-limiting example of an ex vivo two-photon fluorescence image of biopsied tissue from a patient to which no contrast agent was administered.

[0024] [Figure 5] FIG. 5 provides a non-limiting example of a two-photon fluorescence emission spectrum for a brain tissue sample dosed with 5-ALA as an imaging agent.

[0025] [Figure 6] FIG. 6 provides a non-limiting example of a one-photon fluorescence emission spectrum (left) acquired for a 5-ALA-dosed tissue sample (image on the right).

[0026] [Figure 7] FIG. 7 provides an energy level diagram and ground state schematic for the vibrational or electronic state transitions that occur for stimulated Raman and two-photon fluorescence processes.

[0027] [Figure 8A] FIG. 8A provides a non-limiting example of a stimulated Raman scattering image of a brain cancer tissue sample.

[0028] [Figure 8B] FIG. 8B provides a non-limiting example of a two-photon fluorescence image showing the distribution of an imaging agent (5-ALA) within the same brain cancer tissue sample depicted in FIG. 8A.

[0029] [Figure 9A] FIG. 9A illustrates the automated detection of nuclei or cells (circles) within the stimulated Raman scattering image of FIG. 8A.

[0030] [Figure 9B] FIG. 9B illustrates automated measurements of fluorescence intensity within defined cell areas (circles) centered on nuclei or cells detected in the image shown in FIG. 8B.

[0031] [Figure 10A]FIG. 10A provides a first diagram of a dual-channel non-descanned detector design with dichroic filters used to separate emission bands and direct the signals towards different photomultiplier tubes (PMTs), which can be individually filtered to detect specific emission bands.

[0032] [Figure 10B] FIG. 10B provides a second view of the dual channel non-descanned detector design illustrated in FIG. 10A. DETAILED DESCRIPTION OF THE INVENTION

[0033] Identification of neoplastic tissue during surgery is important for making appropriate surgical decisions and achieving maximum safe resection in patients. In recent years, intraoperative fluorescent contrast agents such as fluorescein, 5-aminolevulinic acid (5-ALA) (NX Development Corp, Lexington, KY), BLZ-100 (Blaze Biosciences, Inc., Seattle, WA), and LUM015 (Lumicell, Inc., Newton, MA) have gained approval and popularity. These contrast agents are typically given preoperatively (e.g., orally or by injection) and can be visualized using a conventional fluorescence surgical microscope (e.g., Zeiss OPMI Pentero 800 (Carl Zeiss Meditec, Inc. (Dublin, CA)), Leica PROvido-FL560 (Leica Microsystems Inc. (Buffalo Grove, IL)), or Synaptive Modus V (Synaptive Medical (Totono, ON)). Figure 1A provides an example of a visible light image of brain tissue treated with 5-ALA during a surgical procedure to remove a glioma. Figure 1B shows the corresponding fluorescence image. In current practice, surgeons would resect all fluorescent tissue.

[0034] Limitations of this approach, which are well known in the art, include (i) limited sensitivity at the tumor margin, where concentrations of contrast agent accumulation may be low due to a small number of tumor cells, and (ii) limited specificity due to autofluorescent background signal, nonspecific staining, and non-uniform delivery.

[0035] To overcome these limitations, the methods and systems disclosed herein use high-resolution optical imaging of freshly harvested tissue samples, either ex vivo or in vivo, to resolve dye distribution at the cellular or subcellular level in near real time, in the operating room. This approach relies on optical thinning to enable imaging of freshly harvested tissue samples. Suitable high-resolution optical imaging techniques that can be used to visualize fluorescent contrast agents in thick tissue samples without physically thinning the tissue include, but are not limited to, two-photon fluorescence (2P) microscopy, confocal fluorescence microscopy (CM), light sheet microscopy (LSM), and structured illumination microscopy (SIM). Such imaging modalities rely on optical thinning to suppress out-of-focus signals and can have a resolution equal to or better than 10 microns axially and equal to or better than 5 microns laterally, i.e., sufficient resolution to image cellular and subcellular features. An ex vivo approach may be preferred to achieve the best image quality and sensitivity. In vivo approaches may be preferable from a clinical workflow standpoint, as they do not require tissue biopsies.

[0036] Definitions: Unless defined otherwise, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0037] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Any reference to "or" herein is intended to include "and / or" unless stated otherwise.

[0038] As used herein, the term "about" refers to a number plus or minus 10% of that number. When used in the context of a range, the term "about" refers to a range from -10% of the lowest value to +10% of the highest value.

[0039] Tissue Samples: In some cases, the disclosed systems and methods may be used for the characterization of any of a variety of tissue samples known to those of skill in the art. Examples include, but are not limited to, connective tissue, epithelial tissue, muscle tissue, lung tissue, pancreatic tissue, breast tissue, kidney tissue, liver tissue, prostate tissue, thyroid tissue, and neural tissue samples. In some cases, the tissue sample may be derived from any organ or component of a plant or animal. In some cases, the tissue sample may be derived from any organ or other component of the human body, including, but not limited to, the brain, heart, lung, kidney, liver, stomach, bladder, intestine, skeletal muscle, smooth muscle, breast, prostate, pancreas, thyroid, etc. In some cases, the tissue sample may comprise a sample collected from a patient exhibiting an abnormality or disease, such as a tumor or cancer (e.g., sarcoma, carcinoma, glioma, etc.).

[0040] Tissue Sample Collection: Procedures for obtaining tissue samples from individuals are well known in the art. For example, procedures for drawing and processing tissue samples, such as from fine needle aspiration biopsy, core needle biopsy, or surgical biopsy, are well known and can be employed to obtain tissue samples for analysis using the disclosed systems and methods. Typically, for collection of such tissue samples, a thin, hollow needle is inserted into a mass, such as a tumor mass, or the mass is surgically exposed for sampling of tissue, which will be stained and then examined under a microscope.

[0041] Contrast Agent: In some cases, the disclosed methods and systems include the use of an imaging agent to enhance and / or distinguish the appearance of tumorous, benign, and malignant tissue in an image of a tissue sample. As used herein, the terms "imaging agent" and "optical imaging agent" are used interchangeably. As used herein, the terms "fluorescent imaging agent" and "phosphorescent imaging agent" refer to a portion of an optical imaging agent that emits fluorescence or phosphorescence, respectively, when appropriately excited. Examples of fluorescent imaging agents include, but are not limited to, 5-aminolevulinic acid hydrochloride (5-ALA), BLZ-100, and LUM015. In some cases, the disclosed methods and systems include measuring a signal derived from an imaging agent (e.g., a cell-bound imaging agent), which may be a signal generated by the imaging agent itself or its metabolic or processed forms.

[0042] 5-ALA is a compound that is metabolized intracellularly to form the fluorescent prostaglandin IX (PPIX) molecule. Exogenous application of 5-ALA leads to highly selective accumulation of PPIX in tumor cells and epithelial tissues. PPIX is excited with blue light (excitation maxima at approximately 405 nm and 442 nm) and emits in the red (emission maxima at approximately 630 nm and 690 nm) (see, e.g., Wang, et al. (2017), “Enhancement of 5-aminolevulinic acid-based fluorescence detection of side population-defined glioma stem cells by iron chelation,” Nature Scientific Reports 7:42070).

[0043] BLZ-100, which comprises the tumor ligand chlorotoxin (CTX) conjugated to indocyanine green (ICG), has shown potential as a targeted imaging agent for brain tumors (see, e.g., Butte, et al. (2014), "Near-infrared imaging of brain tumors using the Tumor Paint BLZ-100 to achieve near-complete resection of brain tumors," Neurosurg Focus 36(2):E1). BLZ-100 is typically excited in the near-infrared at approximately 780 nm (a broad ICG absorption peak centered at approximately 800 nm), and its broad fluorescence emission spectrum (with an emission maximum at approximately 810 nm to 830 nm) nearly overlaps the absorption spectrum.

[0044] LUM015 is a protease-activated imaging agent that comprises a commercially available fluorescent quencher molecule (QSY® 21) attached to 20-kD polyethylene glycol (PEG) and a cyanine dye 5 (Cy5) fluorophore via a Gly-Gly-Lys-Arg (GGRK) peptide. The intact molecule is optically inactive, but upon proteolytic cleavage by cathepsins K, L, or S, the quencher is released, generating optically active fragments (see, e.g., Whitley, et al. (2016) "A mouse-human phase 1 co-clinical trial of a protease-activated fluorescent probe for imaging cancer," Science Translational Medicine 8(320) pp. 320ra4). The Cy5-labeled fragment has a fluorescence excitation peak at approximately 650 nm and an emission peak at 670 nm.

