Systems and methods for tissue characterization - Patents.com

Abstract

The present disclosure relates, inter alia, to medical systems, devices, and methods for facilitating the treatment of cancer. Provided herein is a system and method for tissue characterization that can be utilized for intraoperative cancer margin assessment. A tissue sample is excised from a patient and then placed in the system, where it is imaged for autofluorescence properties to help identify areas of interest, such as cancerous tissue margins. Based on the imaging results and tissue characterization, the resection of the tissue in the subject can be adjusted.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 278,255, filed November 11, 2021, which is incorporated by reference in its entirety.

[0002] The present disclosure relates, inter alia, to medical systems, devices and methods for facilitating the treatment of cancer. [Background technology]

[0003] Once cancer has progressed to a certain extent, the most appropriate treatment then becomes surgical resection of the tumor that has metastasized or is affecting nearby organ systems. Often, during surgical dissection or resection of cancerous tissue, the surgeon will dissect a small section of tissue to be provided as a frozen biopsy sample. The frozen biopsy sample is then analyzed by a frozen histology microtome and interpreted by a specialized pathologist reader. This process can be imprecise and result in a substantial increase in operative time for the patient, exposing the patient to additional risks of complications. In light of this, there is an unmet need for a comprehensive and rapid approach for intraoperative analysis of resected cancer samples.

[0004] The following references are incorporated herein by reference: U.S. Patent Nos. 10,980,420, 10,983,060, 10,656,089, 10,605,736, 10,325,366, 10,094,784, 9,677,869, 9,451,882, 8,649,849, 7,890,157, 6,641,835, 6,427,082, 6,405,070, 6,17 Nos. 4,291, 5,601,087, U.S. Publication Nos. 2020 / 0319108, 2020 / 0367818, 2020 / 0096447, 2019 / 0223728, 2019 / 0378292, 2017 / 0367583, 2017 / 0290515, 2013 / 0237842, 2007 / 0093703, 2002 / 0007122, and PCT Publication Nos. WO Attention may be directed to WO 2020 / 148724 A1, WO 2017 / 177194 A1, WO 2017 / 173315 A1, WO 2017 / 075176 A1 and WO 2005 / 019800 A2. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 10,980,420 [Patent Document 2] U.S. Patent No. 10,983,060 Summary of the Invention [Means for solving the problem]

[0006] Provided herein are systems and methods that address the aforementioned unmet need for systems and methods that allow comprehensive and rapid analysis and characterization of sub-tissue types in tissue.

[0007] In some aspects, the invention disclosed herein comprises a device for determining the presence of a tissue or cell type of interest in an excised tissue sample. In some embodiments, the device comprises: (a) a surface for receiving a tissue sample excised from a subject; (b) a light source configured to emit an excitation signal; (c) an optical assembly in optical communication with the light source for directing the excitation signal to the tissue sample received on the surface and collecting autofluorescence light emitted from the tissue sample in response; (d) a detector in communication with the optical assembly configured to capture the autofluorescence light emitted from the tissue sample; and / or (e) a processor in communication with the detector for generating at least one image of the autofluorescence light emitted from the tissue sample. In some embodiments, the subject is afflicted or suspected of being afflicted with a disease. In some embodiments, a pulse signal from a laser is detected. In some embodiments, signal timing jitter is reduced by using a step of detecting the laser signal and triggering a digitizer that collects the autofluorescence signal. In some embodiments, the subject requires surgical intervention, whereby the surgeon needs to be able to distinguish between different types of tissue. In some embodiments, the tissue or cell type of interest comprises diseased tissue or diseased cells. In some embodiments, the diseased tissue or diseased cells comprise cancerous tissue or cancerous cells. In some embodiments, the processor is configured to determine the presence of disease in the excised tissue sample based on the at least one image generated. In some embodiments, the processor is configured to determine the presence of disease in the excised tissue sample based on one or more autofluorescence characteristics of the at least one image generated. In some embodiments, the one or more autofluorescence characteristics comprise an autofluorescence lifetime characteristic. In some embodiments, the autofluorescence lifetime characteristic comprises a plurality of fluorescence exponential decay characteristics of a plurality of regions of the excised tissue. In some embodiments, the processor is configured to determine the presence of disease in the tissue sample based on multiple fluorescent light emitted from the tissue sample using a probabilistic model.In some embodiments, the processor is configured to determine the presence of disease in the multiple margins of the excised tissue sample based on the generated at least one image. In some embodiments, the device further comprises a mechanical stage. In some embodiments, the device further comprises a scan controller in electrical communication with the mechanical stage, the detector, the optical scanning element (e.g., one or more galvanic scanning mirrors), and the light source to operatively control the mechanical stage, the detector, the one or more galvanic scanning mirrors, and / or the light source. In some embodiments, the scan controller may be electrically coupled to and / or in communication with a galvanic scanning mirror driver configured to actuate and scan the light source using the one or more galvanic scanning mirrors. In some embodiments, the galvanic scanning mirror driver may comprise a linear and / or analog motor driver to prevent coupling noise into sensitive electrical amplification, attenuation, analog-to-digital signal conversion, and / or signal digitization. In some embodiments, the scan controller may be configured to synchronize drive signals to actuate one or more galvanic scanning mirrors and translate a motorized stage, e.g., a motorized stage that drives the scanning motion of the optical scanning element. In some embodiments, the scan controller may be configured to synchronize a gain controller (e.g., a gain microcontroller) with a pulse controller of the light source and / or a clock and / or trigger synchronization signal of the seed laser.