[0045] The imaging agent may be applied to a tissue sample for ex vivo imaging using any of a variety of techniques known to those of skill in the art, e.g., by applying a solution of the imaging agent to a tissue section as a staining reagent. The imaging agent may be administered to a subject, e.g., a patient, for in vivo imaging by any of a variety of techniques known to those of skill in the art, including, but not limited to, orally, intravenous injection, etc., the choice of technique may depend on the particular imaging agent.

[0046] For any of the imaging methods and systems disclosed herein, the tissue sample may, in some cases, be stained with one or more optical contrast agents, e.g., in some cases, the tissue sample may be stained with one, two, three, four, five or more different optical contrast agents.

[0047] As described, in some cases, the one or more optical imaging agents may comprise a fluorescent imaging agent that emits a fluorescent signal. In some cases, the one or more optical imaging agents may comprise a phosphorescent imaging agent that emits a phosphorescent signal. In some cases, the one or more optical imaging agents may comprise an imaging agent specifically associated with cells in a tissue sample. In some cases, the one or more optical imaging agents may comprise an imaging agent specifically associated with one or more particular types of cells (e.g., "targeted" cells). In some cases, the one or more optical imaging agents may comprise, for example, an antibody conjugated to a fluorophore, a quantum dot, a nanoparticle, or a phosphorescent material. In some cases, the cell-bound imaging agent may comprise a fluorogenic enzyme substrate designed to target cell types that express a particular enzyme.

[0048] Intraoperative Use: In some cases, the disclosed methods and systems may be used intraoperatively, for example, to guide a surgical procedure, to identify a location for performing a biopsy, or to determine whether a resection is complete. In some cases, the disclosed systems may be deployed within an operating room environment to provide real-time or near-real-time in-vivo imaging capabilities and guidance to surgeons performing surgical procedures.

[0049] High-resolution optical slice fluorescence and related imaging microscopy: High-resolution optical imaging techniques suitable for visualizing the distribution of fluorescent contrast agents in thick tissue samples without physically slicing the tissue include, but are not limited to, two-photon fluorescence (2P or TPF) microscopy, three-photon fluorescence (3P) microscopy, confocal fluorescence microscopy (CFM), light-sheet fluorescence microscopy (LSFM), and structured illumination microscopy (SIM). Generally, these techniques rely on confocal optics and / or tight focusing of the excitation laser beam required to stimulate, for example, two-photon fluorescence and other optical processes, to achieve the small depth of field that enables optically sliced ​​imaging of thick tissue samples. Some of these techniques are compatible with other optical emission modes, such as phosphorescence and fluorescence.

[0050] Two-photon fluorescence microscopy: Two-photon (2P) fluorescence microscopy is a fluorescence imaging technique in which the absorption of two excitation photons by a dye molecule triggers an electron transition that results in the emission of a single emission photon with a wavelength shorter than that of the excitation light. Two-photon fluorescence microscopy typically uses near-infrared (NIR) excitation light, which minimizes scattering in tissue samples. The multiphoton absorption process suppresses background signals (due to nonlinear interactions with the tissue sample, which confines the fluorescent excitation primarily to the focal plane) and contributes to increased tissue penetration depth (up to approximately 1 millimeter thick). In addition to deeper tissue penetration, two-photon excitation has the advantages of efficient photodetection and reduced photobleaching (see, e.g., Denk, et al., (1990), "Two-Photon Laser Scanning Fluorescence Microscopy," Science 248:4951:73-76).

[0051] Confocal Fluorescence Microscopy: Confocal fluorescence microscopy (CFM) is an imaging technique that provides three-dimensional optical resolution by actively suppressing laser-induced fluorescence signals arising from out-of-focus planes. This is typically achieved by using a pinhole in front of the detector, where light originating from in-focus planes is imaged by the microscope objective and passes through the pinhole, while light originating from out-of-focus planes is primarily blocked by the pinhole (see, e.g., Combs (2010), “Fluorescence Microscopy: A Concise Guide to Current Imaging Methods”, Curr. Protocols in Neurosci. 50(1):2.1.1-2.1.14; Sanai, et al. (2011), “Intraoperative confocal microscopy in the visualization of 5-aminolevulinic acid fluorescence in low-grade gliomas”, J. Neurosurg. 115(4):740-748; Liu, et al. (2014), “Trends in Fluorescence Image-guided Surgery for Gliomas”, Neurosurgery 75(1):61-71).

[0052] Light Sheet Fluorescence Microscopy: Light sheet fluorescence microscopy (LSFM) uses a plane of light (typically produced by expanding a laser beam and filling a cylindrical lens and / or a slit aperture) to optically slice tissue, allowing for visualization and imaging deep within transmitted tissue with subcellular resolution. Because tissue is exposed to a thin sheet of light, photobleaching and phototoxicity are minimized compared to wide-field fluorescence, confocal, or multiphoton microscopy techniques (see, e.g., Santi (2011), “Light Sheet Fluorescence Microscopy: A Review”, J. of Histochem. & Cytochem. 59(2):129-138; Meza, et al. (2015), “Comparing high-resolution microscopy techniques for potential intraoperative use in guiding low-grade glioma resections”, Lasers in Surg. And Med. 47(4):289-295; Glaser, et al. (2017), “Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens”, Nat Biomed Eng. 1(7):0084).

[0053] Structured illumination fluorescence microscopy: Structured illumination fluorescence microscopy (SIM) is a method of obtaining optical sections in a conventional wide-field microscope by projecting a single spatial frequency grid pattern of excitation light onto a sample. For example, images taken at three spatial positions of the grid are processed to produce optically thinned images similar to those obtained using a confocal microscope (see, e.g., Neil, et al. (1997), "Method of Obtaining Optical Sectioning By Using Structured Light In A Conventional Microscope," Optics Lett. 22(24):1905-1907).

[0054] High-resolution optical thinning non-fluorescence imaging microscopy: High-resolution optical thinning non-fluorescence imaging microscopy suitable for imaging tissue morphology includes, but is not limited to, stimulated Raman scattering (SRS) microscopy, coherent anti-Stokes Raman scattering (CARS) microscopy, confocal reflectance (CR) microscopy, second harmonic generation (SHG) microscopy, and third harmonic generation (THG) microscopy. Generally, these techniques rely on tight focusing of the excitation laser beam required to stimulate SRS, CARS, SHG, or THG scattering or emission to achieve a small depth of field that enables optically thinned imaging of thick tissue samples.

[0055] Stimulated Raman Scattering (SRS) Microscopy: Stimulated Raman Scattering (SRS) microscopy is an imaging technique that provides fast, label-free, high-resolution microscopic imaging of intact tissue samples. SRS microscopy requires two laser pulse trains that are overlapped in time such that the time mismatch is less than the pulse duration (e.g., less than 100 femtoseconds) and spatially overlapped by less than the focal spot size (e.g., less than 100 nm). Imaging of stimulated Raman scattering induced in a sample by paired spatially and temporally synchronized laser pulse trains can be achieved by, for example, detecting the CH vibrations (2,850 cm) of lipid molecules. -1 ) or CH vibrations of protein and nucleic acid molecules (2,930 cm -1 ), enabling mapping of different molecular components in tissue samples based on image acquisition at vibrational frequencies (or wavenumbers) corresponding to the respective wavelengths (e.g., Freudiger, et al. (2008), “Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy”, Science 322:1857-186 and Orringer, et al. al. (2017), “Rapid Intraoperative Histology of Unprocessed Surgical Specimens via Fiber-Laser-Based Stimulated Raman Scattering Microscopy”, Nature Biomed. Eng. 1:0027).