[0008] In some embodiments, the mechanical stage is coupled to the surface or the light source. In some embodiments, the mechanical stage is configured to move in three dimensions. In some embodiments, the device further comprises a scanning element coupled to the optical assembly for scanning the excitation signal across a plurality of locations on the tissue sample. In some embodiments, the device further comprises a scanning element coupled to the optical assembly for scanning the excitation signal across a plurality of locations on the tissue sample. In some embodiments, the excised tissue sample is unstained prior to imaging. In some embodiments, the tissue sample is exposed to a crosslinking agent prior to imaging. In some embodiments, the tissue sample comprises breast tissue. In some embodiments, the surface comprises a disposable tray. In some embodiments, the disposable tray is sterile. In some embodiments, the light source is a pulsed laser. In some embodiments, the pulsed laser comprises a fiber laser. In some embodiments, the pulsed laser is a Q-switched laser. In some embodiments, the light source is a mode-locked laser. In some embodiments, the pulsed laser is two-photon. In some embodiments, the pulsed laser emits a wavelength of about 300 nanometers (nm) to about 400 nm. In some embodiments, the pulsed laser comprises a pulse energy of about 1 microjoule (μJ) to about 3 μJ. In some embodiments, the pulsed laser comprises a pulse rate of about 10 kilohertz (kHz) to about 1 MHz. In some embodiments, the pulse width may comprise 100 femtoseconds and 2 nanoseconds. In some embodiments, the optical assembly comprises a partially reflective mirror and a plurality of optical elements, the plurality of optical elements comprising one or more of plano-convex, bi-convex, bi-concave, plano-concave, or any combination thereof lenses. In some embodiments, the plurality of optical elements comprises fused silica optics. In some embodiments, the detector comprises one or more photomultiplier tubes, semiconductor (e.g., GaAs, InGaAs, or silicon) based sensors, or avalanche photodiodes.In some embodiments, the detector comprises one or more dichroic filters. In some embodiments, the device further comprises one or more amplifiers electrically coupled to the detector configured to amplify an electrical signal generated when the detector detects the autofluorescence light emitted from the tissue sample. In some embodiments, the one or more amplifiers comprise a programmable attenuator, a radio frequency amplifier, a fixed attenuator, or any combination thereof. In some embodiments, the processor comprises a field programmable gate array (FPGA).

[0009] In some aspects, the disclosure provided herein comprises a method for determining the presence of a tissue or cell type of interest in a tissue sample. In some embodiments, the method comprises: (a) receiving a tissue sample excised from a subject in a fluorescence imaging system; (b) imaging the excised tissue sample and determining one or more autofluorescence properties of the excised tissue sample; and / or (c) determining the presence of the tissue or cell type of interest in the excised tissue sample based on the imaged excised tissue. In some embodiments, the excised tissue sample is unstained prior to imaging. In some embodiments, the one or more autofluorescence properties comprise an autofluorescence lifetime property. In some embodiments, the excised tissue sample is exposed to a crosslinking agent prior to imaging. In some embodiments, the autofluorescence lifetime property comprises a plurality of fluorescence exponential decay properties of a plurality of regions of the excised tissue sample. In some embodiments, the tissue or cell type of interest comprises a diseased tissue or diseased cell. In some embodiments, the diseased tissue or diseased cell comprises a cancerous tissue or cancerous cell. In some embodiments, the tissue sample comprises tissue from the colon, breast, prostate, skin, vascular, or any combination thereof. In some embodiments, determining the presence of disease in the resected tissue comprises characterizing one or more margins in the resected tissue sample as diseased or non-diseased. In some embodiments, the fluorescence imaging system comprises a pulsed fluorescent light source. In some embodiments, the imaging comprises detecting autofluorescent light emitted from the tissue sample in response to the pulsed fluorescent light source providing an excitation signal to the tissue sample. In some embodiments, the pulsed fluorescent light source is a pulsed fiber laser fluorescent light source. In some embodiments, the method further comprises informing the surgeon to resect a second tissue sample from the subject. In some embodiments, the informing comprises a sound, a visual display, or any combination thereof, directed toward the surgeon. In some embodiments, steps (b) and (c) of the method are completed in a maximum of 5 minutes.In some embodiments, the step of determining the presence of disease in the tissue sample is completed by a probability-based model. In some embodiments, the probability-based model comprises clustering, scalar vector machine, kernel SVM, linear discriminant analysis, quadratic discriminant analysis, neighborhood component analysis, manifold learning, convolutional neural network, reinforcement learning, random forest, naive Bayes, Gaussian mixture, hidden Markov model, Monte Carlo, restricted Boltzmann machine, linear regression, or any combination thereof. In some embodiments, the subject is suspected of having or suffering from a disease. In some embodiments, the tissue sample is placed on a surface of a tissue sample carrier of a fluorescence imaging system prior to imaging. In some embodiments, the tissue sample carrier is configured to mechanically couple to a tissue sample barrier. In some embodiments, the tissue sample carrier and the tissue sample barrier are mechanically coupled to fix at least two degrees of freedom of the tissue sample carrier.

[0010] In some aspects, the disclosure provided herein comprises a method for determining the presence of a tissue or cell type of interest in a tissue sample. In some embodiments, the method comprises: (a) excising a tissue sample from a subject; (b) placing the tissue sample in a fluorescence imaging system; (c) imaging the excised tissue sample with the aid of the fluorescence imaging system and determining one or more autofluorescence properties of the excised tissue sample; and / or (d) receiving from the fluorescence imaging system a determination of the presence of the tissue or cell type of interest in the excised tissue sample based on the imaged excised tissue. In some embodiments, the excised tissue sample is unstained prior to imaging. In some embodiments, the tissue sample is exposed to a crosslinking agent prior to imaging. In some embodiments, the one or more autofluorescence properties comprise an autofluorescence lifetime property. In some embodiments, the autofluorescence lifetime property comprises a plurality of fluorescence exponential decay properties of a plurality of regions of the excised tissue sample. In some embodiments, the tissue or cell type of interest comprises a diseased tissue or diseased cell. In some embodiments, the diseased tissue or diseased cell comprises a cancerous tissue or cancerous cell. In some embodiments, the tissue sample includes tissue from the colon, breast, prostate, skin, vascular, or any combination thereof. In some embodiments, determining the presence of disease in the resected tissue comprises characterizing one or more margins in the resected tissue sample as diseased or non-diseased. In some embodiments, the fluorescence imaging system comprises a pulsed fluorescent light source. In some embodiments, the pulsed fluorescent light source comprises a pulsed fiber laser. In some embodiments, the imaging step comprises detecting autofluorescent light emitted from the tissue sample in response to the pulsed fluorescent light source providing an excitation signal to the tissue sample. In some embodiments, the method further comprises informing the surgeon to excise a second tissue sample from the subject. In some embodiments, the informing step comprises a sound, a visual display, or any combination thereof, directed toward the surgeon.In some embodiments, steps (c) and (d) of the method are completed in up to 5 minutes. In some embodiments, the determination of the presence of disease in the tissue sample is completed by a probability-based model. In some embodiments, the probability-based model comprises clustering, scalar vector machine, kernel SVM, linear discriminant analysis, quadratic discriminant analysis, neighborhood component analysis, manifold learning, convolutional neural network, reinforcement learning, random forest, naive Bayes, Gaussian mixture, hidden Markov model, Monte Carlo, restricted Boltzmann machine, linear regression, or any combination thereof. In some embodiments, the subject is suspected of having or suffering from a disease. In some embodiments, the tissue sample is placed on a surface of a tissue sample carrier of a fluorescence imaging system prior to imaging. In some embodiments, the tissue sample carrier is configured to mechanically couple to a tissue sample barrier. In some embodiments, the tissue sample carrier and the tissue sample barrier are mechanically coupled to fix at least two degrees of freedom of the tissue sample carrier.