[0056] Coherent anti-Stokes Raman scattering (CARS) microscopy: Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free imaging technique that creates images of structures in a sample, e.g., a tissue sample, by displaying characteristic vibrational contrast arising from the sample's molecular components (see, e.g., Camp, et al. (2014), "High-speed coherent Raman fingerprint imaging of biological tissues," Nat. Photon. 8, 627-634 and Evans, et al. (2007), "Chemically-selective imaging of brain structures with CARS microscopy," Opt. Express. 15, 12076-12087). The technique uses two high-power lasers to illuminate the sample; the frequency of the first laser is typically held constant, while the frequency of the second is adjusted so that the frequency difference between the two lasers is equal to the frequency of the Raman-active mode of interest. CARS is several orders of magnitude more powerful than typical Raman scattering.

[0057] Confocal reflectance (CR) microscopy: Performed using a confocal microscope operating in reflectance mode, rather than fluorescence mode, to take advantage of the optical thinning capabilities of confocal optics, confocal reflectance microscopy can be used to image unstained tissue or tissue labeled with a light-reflecting probe. Near-infrared confocal laser scanning microscopy, for example, uses a relatively low-power laser beam tightly focused onto a specific point in the tissue. Only light backscattered from the focal plane is detected using contrast caused by inherent variations in the refractive index of tissue microstructure (see, e.g., Gonzalez, et al. (2002), "Real-time, in vivo confocal reflectance microscopy of basal cell carcinoma," J. Am. Acad. Dermatol. 47(6):869-874).

[0058] Second Harmonic Generation (SHG) Microscopy: SHG microscopy is a non-fluorescent multiphoton imaging technique that utilizes the nonlinear optical process of second harmonic generation, in which two excitation photons of a given wavelength interact with a material with a non-centrosymmetric structure and are "converted" to form an emission photon with half the wavelength of the excitation light. A laser source focused to a tight focal spot is typically required to excite the second harmonic light, and imaging of the generated light allows for the acquisition of high-resolution optically thinned images of biological tissues, such as those containing collagen fibers (see, for example, Bueno, et al. (2016), "Second Harmonic Generation Microscopy: A Tool for Quantitative Analysis of Tissues," Chapter 5 in Microscopy and Analysis, Stefan Stanciu, Ed., InTech Open).

[0059] Third Harmonic Generation (THG) Microscopy: THG microscopy is also a non-fluorescent multiphoton imaging technique that combines the advantages of label-free imaging with the restriction of signal generation to the focal spot of a scanning laser. Third harmonic generation is a process in which three excitation photons interact with matter to produce a single emission photon with a wavelength one-third that of the excitation light. It enables high-resolution, optically thinned imaging of refractive index mismatches in biological tissues (see, for example, Dietzel (2014), “Third harmonic generation microscopy,” Wiley Analytical Science, Imaging and Microscopy, November 11, 2014).

[0060] Multispectral and / or multimodal imaging methods and systems for qualitative and quantitative detection of cell-bound contrast agents: The disclosed imaging methods and systems involve the use of novel combinations of multispectral and / or multimodal in vivo or ex vivo imaging to detect the distribution of optical contrast agents, such as cell-bound fluorescent contrast agents, in tissue samples and provide qualitative and / or quantitative measures of signals resulting from the optical contrast agents (see, e.g., Yue, et al. (2011), “Multimodal nonlinear optical microscopy,” Laser and Photonics Review 5(4):496-512 for a review of conventional multimodal imaging techniques). The disclosed multispectral and / or multimodal imaging methods and systems provide more accurate quantitative measures of signals resulting from the optical contrast agents by using image interpretation algorithms to correct for background signals (e.g., autofluorescent background and / or hyperfluorescent particles) and resolve the measured signals at the cellular or subcellular level. The unexpected ability to resolve and measure signals arising from cell-bound optical contrast agents at the cellular or subcellular level (due in part to improved detection sensitivity (as described in U.S. Pat. Nos. 8,792,156, 9,104,030, and 9,634,454) and improved image contrast achieved through the use of novel dual-wavelength fiber laser systems) leads to significant improvements in quantification. In some cases, the disclosed multispectral and / or multimode imaging methods and systems enable the generation of pseudocolor images that can be overlaid on images, e.g., two-photon fluorescence and stimulated Raman scattering images, to provide enhanced contrast and / or facilitate direct human interpretation.

[0061] In a first aspect, the disclosed imaging method (and a system configured to perform the method) may include the use of high-resolution optical thin-sectioning microscopy techniques, including, but not limited to, two-photon microscopy, confocal fluorescence microscopy, light sheet microscopy, or structured illumination microscopy, to obtain images at one, two, or more detection wavelengths (or within one, two, or more detection wavelength ranges) using excitation light at one, two, or more excitation wavelengths (or within one, two, or more excitation wavelength ranges). The disclosed imaging method (and a system configured to perform the method) then applies an image interpretation algorithm to detect contrast agent-positive cells based on size, shape, pattern, or intensity, filter out background signal (e.g., highly fluorescent particles) based on size, shape, pattern, or intensity, and provide a quantitative measure of the signal resulting from the cell-bound contrast agent, the quantitative measure of the signal being resolved at the cellular level. Specific examples of this embodiment will be described in further detail below. In some cases, the image interpretation algorithm may provide qualitative and / or quantitative measures derived from signals associated with one or more optical contrast agents. For example, in some cases, the image interpretation algorithm may provide a "cellularity score," e.g., a determination of the number of cells (or cell nuclei). Results may also be presented as density per unit area of ​​the imaged tissue sample. In some cases, the image interpretation algorithm may provide an average signal derived from the cell-bound contrast agent. In some cases, the image interpretation algorithm may provide an average signal for contrast-positive cells after subtracting background signal averaged over the entire image. In some cases, the image interpretation algorithm may provide the number of cells (the signal for which exceeds a specified signal threshold, the signal threshold defining a contrast-positive cell). In some cases, the image interpretation algorithm may provide the percentage of total cells in the image that are contrast-positive. In some cases, the disclosed methods and systems may be used during surgical procedures to identify a location for performing a biopsy or to determine whether a resection is complete.

[0062] In a second aspect, the disclosed method (and a system configured to implement the method) combines a first optical sectioning high-resolution microscopy (e.g., 2P, CFM, LSM, or SIM) imaging modality, i.e., cellular imaging of one or more contrast agents using a first imaging modality, with a second optical sectioning high-resolution microscopy (e.g., SRS, CARS, CR, SHG, or THG) imaging modality, i.e., a second imaging modality, used to image tissue morphology within the same optical focal plane of the tissue sample, independent of the presence of the one or more contrast agents. The instrument then processes the images using an image interpretation algorithm to provide the location and / or area of ​​one or more individual cells based on the images obtained using the second imaging modality, and provides a quantitative measure of the signal resulting from one or more contrast agents in the tissue sample at locations and / or areas corresponding to one or more cells based on the images obtained using the first modality, such that the quantitative measure of the signal is resolved at the cellular level. Specific examples of this embodiment will be described in further detail below. In some cases, the instrument processes the images using an image interpretation algorithm to provide the location and / or size of one or more individual cells based on the images obtained using the first imaging modality, and to provide a quantitative measure of signals resulting from one or more contrast agents in the tissue sample at locations and / or areas corresponding to the one or more cells based on the images obtained using the first modality. In some cases, the quantitative measure may be derived from signals resulting from one or more contrast agents at locations and / or areas corresponding to the one or more cells, or from signals, e.g., SRS signals, derived from images obtained using a second imaging modality at locations and / or areas corresponding to the one or more cells, or from a combination of both signals resulting from the one or more contrast agents and signals derived from images obtained using the second imaging modality at locations and / or areas corresponding to the one or more cells. In some cases, the image interpretation algorithm may provide qualitative and / or quantitative measures derived from signals associated with one or more optical contrast agents.For example, in some cases, the image interpretation algorithm may provide (i) a "cellularity score" as discussed above, (ii) the average signal derived from the cell-bound contrast agent, (iii) the average signal for contrast-positive cells after subtraction of background signal averaged over the entire image, (iv) the number of cells whose signal exceeds a defined signal threshold that defines contrast-positive cells, (v) the percentage of total cells in the image that are contrast-positive, or any combination thereof. In some cases, the disclosed methods and systems may be used during surgical procedures to identify a location for performing a biopsy or to determine whether a resection is complete.