[0011] Aspects of the disclosure provided herein include a device for determining the presence of a tissue or cell type of interest in an excised tissue sample. In some embodiments, the device comprises: (a) a surface for receiving a tissue sample excised from a subject; (b) a light source configured to emit an excitation signal; (c) an optical assembly in optical communication with the light source for directing the excitation signal to the tissue sample received on the surface and collecting fluorescent light emitted from the tissue sample in response; (d) a detector in optical communication with the optical assembly configured to collect fluorescent light emitted from the tissue sample; and / or (e) a processor in communication with the detector for characterizing at least a portion of the tissue sample with respect to the tissue or cell type of interest based on a fluorescence lifetime characteristic of the collected fluorescent light. In some embodiments, the processor is configured to determine the presence of disease in the excised tissue sample based on the at least one image generated. In some embodiments, the autofluorescence lifetime characteristic comprises a plurality of fluorescence exponential decay characteristics of a plurality of regions of the excised tissue. In some embodiments, the processor is configured to use a probabilistic model to determine the presence of disease in the tissue sample based on the fluorescent light emitted from the tissue sample. In some embodiments, the processor is configured to determine the presence of disease in the multiple margins of the excised tissue sample based on the generated at least one image. In some embodiments, the device further comprises a mechanical stage. In some embodiments, the device further comprises a scanning controller in electrical communication with the mechanical stage, the detector, and the light source for operatively controlling the mechanical stage, the detector, and the light source. In some embodiments, the mechanical stage is coupled to the surface or the light source. In some embodiments, the mechanical stage is configured to move in three dimensions. In some embodiments, the device further comprises a scanning element coupled to the optical assembly for scanning the excitation signal across multiple locations on the tissue sample. In some embodiments, the device further comprises a scanning element coupled to the optical assembly for scanning the excitation signal across multiple locations on the tissue sample.In some embodiments, the excised tissue sample is unstained prior to imaging. In some embodiments, the tissue is exposed to a crosslinking agent prior to imaging. In some embodiments, the tissue sample comprises breast tissue. In some embodiments, the tissue or cell type of interest comprises diseased tissue or diseased cells. In some embodiments, the diseased tissue or diseased cells comprise cancerous tissue or cancerous cells. In some embodiments, the surface comprises a disposable tray. In some embodiments, the disposable tray is sterile. In some embodiments, the light source is a pulsed laser. In some embodiments, the pulsed laser is a Q-switched laser. In some embodiments, the pulsed laser is a passive Q-switched laser. In some embodiments, the pulsed laser is a two-photon laser. In some embodiments, the pulsed laser emits a wavelength of about 300 nanometers (nm) to about 400 nm. In some embodiments, the pulsed laser comprises a pulse energy of about 1 microjoule (μJ) to about 3 μJ. In some embodiments, the pulsed laser comprises a pulse rate of about 10 kilohertz (kHz) to about 50 kHz. In some embodiments, the optical assembly comprises a partially reflective mirror and a plurality of optical elements, the plurality of optical elements comprising one or more of plano-convex, bi-convex, bi-concave, plano-concave, or any combination thereof, lenses. In some embodiments, the plurality of optical elements comprises fused silica optics. In some embodiments, the detector comprises one or more photomultiplier tubes. In some embodiments, the detector comprises one or more dichroic filters. In some embodiments, the device further comprises one or more amplifiers electrically coupled to the detector configured to amplify an electrical signal generated when the detector detects fluorescent light emitted from the tissue sample. In some embodiments, the one or more amplifiers comprise a programmable attenuator, a radio frequency amplifier, a fixed attenuator, or any combination thereof. In some embodiments, the processor comprises a field programmable gate array (FPGA).In some embodiments, the subject has or is suspected of having a disease.

[0012] Aspects of the disclosure provided herein comprise a method for determining the presence of a tissue or cell type of interest in a tissue sample. In some embodiments, the method comprises: (a) receiving a tissue sample excised from a subject in a fluorescence imaging system; (b) directing an excitation signal to the tissue sample; (c) collecting fluorescent light emitted from the tissue sample in response to the excitation signal; and (d) characterizing at least a portion of the tissue sample for the tissue or cell type of interest based on a fluorescence lifetime characteristic of the collected fluorescent light. In some embodiments, the excised tissue sample is unstained prior to imaging. In some embodiments, the tissue sample is exposed to a crosslinking agent prior to imaging. In some embodiments, the autofluorescence lifetime characteristic comprises a plurality of fluorescence exponential decay characteristics of a plurality of regions of the excised tissue sample. In some embodiments, the tissue or cell type of interest comprises a diseased tissue or cell. In some embodiments, the diseased tissue or cell comprises a cancerous tissue or cell. In some embodiments, the tissue sample comprises tissue from the colon, breast, prostate, skin, vascular, or any combination thereof. In some embodiments, the characterizing step includes characterizing one or more margins in the excised tissue sample as diseased or non-diseased. In some embodiments, the fluorescence imaging system includes a pulsed fluorescent light source. In some embodiments, the pulsed fluorescent light source includes a pulsed fiber laser. In some embodiments, the collecting step includes detecting emitted fluorescent light from the tissue sample in response to the pulsed fluorescent light source providing an excitation signal to the tissue sample. In some embodiments, the method further includes informing the surgeon to excise a second tissue sample from the subject. In some embodiments, the informing step includes a sound, a visual display, or any combination thereof, directed toward the surgeon. In some embodiments, steps (c) and (d) are completed in a maximum of 5 minutes. In some embodiments, the characterization is completed by a probability-based model.In some embodiments, the probability-based model comprises clustering, scalar vector machine, kernel SVM, linear discriminant analysis, quadratic discriminant analysis, neighborhood component analysis, manifold learning, convolutional neural network, reinforcement learning, random forest, naive Bayes, Gaussian mixture, hidden Markov model, Monte Carlo, restricted Boltzmann machine, linear regression, or any combination thereof. In some embodiments, the subject is afflicted with or suspected of afflicting a disease. In some embodiments, the tissue sample is placed on a surface of the fluorescent tissue sample carrier prior to directing an excitation signal to the tissue sample. In some embodiments, the tissue sample carrier is configured to mechanically couple to the tissue sample barrier. In some embodiments, the tissue sample carrier and the tissue sample barrier are mechanically coupled to fix at least two degrees of freedom of the tissue sample carrier.