[0063] In a third aspect, the imaging system may be, for example, a multichannel imaging microscope that simultaneously acquires a first image at the emission wavelength of the contrast agent and a second image outside the emission wavelength of the contrast agent, and then provides the multichannel emission signals to an image interpretation algorithm that detects cells in the first image and suppresses nonspecific background signals (e.g., autofluorescent background or hyperfluorescent particles) based on the measured spectral characteristics (e.g., by ratioing or thresholding the first image using signal data acquired in the second image (e.g., at locations corresponding to one or more cells detected in the first image), or otherwise correcting the signals measured in the first image by background values ​​determined from the second image). In some cases, a multicolor image can be generated based on the application of a pseudocolor algorithm to the multichannel image data (e.g., by assigning signals associated with the contrast agent red and background signals green), simplifying human interpretation of the image. The latter approach may avoid the need for a computer-assisted interpretation algorithm. Examples of this embodiment of the disclosed imaging method and system will be described in further detail below.

[0064] Image Acquisition Parameters: For any of the imaging methods and systems disclosed herein, images of a tissue sample may be acquired using various image acquisition parameter settings, including, but not limited to, effective image resolution, number of excitation wavelengths used, number of emission wavelengths at which images are acquired, number of images acquired over a defined period of time, and number of different imaging modalities used to acquire the images, and the images are then processed and / or combined to (i) generate multi-color or enhanced contrast images that facilitate image interpretation and identification of neoplastic tissue, for example, if present in the tissue sample, and / or (ii) generate quantitative measures based on signals derived from one or more cell-bound contrast agents and / or signals derived from a second imaging modality, e.g., a non-fluorescent imaging modality such as SRS.

[0065] In some cases, the disclosed imaging systems may comprise a laser scanning system, e.g., a system in which an image is obtained in two dimensions by scanning or rastering a laser spot across the optical focal plane of the imaging system, and emitted or scattered light, e.g., two-photon fluorescence or stimulated Raman scattered light, is directed through an optical system to one or more photodetectors, e.g., photomultipliers, avalanche photodiodes, solid-state near-infrared detectors, etc. In some cases, when the imaging system comprises two or more photodetectors, the two or more detectors may be of the same type or different types. In some cases, the two or more detectors of different types may differ in terms of size (diameter or cross-sectional area), integration time, signal-to-noise ratio, sensitivity, etc.

[0066] In some cases, the disclosed imaging systems may include laser scanning systems that utilize one or more image sensors or cameras. For example, in some cases, the disclosed imaging systems may include one, two, three, four, or five or more image sensors or cameras. In some cases, for example, when an imaging system includes two or more image sensors or cameras, the image sensors or cameras may be the same or may differ in terms of pixel size, pixel count, dark current, signal-to-noise ratio, detection sensitivity, etc. Images so obtained by the two or more image sensors or cameras may therefore have different image resolutions. In some cases, the one or more image sensors may have a pixel count of approximately 0.5 megapixels, 1 megapixel, 2 megapixels, 4 megapixels, 6 megapixels, 8 megapixels, 10 megapixels, 20 megapixels, 50 megapixels, 80 megapixels, 100 megapixels, 200 megapixels, 500 megapixels, or 1,000 megapixels (or any pixel count within a range spanning these values). In some cases, the size of a pixel in a given image sensor may be approximately 20 μm, 10 μm, 5 μm, 3.5 μm, 2 μm, 1 μm, 0.5 μm, or 0.1 μm (or any pixel size within a range spanning these values). In some cases, one or more image sensors or cameras may be configured to bin groups of individual pixels to vary the effective resolution of the images so obtained.

[0067] In some cases, the disclosed imaging systems (either scanning systems or image sensor-based systems) can be configured to acquire a single image for each of one or more defined excitation wavelengths, emission wavelengths, and / or imaging modalities. In some cases, the disclosed systems can be configured to acquire 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 images (or any number of images within this range) for each of one or more defined excitation wavelengths, emission wavelengths, and / or imaging modalities. In some cases, the disclosed systems can be configured to acquire video data for each of one or more defined excitation wavelengths, emission wavelengths, and / or imaging modalities.

[0068] In some cases, the disclosed imaging systems can be configured to acquire one or more images within a specified time period for each of one or more defined excitation wavelengths, emission wavelengths, and / or imaging modalities. For example, in some cases, the disclosed systems can acquire one or more images within a specified time period for each of one or more defined excitation wavelengths, emission wavelengths, and / or imaging modalities. For example, in some cases, the disclosed systems can acquire one or more images within a specified time period for each of one or more defined excitation wavelengths, emission wavelengths, and / or imaging modalities. One or more images may be configured to be acquired every millisecond, 900 milliseconds, 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours (or any period within this range). In some cases, the disclosed systems may be configured to acquire video data for each of one or more defined excitation wavelengths, emission wavelengths, and / or imaging modalities.

[0069] In some cases, the disclosed imaging systems may be configured to acquire images using exposure times (or integration times or image capture times) of about 1 millisecond, 5 milliseconds, 10 milliseconds, 25 milliseconds, 50 milliseconds, 75 milliseconds, 100 milliseconds, 250 milliseconds, 500 milliseconds, 750 milliseconds, 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, or more than 60 seconds (or any exposure time, image capture time, or integration time in a range spanning these values).

[0070] In some cases, including but not limited to those where two-photon fluorescence imaging is utilized, images may be obtained using one, two, three, four, or five or more different excitation wavelengths (or wavelength ranges). In some cases, images may be obtained using wavelengths of about 360 nm, 380 nm, 400 nm, 420 nm, 440 nm, 460 nm, 480 nm, 500 nm, 520 nm, 540 nm, 560 nm, 580 nm, 600 nm, 620 nm, 640 nm, 660 nm, 680 nm, 700 nm, 720 nm, 740 nm, 760 nm, 780 nm, 800 nm, 820 nm, 840 nm, 860 nm, 880 nm, 900 nm, 920 nm, 940 nm, 960 nm, 9 ... The excitation wavelengths may be obtained using excitation light at wavelengths of 0 nm, 920 nm, 940 nm, 960 nm, 980 nm, 1000 nm, 1020 nm, 1040 nm, 1060 nm, 1080 nm, 1100 nm, 1120 nm, 1140 nm, 1160 nm, 1180 nm, or 1200 nm, and the excitation wavelengths used will typically be selected based on the choice of optical contrast agent used to stain the tissue sample. In some cases, light at one, two, three, four, or five or more excitation wavelengths may be provided by one, two, three, four, or five or more lasers or other light sources. In some cases, the excitation wavelengths (or wavelength ranges) may be selected using optical glass filters, bandpass filters, interference filters, long-band filters, short-band filters, dichroic reflectors, monochromators, or any combination thereof.

[0071] In some cases, including but not limited to those where two-photon fluorescence imaging is utilized, images may be obtained for one, two, three, four, five, or more different emission (or detection) wavelengths (or emission (or detection) wavelength ranges). In some cases, images may be obtained for wavelengths of about 360 nm, 380 nm, 400 nm, 420 nm, 440 nm, 460 nm, 480 nm, 500 nm, 520 nm, 540 nm, 560 nm, 580 nm, 600 nm, 620 nm, 640 nm, 660 nm, 680 nm, 700 nm, 720 nm, 740 nm, 760 nm, 780 nm, 800 nm, 820 nm, 840 nm, 860 nm, 880 nm, 900 nm, The detection (emission) wavelength used may be obtained for light emitted at 920 nm, 940 nm, 960 nm, 980 nm, 1000 nm, 1020 nm, 1040 nm, 1060 nm, 1080 nm, 1100 nm, 1120 nm, 1140 nm, 1160 nm, 1180 nm, or 1200 nm, and the detection (emission) wavelength used will typically be selected based on the choice of optical contrast agent used to stain the tissue sample. In some cases, the emission (or detection) wavelength (or emission (or detection) wavelength range) may be selected using optical glass filters, bandpass filters, interference filters, long-band filters, short-band filters, dichroic reflectors, monochromators, or any combination thereof.

[0072] In some cases, images may be obtained using excitation and / or emission (detection) wavelength ranges having a bandwidth of about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, or greater than 100 nm. In some cases, the bandpass for the excitation and / or emission wavelength ranges may be selected using optical glass filters, bandpass filters, interference filters, long-band filters, short-band filters, dichroic reflectors, monochromators, or any combination thereof.