[0013] Aspects of the disclosure provided herein comprise a method for determining the presence of a tissue or cell type of interest in a tissue sample. In some embodiments, the method comprises: (a) excising a tissue sample from a subject; (b) placing the tissue sample in a fluorescence imaging system, the fluorescence imaging system directing an excitation signal to the tissue sample and collecting fluorescent light emitted from the sample in response; and (c) receiving from the fluorescence imaging system a characterization of at least a portion of the tissue sample with respect to the tissue or cell type of interest based on a fluorescence lifetime characteristic of the collected fluorescent light. In some embodiments, the excised tissue sample is unstained prior to imaging. In some embodiments, the tissue sample is exposed to a crosslinking agent prior to placing the tissue sample in the fluorescence imaging system. In some embodiments, the autofluorescence lifetime characteristic comprises a plurality of fluorescence exponential decay characteristics of a plurality of regions of the excised tissue sample. In some embodiments, the tissue or cell type of interest comprises a diseased tissue or diseased cell. In some embodiments, the diseased tissue or diseased cell comprises a cancerous tissue or cell. In some embodiments, the tissue sample includes tissue from the colon, breast, prostate, skin, vascular, or any combination thereof. In some embodiments, the characterization comprises characterization of one or more margins in the resected tissue sample as diseased or non-diseased. In some embodiments, the fluorescence imaging system comprises a pulsed fluorescent light source. In some embodiments, the pulsed fluorescent light source comprises a pulsed fiber laser. In some embodiments, the receiving step comprises detecting emitted fluorescent light from the tissue sample in response to the pulsed fluorescent light source providing an excitation signal to the tissue sample. In some embodiments, the method further comprises informing the surgeon to resect a second tissue sample from the subject. In some embodiments, the informing step comprises a sound, a visual display, or any combination thereof, directed toward the surgeon. In some embodiments, steps (b) and (c) are completed in a maximum of 5 minutes. In some embodiments, the characterization is completed by a probability-based model.In some embodiments, the probability-based model comprises clustering, scalar vector machine, kernel SVM, linear discriminant analysis, quadratic discriminant analysis, neighborhood component analysis, manifold learning, convolutional neural network, reinforcement learning, random forest, naive Bayes, Gaussian mixture, hidden Markov model, Monte Carlo, restricted Boltzmann machine, linear regression, or any combination thereof. In some embodiments, the subject is afflicted with or suspected of afflicting a disease. In some embodiments, the tissue sample is placed on a surface of a fluorescent tissue sample carrier prior to placing the tissue sample in a fluorescent imaging system. In some embodiments, the tissue sample carrier is configured to mechanically couple to a tissue sample barrier. In some embodiments, the tissue sample carrier and the tissue sample barrier are mechanically coupled to fix at least two degrees of freedom of the tissue sample carrier. [Brief description of the drawings]

[0014] 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.

[0015] [Figure 1] FIG. 1 illustrates a block diagram of an exemplary system for analyzing excised tissue, as described in certain embodiments herein.

[0016] [Diagram 2] FIG. 2 illustrates a block diagram of an exemplary system for analyzing excised tissue, detailing the subsystems optical, optoelectronic, and signal processing controller, as described in certain embodiments herein.

[0017] [Figure 3A]3A-3B illustrate a representative diagram (FIG. 3A) and show an image (FIG. 3B) of an example of a system for intraoperative imaging of a surgically resected cancer sample, as described in some embodiments herein. [Figure 3B] 3A-3B illustrate a representative diagram (FIG. 3A) and show an image (FIG. 3B) of an example of a system for intraoperative imaging of a surgically resected cancer sample, as described in some embodiments herein.

[0018] [Figure 4A] 4A-4B illustrate a representative schematic diagram (FIG. 4A) of an exemplary system user interface for an intraoperative imaging system as described in certain embodiments herein, and show an image thereof (FIG. 4B). [Figure 4B] 4A-4B illustrate a representative schematic diagram (FIG. 4A) of an exemplary system user interface for an intraoperative imaging system as described in certain embodiments herein, and show an image thereof (FIG. 4B).

[0019] [Diagram 5] FIG. 5 provides a graph showing various fluorescence lifetime signals of multiple time-resolved fluorescence imaging channels of the system, as described in certain embodiments herein.

[0020] [Figure 6A] 6A-6B illustrate a workflow diagram for a method of determining the presence or absence of cancer by analyzing the autofluorescence signal emitted by an excised tissue sample using the system, as described in certain embodiments herein. [Figure 6B] 6A-6B illustrate a workflow diagram for a method of determining the presence or absence of cancer by analyzing the autofluorescence signal emitted by an excised tissue sample using the system, as described in certain embodiments herein.

[0021] [Figure 7A] 7A-7B illustrate a workflow diagram for a method of determining the presence or absence of cancer by analyzing the fluorescent lifetime signals emitted by an excised tissue sample using the present system, as described in certain embodiments herein. [Figure 7B] 7A-7B illustrate a workflow diagram for a method of determining the presence or absence of cancer by analyzing the fluorescent lifetime signals emitted by an excised tissue sample using the present system, as described in certain embodiments herein.

[0022] [Figure 8] FIG. 8 illustrates a system diagram of a computer system comprising a processor configured to obtain and analyze autofluorescence and / or fluorescence lifetime data of a tissue sample as described in certain embodiments herein.

[0023] [Figure 9A] 9A-9C illustrate a workflow diagram for system setup and / or preparation operations, including device power on (FIG. 9A), password authorization (FIG. 9B), and tray placement (FIG. 9C), as described in certain embodiments herein. [Figure 9B] 9A-9C illustrate a workflow diagram for system setup and / or preparation operations, including device power on (FIG. 9A), password authorization (FIG. 9B), and tray placement (FIG. 9C), as described in certain embodiments herein. [Figure 9C] 9A-9C illustrate a workflow diagram for system setup and / or preparation operations, including device power on (FIG. 9A), password authorization (FIG. 9B), and tray placement (FIG. 9C), as described in certain embodiments herein.

[0024] [Figure 10A]10A-10C illustrate a workflow diagram for sample preparation and / or placement operations, including steps of preparing a tissue sample (FIG. 10A), placing the tissue sample (FIG. 10B), and selecting a new patient (FIG. 10C), as described in several embodiments herein. [Figure 10B] 10A-10C illustrate a workflow diagram for sample preparation and / or placement operations, including steps of preparing a tissue sample (FIG. 10A), placing the tissue sample (FIG. 10B), and selecting a new patient (FIG. 10C), as described in several embodiments herein. [Figure 10C] 10A-10C illustrate a workflow diagram for sample preparation and / or placement operations, including steps of preparing a tissue sample (FIG. 10A), placing the tissue sample (FIG. 10B), and selecting a new patient (FIG. 10C), as described in several embodiments herein.

[0025] [Figure 11A] 11A-11D illustrate a workflow diagram for a sample scanning operation, including steps of scan area selection (FIG. 11A), initiating a sample scan (FIG. 11B), repositioning and / or repositioning the sample (FIG. 11C), and aborting the scan (FIG. 11D), as described in certain embodiments herein. [Figure 11B] 11A-11D illustrate a workflow diagram for a sample scanning operation, including steps of scan area selection (FIG. 11A), initiating a sample scan (FIG. 11B), repositioning and / or repositioning the sample (FIG. 11C), and aborting the scan (FIG. 11D), as described in certain embodiments herein. [Figure 11C]11A-11D illustrate a workflow diagram for a sample scanning operation, including steps of scan area selection (FIG. 11A), initiating a sample scan (FIG. 11B), repositioning and / or repositioning the sample (FIG. 11C), and aborting the scan (FIG. 11D), as described in certain embodiments herein. [Figure 11D] 11A-11D illustrate a workflow diagram for a sample scanning operation, including steps of scan area selection (FIG. 11A), initiating a sample scan (FIG. 11B), repositioning and / or repositioning the sample (FIG. 11C), and aborting the scan (FIG. 11D), as described in certain embodiments herein.

[0026] [Figure 12A] 12A-12B illustrate a workflow diagram for a results viewing operation, including steps of selecting a scan result (FIG. 12A) and reviewing the scan (FIG. 12B), as described in certain embodiments herein. [Figure 12B] 12A-12B illustrate a workflow diagram for a results viewing operation, including steps of selecting a scan result (FIG. 12A) and reviewing the scan (FIG. 12B), as described in certain embodiments herein.