[0073] In some cases, images obtained by the disclosed imaging systems may have a lateral resolution of less than 20 μm, 15 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, 0.5 μm, or 0.25 μm. In some cases, the lateral resolution of the disclosed imaging systems may be limited by the size of the focused laser spot.

[0074] In some cases, images obtained by the disclosed imaging systems may have an axial resolution of less than 50 μm, 20 μm, 15 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, or 0.5 μm. In some cases, the axial resolution of the disclosed imaging systems may be limited by the size of the focused laser spot. In some cases, the axial resolution of the disclosed imaging systems may be limited by the diameter of the pinhole aperture in a confocal optical system.

[0075] In some cases, the disclosed imaging systems are configured to obtain images of a tissue sample at the same focal plane for two or more imaging modalities. In some cases, the focal planes for two different imaging modalities may be considered "the same" (or coplanar) if they are offset from one another by less than 10 μm, less than 9 μm, less than 8 μm, less than 7 μm, less than 6 μm, less than 5 μm, less than 4 μm, less than 3 μm, less than 2 μm, less than 1 μm, less than 0.5 μm, or less than 0.25 μm.

[0076] In some cases, including but not limited to those where SRS imaging is utilized, images may be obtained at one or more selected wavenumbers (or wavenumber spectral ranges) corresponding to Raman shifts for different chemical groups. In some cases, images may be obtained at one, two, three, four, five, or more different wavenumbers (or wavenumber spectral ranges). Examples of wavenumber ranges corresponding to Raman shifts for particular chemical groups include, but are not limited to, those listed in Table 1. [Table 1]

[0077] In some cases, including but not limited to those where SRS imaging is utilized, the image is taken at a depth of approximately 100 cm. -1 ~Approx. 3,000cm -1 In some cases, the image may be obtained in a spectral range spanning at least 100 cm -1 , at least 125cm -1 , at least 150cm -1 , at least 200cm -1 , at least 250cm -1 , at least 300cm -1 , at least 350cm -1 , at least 400cm -1 , at least 450cm -1 , at least 500 cm -1 , at least 550cm -1 , at least 600 cm -1 , at least 650cm -1 , at least 700 cm -1 , at least 750cm -1 , at least 800 cm -1 , at least 900 cm -1 , at least 1,000 cm -1 , at least 1,100 cm -1 , at least 1,200 cm -1 , at least 1,300 cm -1 , at least 1,400 cm -1 , at least 1,500 cm -1 , at least 1,750 cm -1 , at least 2,000 cm -1 , at least 2,250 cm -1 , at least 2,500 cm -1 , at least 2,750 cm -1 , or at least 3,000 cm -1 In some cases, images can be obtained in a spectral range spanning up to 3,000 cm -1 , maximum 2,750 cm -1, up to 2,500 cm -1 , maximum 2,250 cm -1 , up to 2,000 cm -1 , up to 1,750 cm -1 , up to 1,500 cm -1 , up to 1,400 cm -1 , up to 1,300 cm -1 , up to 1,200 cm -1 , up to 1,100 cm -1 , up to 1,000 cm -1 , up to 900cm -1 , up to 800cm -1 , up to 750cm -1 , up to 700cm -1 , maximum 650cm -1 , up to 600cm -1 , maximum 550cm -1 , up to 500cm -1 , up to 450cm -1 , up to 400cm -1 , maximum 350cm -1 , up to 300cm -1 , up to 250cm -1 , up to 200cm -1 , or up to 150cm -1 In some cases, images can be obtained in a spectral range spanning, for example, about 250 cm -1 Those skilled in the art will appreciate that an image can be obtained using a spectral range that falls anywhere within any range bounded by any of these values ​​(e.g., about 200 cm). -1 Range: approx. 760cm -1 It will be understood that the invention may be obtained within the range of

[0078] Multispectral and / or multimode imaging system components: For any of the embodiments described herein, the disclosed imaging systems may include one or more excitation light sources (e.g., solid-state lasers, fiber lasers, etc.), one or more image sensors or photodetectors (e.g., photomultipliers, avalanche photodiodes, solid-state near-infrared detectors, charge-coupled device (CCD) sensors or cameras, CMOS image sensors or cameras, etc.), one or more scanning mirrors or translation stages, additional optical components (e.g., objective lenses, additional lenses, mirrors, prisms, optical filters, colored glass filters, narrow-band interference filters, broad-band interference filters, dichroic reflectors, diffraction gratings, monochromators, apertures, optical fibers, optical waveguides, etc., or any combination thereof, used to collimate, focus, and / or image the excitation and / or emission light beams). In some cases, the disclosed imaging systems may include one, two, three, four, or five or more lasers providing excitation light at one, two, three, four, or five or more excitation wavelengths. In some cases, excitation light at one, two, three, four, or more excitation wavelengths can be delivered to the optical focal plane through an objective lens used to image the tissue sample, for example, by using an appropriate combination of mirrors, dichroic reflectors, or beam splitters. In some cases, excitation light at one, two, three, four, or more excitation wavelengths can be delivered to the optical focal plane using an optical path that does not include the objective lens used to image the sample. In some cases, the disclosed imaging systems are configured to obtain images for two or more imaging modalities from the same optical plane (or focal plane) in the tissue sample. In some cases, the disclosed imaging systems are configured to obtain images for two or more imaging modalities for the same field of view in the tissue sample. In some cases, the disclosed imaging systems may include one or more processors or computers, as will be discussed in further detail below.In some cases, an instrument designed to acquire images using a first imaging modality, e.g., SRS imaging or 2P imaging, may be modified to allow for simultaneous or sequential acquisition of images using a second imaging modality, e.g., 2P imaging or SRS imaging, respectively.

[0079] A non-limiting example of a stimulated Raman scattering (SRS) imaging system is described in Orringer, et al. (2017), “Rapid intraoperative histology of unprocessed surgical specimens via fiber-laser-based stimulated Raman scattering microscopy,” Nature Biomed. Eng. 1:0027. The fully integrated SRS imaging system comprises five main components: (1) a fiber-coupled microscope with a motorized stage, (2) a dual-wavelength fiber-laser module, (3) a laser control module, (4) a microscope control module, and (5) a computer for image acquisition, display, and processing. The dual-wavelength fiber-laser design exploits the fact that the frequency difference between the two main fiber gain media, i.e., erbium and ytterbium, overlaps with the high-wavenumber region of the Raman spectrum. The two synchronized narrowband laser pulse trains required for SRS imaging were generated by narrowband filtering of a broadband supercontinuum derived from a single fiber oscillator, followed by amplification in the respective gain media. The development of an all-fiber system based on polarization-maintaining components provides greater laser stability than previous non-polarization-maintaining implementations. To enable high-speed diagnostic-quality imaging (e.g., 1-megapixel images acquired in approximately 2 seconds per wavelength) with signal-to-noise ratios comparable to those achievable using solid-state lasers, the laser output power was scaled to approximately 120 mW for the fixed-wavelength 790 nm pump beam and approximately 150 mW for the tunable Stokes beam over the entire tuning range of 1,010 to 1,040 nm at a 40 MHz laser pulse repetition rate and a 2 picosecond transform-limited laser pulse duration. Custom laser controller electronics were developed to tightly control the operating settings of the laser system using a microcontroller. A noise cancellation scheme based on auto-balanced detection was used to further improve image quality. In this noise cancellation scheme, a portion of the laser beam is sampled to provide a measure of laser noise, which can then be subtracted in real time.In some cases, systems such as the described SRS imaging systems can be modified to obtain, for example, two-photon fluorescence images simultaneously or sequentially by providing at least one additional excitation laser of an appropriate wavelength and at least one additional image sensor or camera, and the at least one additional excitation beam and emitted two-photon fluorescence are combined with the SRS imaging system using a combination of dichroic reflectors, beam splitters, etc.

[0080] Image Processing and Image Interpretation: In some cases, the disclosed imaging methods may include the use of image processing and / or image interpretation algorithms to process acquired images and provide qualitative and / or quantitative measures derived from signals associated with one or more cell-bound contrast agents and / or signals derived from non-fluorescence imaging modalities. In some cases, the image processing and / or image interpretation algorithms may process images acquired using a first imaging modality, e.g., a first optical thinning imaging modality, to identify cells and determine their location. In some cases, the image processing and / or image interpretation algorithms may process images acquired using a second imaging modality, different from the first, e.g., a second optical thinning imaging modality, to identify cells and determine their location. In some cases, the image processing and / or image interpretation algorithms may process images acquired using one or both of the first and second imaging modalities to identify cells and determine their location.

[0081] In some cases, the image interpretation algorithm may comprise any of a variety of conventional image processing algorithms known to those skilled in the art. Examples include, but are not limited to, Canny edge detection, Canny-Delicia edge detection, first-order gradient edge detection (e.g., Sobel operator), second-order derivative edge detection, phase coherence edge detection, other image segmentation algorithms (e.g., intensity thresholding, intensity clustering, intensity histogram-based methods, etc.), feature and pattern recognition algorithms (e.g., generalized Hough transform for detecting arbitrary shapes, circle Hough transform, etc.), and mathematical analysis algorithms (e.g., Fourier transform, fast Fourier transform, wavelet analysis, autocorrelation, etc.), or any combination thereof. In some cases, such image processing algorithms may be used to detect individual cells within an image based on, for example, feature size, shape, pattern, intensity, or any combination thereof.

[0082] In some cases, the image interpretation algorithm may comprise an artificial intelligence or machine learning algorithm trained to, for example, further refine the capabilities of the image processing and / or image interpretation algorithm, identify individual cells within an image, and / or distinguish between normal and non-normal tissue (e.g., neoplastic tissue) based on qualitative and / or quantitative measures derived from signals derived from one or more contrast agents and / or signals derived from non-fluorescence imaging modalities. Any of a variety of machine learning algorithms may be used in implementing the disclosed methods and systems. Examples include, but are not limited to, supervised learning algorithms, unsupervised learning algorithms, semi-supervised learning algorithms, deep learning algorithms, or any combination thereof. In some cases, the machine learning algorithm may comprise an artificial neural network algorithm, a deep convolutional neural network algorithm, a deep recurrent neural network, a generative adversarial network, a support vector machine, a hierarchical clustering algorithm, a Gaussian process regression algorithm, a decision tree algorithm, a logistic model tree algorithm, a random forest algorithm, a fuzzy classifier algorithm, a k-means algorithm, an expectation maximization algorithm, a fuzzy clustering algorithm, or any combination thereof.

[0083] As a non-limiting example, in some cases, machine learning algorithms used to further refine the capabilities of image processing and / or image interpretation algorithms, identify individual cells in images, and / or distinguish between normal and non-normal tissue (e.g., tumorous tissue), may comprise artificial neural networks (ANNs), e.g., deep learning algorithms. Artificial neural networks generally comprise an interconnected group of "nodes" (or "neurons") organized into multiple layers. A typical ANN architecture may comprise an input layer, at least one or more hidden layers, and an output layer. ANNs may comprise any total number of layers and any number of hidden layers, with the hidden layers functioning as trainable feature extractors that enable the mapping of sets of input data to output values ​​or sets of output values. As described, each layer of a neural network comprises multiple nodes. A node receives inputs resulting from either direct input data (e.g., raw image data and / or preprocessed image data) or the outputs of nodes in previous layers, and performs a particular operation, e.g., a summation operation. In some cases, connections from inputs to nodes are associated with weights (or weight coefficients). In some cases, for example, a node receives input x i and its associated weight w i The weighted sum may be summed over all pairs of products of . In some cases, the weighted sum is offset by a bias b. In some cases, the output of the neuron may be gated using a threshold or an activation function f, which may be a linear or nonlinear function. The activation function may be, for example, a rectified linear unit (ReLU) activation function or other functions, such as saturated bipolar tangent, identity, binary step, logistic, arctangent, soft sine, parametric rectified linear unit, exponential linear unit, soft plus, bent identity, soft exponential, sinusoidal, sine, Gaussian, or sigmoid function, or any combination thereof.

[0084] The weighting coefficients, bias values, and thresholds, or other calculated parameters of the neural network, can be "taught" or "learned" in a training phase using one or more sets of training data. For example, the parameters can be trained using input data (e.g., raw image data and / or preprocessed image data) from a training dataset and gradient descent or backpropagation techniques, such that the output value calculated by the ANN (e.g., the classification of a given tissue sample as comprising neoplastic tissue) is consistent with the examples contained in the training dataset.

[0085] In some cases, the machine learning algorithm may be trained using one or more training datasets comprising, for example, imaging data obtained for archived histopathological tissue samples (e.g., formalin-fixed or freshly frozen tissue samples), imaging data obtained for freshly collected histopathological tissue samples, or any combination thereof. In some cases, the training dataset may be continuously, periodically, or randomly updated with imaging data obtained by two or more systems deployed for use in the same or different facilities. In some cases, the two or more systems are deployed in different facilities, and the training dataset optionally resides in a cloud-based database and / or is continuously, periodically, or randomly updated via an internet connection.

[0086] As described, in some cases, the image interpretation algorithm provides a "cellularity score," e.g., a determination of the number of cells (or cell nuclei) identified per unit area of ​​the imaged sample. In some cases, the image interpretation algorithm provides an average signal derived from the cell-bound contrast agent. In some cases, the image interpretation algorithm provides an average signal for contrast-positive cells after subtracting background signal averaged over the entire image. In some cases, the image interpretation algorithm provides the number of cells (whose signal exceeds a specified signal threshold that defines a contrast-positive cell). In some cases, the image interpretation algorithm provides the percentage of total cells in the image that are contrast-positive. As mentioned above, in some cases, the image interpretation algorithm may be configured to generate a quantitative measure based on signals derived from one or more cell-bound contrast agents and / or signals derived from a second imaging modality, e.g., a non-fluorescence imaging modality such as SRS or one of the other non-fluorescence imaging modalities described herein.

[0087] In some cases, the imaging methods and systems of the present disclosure may comprise the use of a pseudocolor algorithm to transform images obtained using any of one or more imaging modalities, for example, to provide enhanced contrast for tissue structures (e.g., to provide enhanced detection of neoplastic tissue) and / or to generate multicolor images that facilitate human interpretation of the images. In some cases, the use of a pseudocolor algorithm to transform images obtained using any of one or more imaging modalities may facilitate human interpretation of the images without the need to implement additional image interpretation algorithms. In some cases, the pseudocolor images generated from images obtained using any of one or more imaging modalities may then be combined (e.g., subjected to linear or nonlinear algebraic operations), for example, to provide enhanced contrast for tissue structures (e.g., to provide enhanced detection of neoplastic tissue) and / or to facilitate human interpretation of the images.

[0088] Software and Computer-Readable Media: Various aspects of the disclosed algorithms (or computer-implemented methods) may be considered as a "product" or "article of manufacture," e.g., a "computer program or software product," typically in the form of processor-executable code and / or associated data stored in some type of computer-readable medium, the processor-executable code comprising a plurality of instructions for controlling a computer or computer system in performing one or more of the methods disclosed herein. Thus, disclosed herein is a computer-readable medium (a "computer program or software product") comprising a set of encoded instructions (i.e., software) that, when executed by a processor, causes the processor to perform a series of logical steps, directing the processor to perform any of the methods disclosed herein. For example, disclosed herein is a computer-readable medium ("computer program or software product") having a set of encoded instructions (i.e., software) that, when executed by a processor, causes the processor to perform a series of logical steps to (i) acquire images using one or more of the imaging modalities disclosed herein; (ii) perform manual, semi-automated, or automated processing of the acquired images to identify one or more individual cells therein; (iii) perform manual, semi-automated, or automated further processing of the acquired images to extract a quantitative measure of a signal derived from the cell-bound contrast agent at the location of one or more individual cells; (iv) perform manual, semi-automated, or automated further processing of the acquired images to extract a quantitative measure of a signal derived from a non-fluorescent image of the same tissue sample at the location of one or more individual cells; or (v) any combination thereof.

[0089] The processor-executable (or machine-executable) code may be stored, for example, in an optical storage unit comprising an optically readable medium, e.g., an optical disk, CD-ROM, DVD, or Blu-Ray® disk. The processor-executable code may be stored in an electronic storage unit, e.g., a memory (e.g., read-only memory, random access memory, flash memory) or on a hard disk. A "storage" type medium includes any or all of the tangible memory of a computer, computer system, or the like, or its associated modules, e.g., various semiconductor memory chips, optical drives, tape drives, disk drives, and the like, which may provide non-transitory storage from time to time for software encoding the methods and algorithms disclosed herein.