[0027] [Figure 13] FIG. 13 illustrates a workflow diagram for a sample removal operation as described in certain embodiments herein.

[0028] [Figure 14] FIG. 14 illustrates a workflow diagram for a system timeout operation as described in certain embodiments herein.

[0029] [Figure 15A] 15A-15B illustrate a workflow diagram for a system shutdown operation as described in certain embodiments herein. [Figure 15B] 15A-15B illustrate a workflow diagram for a system shutdown operation as described in certain embodiments herein.

[0030] [Figure 16] FIG. 16 illustrates a workflow diagram for a chamber cleaning operation as described in certain embodiments herein.

[0031] [Figure 17] FIG. 17 illustrates a workflow diagram for a device transport operation as described in certain embodiments herein.

[0032] [Figure 18A] 18A-18C show image data obtained using the devices and systems described in some embodiments herein. In particular, a visible light sample image, a fluorescence map image, and a corresponding histopathology image are shown for an excised tissue sample. [Figure 18B] 18A-18C show image data obtained using the devices and systems described in some embodiments herein. In particular, a visible light sample image, a fluorescence map image, and a corresponding histopathology image are shown for an excised tissue sample. [Figure 18C] 18A-18C show image data obtained using the devices and systems described in some embodiments herein. In particular, a visible light sample image, a fluorescence map image, and a corresponding histopathology image are shown for an excised tissue sample.

[0033] [Figure 19A] 19A-19B illustrate a schematic diagram of a data processing workflow as described in certain embodiments herein. [Figure 19B]19A-19B illustrate a schematic diagram of a data processing workflow as described in certain embodiments herein.

[0034] [Figure 20A] 20A-20B illustrate scanning patterns implemented by the methods and systems as described in certain embodiments herein. [Figure 20B] 20A-20B illustrate scanning patterns implemented by the methods and systems as described in certain embodiments herein.

[0035] [Figure 21A] 21A-21D illustrate a carrier (FIGS. 21A-21B) and a barrier (FIGS. 21C-21D) of a fluorescence imaging system as described in certain embodiments herein. [Figure 21B] 21A-21D illustrate a carrier (FIGS. 21A-21B) and a barrier (FIGS. 21C-21D) of a fluorescence imaging system as described in certain embodiments herein. [Figure 21C] 21A-21D illustrate a carrier (FIGS. 21A-21B) and a barrier (FIGS. 21C-21D) of a fluorescence imaging system as described in certain embodiments herein. [Figure 21D] 21A-21D illustrate a carrier (FIGS. 21A-21B) and a barrier (FIGS. 21C-21D) of a fluorescence imaging system as described in certain embodiments herein.

[0036] [Figure 22A] 22A-22B illustrate exploded views of the drawers, carriers, barriers, and linear actuators in retracted and extended states, as described in certain embodiments herein. [Figure 22B] 22A-22B illustrate exploded views of the drawers, carriers, barriers, and linear actuators in retracted and extended states, as described in certain embodiments herein.

[0037] [Diagram 23] FIG. 23 illustrates the installation of a depth sensor and a fluorescent imaging system relative to a scanning optics system, as described in certain embodiments herein.

[0038] [Figure 24A] 24A-24C illustrate a fluorescent imaging system drawer, system display, work surface, and system component storage locations (ie, carrier and / or barrier storage locations), as described in certain embodiments herein. [Figure 24B] 24A-24C illustrate a fluorescent imaging system drawer, system display, work surface, and system component storage locations (ie, carrier and / or barrier storage locations), as described in certain embodiments herein. [Figure 24C] 24A-24C illustrate a fluorescent imaging system drawer, system display, work surface, and system component storage locations (ie, carrier and / or barrier storage locations), as described in certain embodiments herein.

[0039] [Diagram 25] FIG. 25 illustrates a block diagram of an imaging system for analyzing a sample as described in certain embodiments herein.

[0040] [Figure 26] FIG. 26 illustrates a block diagram of amplify-attenuate electronic elements and their interaction with other imaging system components, as described in certain embodiments herein.

[0041] [Figure 27] FIG. 27 illustrates a workflow diagram for system transportation and start-up operations as described in certain embodiments herein.

[0042] [Figure 28] FIG. 28 illustrates a workflow diagram of an imaging operation for imaging a sample mounted on a carrier and a barrier in a fluorescent imaging system, as described in certain embodiments herein.

[0043] [Figure 29] FIG. 29 illustrates a workflow diagram of cleaning and system shutdown operations for a fluorescent imaging system as described in certain embodiments herein.