[0090] All or part of the software code may be communicated from time to time via the Internet or various other telecommunications networks. Such communication may, for example, enable loading of software from one computer or processor to another, such as from an administrative server or host computer to an application server computer platform. Accordingly, other types of media that may be used to convey software-encoded instructions include optical, electrical, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical landline networks, and via various ambient telecommunications links. Physical elements that carry such waves, such as wired or wireless links, optical links, or the like, are also considered media that convey software-encoded instructions for implementing the methods disclosed herein.

[0091] Computer Processor: In some cases, the disclosed imaging systems may include one or more processors or computers that are individually or collectively programmed to provide instrument control and / or image processing functionality in accordance with the methods disclosed herein. The one or more processors may comprise a hardware processor, such as a central processing unit (CPU), a graphics processing unit (GPU), a general-purpose processing unit, or a computing platform. The one or more processors may comprise any of a variety of suitable integrated circuits (e.g., application-specific integrated circuits (ASICs) or field-programmable gate arrays (FPGAs)), microprocessors, new next-generation microprocessor designs (e.g., memristor-based processors), logic devices, etc. Although the present disclosure is described with reference to processors, other types of integrated circuits and logic devices may also be applicable. The processor may have any suitable data operation capability. For example, the processor may perform 512-bit, 256-bit, 128-bit, 64-bit, 32-bit, or 16-bit data operations. The one or more processors may be single-core or multi-core processors, or multiple processors configured for parallel processing.

[0092] In some cases, one or more processors or computers used to implement the disclosed imaging methods may be part of a larger computer system and / or may be operably coupled to a computer network ("network") using a communication interface to facilitate data transmission and sharing. The network may be a local area network, an intranet and / or an extranet, an intranet and / or an extranet in communication with the Internet, or the Internet. In some cases, the network is a telecommunications and / or data network. The network may include one or more computer servers, which in some cases enables distributed computing such as cloud computing. In some cases, the network may implement a peer-to-peer network using a computer system, which may allow devices coupled to the computer system to act as clients or servers.

[0093] In some cases, the disclosed imaging systems may also include memory or memory locations (e.g., random access memory, read-only memory, flash memory, etc.), electronic storage units (e.g., hard disks), communication interfaces (e.g., network adapters) for communicating with one or more other imaging systems, and / or peripheral devices such as data storage devices and / or electronic display adapters.

[0094] In some cases, the data storage unit stores files, e.g., drivers, libraries, and saved programs. The storage unit may also store user data, e.g., user-defined preferences, user-defined programs, and image data obtained during testing or use of the disclosed imaging system. The computer system or network may, in some cases, include one or more additional data storage units external to the computer system, such as a data storage unit located on a remote server that communicates with the computer system through an intranet or the Internet. (Example)

[0095] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein. Example 1: Two-photon fluorescence imaging of brain tissue administered with 5-ALA

[0096] We imaged an ex vivo sample of brain tumor tissue treated with 5-ALA as a contrast agent using the 2P imaging mode of the combined 2P / SRS microscope illustrated in Figure 2, with two excitation beams at wavelengths of 790 nm and 1020 nm and a 640 nm / 80 nm bandpass detection filter (Figure 3A). Figure 3B shows the same two-photon fluorescence image of the brain tumor tissue sample shown in Figure 3A after processing the image and identifying individual cells (circles) in the image. To our knowledge, this is the first time that individual cancer cells have been visualized in tissue using 5-ALA, marking a breakthrough in detection sensitivity. We were able to analyze individual cells and found that the contrast agent accumulated within the cell cytoplasm and did not penetrate into the cell nucleus.

[0097] We also found that individual highly fluorescent particles could be seen (Fig. 3A). Surprisingly, these were still visible in control samples from tissues that had not been dosed with 5-ALA and were therefore due to autofluorescent background.

[0098] When we extended our evaluation to in vivo imaging of human tissue samples (e.g., Figure 4A; brain tissue from a patient administered 5-ALA) using our two-photon microscope, we experienced a similar unexpected nonspecificity of the contrast mechanism, in that a 640 nm fluorescent signal was also detected in tissue samples from patients not receiving the 5-ALA contrast agent (Figure 4B). Specifically, it can be seen from Figure 4B that the cell-associated fluorescent signal and general "opacity" visible in Figure 4A is not visible in patients not receiving the contrast agent, although fluorescent particles are visible in both cases.

[0099] This was surprising because this nonspecificity had not been disclosed in previous studies of imaging contrast using conventional (one-photon fluorescence) surgical microscopes. In fact, the US FDA approved this contrast agent for clinical use based on data demonstrating its excellent sensitivity. We hypothesize that such nonspecificity may be due to the nature of two-photon excitation, which is known to be less specific. We investigated the two-photon emission spectra of 5-ALA-dosed brain tumor tissue samples and found that the characteristic spectral peak of 5-ALA (620 nm–650 nm) indeed resides on top of a large and spectrally broad background signal (Figure 5).

[0100] We therefore set up a one-photon confocal fluorescence microscope to image the sample. When the sample was imaged using an open pinhole, i.e., without significant optical thinning (Figure 6, right), we regained the excellent specificity of the contrast agent; the emission peak was visible without a large, spectrally broad background (Figure 6, left). However, when we closed the pinhole to generate the optical thinning (i.e., reduced depth of field) required for imaging single cells within thick, unsectioned tissue samples, this spectral specificity was lost, and only spectrally broad particles were visible, as observed in the two-photon images. We hypothesized that this could be due to the increased photobleaching found with one-photon excitation in the focal plane, where the photon flux is highest and the contrast agent is bleached much faster compared to the background signal. Thus, cellular imaging of contrast agents (such as 5-ALA) using 2P imaging, as opposed to macroscopic imaging (e.g., using a conventional surgical microscope), faces the dilemma that 2P imaging may offer sensitivity for imaging single contrast agent-positive cells but is inherently less specific, while 1P imaging suffers from photobleaching at the focal plane and therefore suffers from limited sensitivity or reduced imaging speed. In either case, measurements of fluorescence intensity averaged over the image will not provide an accurate readout of the tumor burden within the image. Example 2: Dual-mode imaging for increased sensitivity and specificity

[0101] This example combines SRS microscopy for imaging tissue morphology with 2P microscopy for imaging the distribution of a fluorescent contrast agent (Figure 2). A dual-wavelength fiber laser system (e.g., producing light at 1020 nm and 790 nm) was used to excite the sample and detect the SRS signal in transmission using a photodetector (PD). After blocking the excitation light using a filter, we also detect the 2P signal in reflection using a second photodetector (e.g., a photomultiplier (PMT)). Images were obtained by scanning the laser focus through the sample point-by-point using a computer-controlled galvanometer scanning mirror. With such a multimode microscope, both SRS and 2P images can be acquired simultaneously.

[0102] Energy level diagrams for the two techniques are shown in Figure 7. In SRS, molecules are excited from the ground state to a vibrational state upon stimulated excitation by a pump photon (Figure 7, left, up arrow) and a Stokes photon (Figure 7, left, down arrow) when the energy difference matches that of the molecular vibration. In 2P, the electronic state of the contrast agent is excited by the simultaneous absorption of two photons (e.g., two pump photons, or two Stokes photons, or one pump and one Stokes photon) (Figure 7, right, up arrow), and the molecule subsequently returns to the ground state while emitting a fluorescence photon that can be detected (Figure 7, right, down arrow).

[0103] The image in Figure 8A shows the CH vibrational frequencies of lipids (e.g., 2850 cm -1 ) shows an SRS image of a brain cancer tissue sample at 1000 Hz. At this wavenumber, the image has a positive signal for the cytoplasm, while the intercellular spaces and nuclei are dark (due to the lack of lipids). This contrast can be used to positively identify cells based on nuclear identification, for example, using information including the size, shape, pattern, and / or signal intensity of features in the image. The CH vibrational frequencies of proteins and nucleic acids (e.g., 2930 cm) are also used. -1It may be advantageous to image the same tissue in a 3D CT image (Figure 1B) to provide additional contrast for the nuclei. The SRS imaging channel is independent of the contrast agent (2P fluorescence) imaging channel. Other label-free approaches to imaging, such as CARS, CR, or THG, can also be used as alternative imaging modalities to SRS.

[0104] The image in Figure 8B shows a 2P fluorescence microscopy image of the distribution of 5-ALA imaging agent obtained as discussed above at the same location as Figure 8A. Comparison of the two images shows that some, but not all, of the cells identified based on the SRS image also have a positive cell signal in the fluorescence channel.

[0105] The imaging system can use automated image processing to determine the location and / or size of nuclei or cells from SRS images (Figure 9A, circle) and measure the fluorescence intensity within individual cell areas (Figure 9B, circle), providing a measure of the fluorescent signal resulting from the cell-bound contrast agent. It is also possible to determine whether the fluorescent signal within a selected area exceeds a defined threshold level and provide a binary output of whether a particular cell is positive or negative for the contrast agent. This allows for the determination of a quantitative measure of the percentage of contrast agent-positive cells within the image.

[0106] In some cases, the disclosed imaging system is -1 and 2,930 cm -1In addition to these images, the disclosed imaging system can provide high-resolution optically sliced ​​Raman images at both wavelengths. In addition to these images, the disclosed imaging system can provide high-resolution optically sliced ​​images of one or more fluorescence channels (emission wavelength ranges). Using the Raman data, an image interpretation algorithm can spatially identify cellular characteristics and determine a cellularity score. The combination of these measurements and the data with the fluorescence channels as input to the image interpretation algorithm generates a set of metrics that correlate the fluorescence and Raman data. Examples of these metrics include, but are not limited to, the number of identified cells, the number of cells associated with positive fluorescence, the percentage of cells associated with positive fluorescence, the ratio of positive fluorescent cells to negative fluorescent cells, and the ratio of positive fluorescent cells to total cells, or any combination thereof. Example 3 (Dual wavelength fluorescence imaging)

[0107] In this example, the imaging system is a multi-channel 2P microscope that simultaneously acquires a first fluorescence image at the emission wavelength of the contrast agent (e.g., 640 nm ± 40 nm for 5-ALA) and a second fluorescence image outside the emission wavelength of the contrast agent (e.g., < 600 nm for 5-ALA) as a measure of non-specific background. The multi-channel emission signals can then be provided to an image processing or interpretation algorithm to suppress the non-specific background signal (e.g., by ratio or thresholding) based on the measured spectral characteristics of the contrast agent-specific and background signals. As described above, multi-color images can be generated based on applying a pseudocolor algorithm to the multi-channel image data to facilitate human interpretation of the image in real time or near real time.

[0108] An example of a dual-channel non-descanned (external) detector design for collecting fluorescence signals in two different emission wavelength ranges is shown in Figures 10A and 10B, where a dichroic filter is used to separate the emission bands and direct the signals to two photomultiplier tubes (PMTs), which individually filter and detect each emission band. This design can be expanded to include additional detectors (>2) to further improve sensitivity and specificity.

[0109] While preferred embodiments of the present invention have been shown and described herein, it will be apparent 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 invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in any combination in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method of operating a system for imaging a tissue sample, the system comprising a first optical subsystem, a second optical subsystem, and a processor, the method comprising: a) the first optical subsystem obtaining one or more high-resolution optically sliced ​​images of a distribution of a contrast agent in the tissue sample using a first imaging modality; b) the second optical subsystem obtaining one or more high-resolution optically sliced ​​images of a tissue sample morphology using a second imaging modality, wherein the first imaging modality and the second imaging modality are configured to image the same optical plane in the tissue sample; and c) the processor processes the one or more high-resolution optical section images of the distribution of the contrast agent and / or the tissue sample morphology, wherein the processing includes the processor using an image interpretation algorithm to (i) identify individual cells and determine the location of the individual cells, (ii) derive a signal from the contrast agent at the location of the individual cells, and (iii) output a quantitative measure of the signal; A method of operation comprising:

2. The method of claim 1 , wherein the first imaging modality comprises two-photon fluorescence microscopy, confocal fluorescence microscopy, light sheet microscopy, or structured illumination microscopy.

3. 3. The method of claim 1 or claim 2, wherein the second imaging modality comprises stimulated Raman scattering microscopy, coherent anti-Stokes Raman scattering microscopy, confocal reflectance microscopy, second harmonic generation microscopy, or third harmonic generation microscopy.

4. The method of any one of claims 1 to 3, wherein the one or more high-resolution optically sliced ​​images have an axial resolution of less than 10 μm.

5. The method of any one of claims 1 to 4, wherein the one or more high-resolution optically sliced ​​images have a lateral resolution of less than 5 μm.

6. An operating method described in any one of claims 1 to 5, wherein the first optical subsystem obtains one or more high-resolution optically thinned images of the distribution of the contrast agent within a first detection wavelength range and a second detection wavelength range.

7. the first detection wavelength range includes an emission peak of the contrast agent; the second detection wavelength range excludes the emission peak of the contrast agent; the first detection wavelength range includes 640 nm light, and the second detection wavelength range includes wavelengths shorter than 600 nm; 7. The method of claim 6, wherein the processing includes the processor using the image interpretation algorithm on the one or more high-resolution optically thinned images of the distribution of the contrast agent in the first detection wavelength range and the one or more high-resolution optically thinned images of the distribution of the contrast agent in the second detection wavelength range to (i) determine a background value at the location of the individual cell, and (ii) correct the signal from the contrast agent at the location of the individual cell using the background value to output the quantitative measure of the signal.

8. The second imaging modality comprises stimulated Raman scattering microscopy, and the one or more high-resolution optically thinned images of the tissue sample form a CH of lipid molecules. ２ 2,850 cm corresponding to vibration －１ The method of any one of claims 3 to 7, wherein the wavelength is obtained at a wave number of .gtoreq.

9. The one or more high resolution optical sliced ​​images of the tissue sample form CH of protein and nucleic acid molecules. ３ 2,930 cm corresponding to the vibration －１ 9. The method of claim 8, wherein the wavelength is also obtained at a wave number of .gtoreq.

10. The method of any one of claims 1 to 9, wherein the contrast agent comprises fluorescein, 5-ALA, BLZ-100, or LUM015.

11. The method of claim 10, wherein the imaging agent comprises 5-aminolevulinic acid (5-ALA).

12. 12. The method of any one of claims 1 to 11, wherein the image interpretation algorithm identifies the individual cells and determines the location of the individual cells based on one or more image features, the one or more image features comprising size, shape, pattern, intensity, or any combination thereof.

13. 13. The method of any one of claims 1 to 12, wherein the image interpretation algorithm comprises a supervised machine learning algorithm, an unsupervised machine learning algorithm, a semi-supervised machine learning algorithm, or any combination thereof.

14. 14. The method of claim 13, wherein the machine learning algorithm adjusts the quantitative measure of the signal based on a training dataset comprising imaging data obtained on archived histopathological tissue samples, imaging data obtained on freshly collected histopathological tissue samples, or any combination thereof.

15. The method of claim 14 , wherein the machine learning algorithm continuously, periodically, or randomly updates the training data set.

16. A method according to any preceding claim, wherein the image interpretation algorithm determines a total or average intensity of the signal from the contrast agent.

17. A method according to any preceding claim, wherein the image interpretation algorithm determines whether the signal from the contrast agent is above a defined threshold level for contrast-positive cells.

18. 18. The method of claim 17, wherein the quantitative measure of the signal indicates the total number of contrast-positive cells, the density of contrast-positive cells, the percentage of contrast-positive cells, or any combination thereof within the one or more high-resolution optically thinned images of the tissue sample.

19. 20. The method of claim 18, wherein the image interpretation algorithm outputs a cellularity score based on the one or more high-resolution optically thinned images of tissue sample morphology.

20. 20. The method of any one of claims 1 to 19, wherein the one or more high-resolution optically thinned images of the distribution of the contrast agent and the one or more high-resolution optically thinned images of tissue sample morphology comprise in vivo images of the tissue sample.

21. 20. The method of any one of claims 1 to 19, wherein the one or more high-resolution optically thinned images of the distribution of the contrast agent and the one or more high-resolution optically thinned images of tissue sample morphology comprise ex-vitro images of the tissue sample.

22. The method of any one of claims 1 to 21, further comprising the processor identifying a location for performing a surgical procedure.

Images