[0044] (Incorporated by reference) All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Detailed Description In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, similar symbols typically identify similar components unless the context dictates otherwise. The illustrative embodiments described in the detailed description, figures, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the scope of the subject matter presented herein. In general, it will be readily understood that the aspects of the present disclosure as described herein and illustrated in the figures may be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0046] Although certain embodiments and examples are disclosed below, the subject matter of the present invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses, and to modifications and equivalents thereof. Thus, the scope of the claims appended hereto is not limited by any of the specific embodiments described below. For example, in any method or process disclosed herein, the acts or operations of the method or process may be performed in any suitable sequence and are not necessarily limited to any particular disclosed sequence. Various operations may be described in sequence as multiple discrete operations in a manner that may be useful in understanding an embodiment, however, the order of description should not be interpreted to imply that these operations are order dependent. In addition, the structures, systems, and / or devices described herein may be embodied as integrated components or as separate components.

[0047] For purposes of comparing various embodiments, certain aspects and advantages of these embodiments are described. Not necessarily all such aspects or advantages are achieved by any particular embodiment. Thus, for example, various embodiments may be performed in a manner that achieves or optimizes one or a group of advantages as taught herein, without necessarily achieving other aspects or advantages as may also be taught or suggested herein. Overview

[0048] The disclosure provided herein comprises systems, methods, and devices capable of characterizing tissue, pharmaceutical, agricultural, industrial (e.g., oil and gas), raw material, or any combination thereof samples. Tissue samples may include solid tissue and / or liquid biopsies (e.g., blood and / or other bodily fluids). The systems, methods, and devices described herein may be used for one or more applications. In some cases, the application may include characterizing tissue samples intraoperatively, e.g., classifying tissues resected from subjects undergoing cancer resection surgery. In some cases, the systems, methods, and devices herein may be configured to determine the extent of presence of a tissue or cell of interest in a tissue stump. In some cases, the tissue or cell of interest may include diseased tissue or cell. In some cases, the diseased tissue or cell may include cancerous tissue or cell. The tissue sample may include cancerous tissue, suspected cancerous tissue, dysplastic tissue, or any combination thereof.

[0049] In some cases, the systems, methods, and devices may provide an indication of the presence or absence of tissue or cells of interest in a resected tissue sample to inform health care personnel who prescribe or guide the course of surgery. In some cases, the tissue or cells of interest may include diseased tissue or cells. In some cases, the diseased tissue or cells may include cancerous tissue or cells. In some cases, applications may include determining the presence or absence of cancer in dermatological skin biopsies or surgically resected samples. In some cases, applications may include screening intravascular atherosclerotic plaques and determining the classification of the plaques (e.g., stable, unstable, type of lipid content, etc.). Applications may include distinguishing between various tissue types (e.g., musculoskeletal tissue, ligaments, etc.).

[0050] Various aspects of the disclosure provided herein may provide the advantage of being able to analyze the entirety of a resected tissue sample specimen several minutes (e.g., 5 minutes or less) after the sample is resected, which provides several advantages over traditional frozen section biopsy. For example, typically, a resected tissue sample sent for frozen section processing may not be analyzed in its entirety. Often, due to time and resource limitations in pathology processing laboratories, up to three sections of the entire tissue sample may be taken for analysis. In this regard, sampling errors that improperly sample the resected tissue to analyze all aspects of the tissue may lead to an inaccurate diagnosis of the presence or absence of cancer in the tissue sample. Such inaccuracies may lead to cancerous tissue not being completely resected from the subject's body, but instead being left in the body after surgery, which may lead to further health complications (e.g., poor oxygenation, jaundice, etc.), leading to cancer recurrence and metastasis. Aspects of the disclosure provided herein comprise systems, methods, and devices that address such shortcomings.

[0051] Additionally, aspects of the disclosure provided herein may include devices and systems configured to detect fluorescence or autofluorescence emissions at real-time imaging rates. In some cases, the systems and devices described herein may obtain one or more points of fluorescence or autofluorescence data across the entirety of a tissue sample. In some cases, the one or more points of fluorescence or autofluorescence data may comprise one or more fluorescence or multiple fluorescence lifetime data measurements. In some cases, the devices and systems described herein may process, classify, and pseudocolor one or more points of fluorescence or autofluorescence data to display to a user or operator of the device and / or system where the presence of the tissue or cell of interest may reside. In some cases, the tissue or cell of interest may include diseased tissue or cell. In some cases, the diseased tissue or cell may include cancerous tissue or cell. In some cases, the real-time imaging rate may comprise at least 30 imaging frames per second. The real-time imaging rate may be enabled by the use of a filter wheel in the system. It may be considered that the system would not be able to achieve real-time imaging speed by incorporating a filter wheel that is intended to physically change the position of the filter in the path of the emitted fluorescent light beam.Aspects of the present disclosure provided herein may comprise optical elements arranged together to provide unexpected results in signal-to-noise ratio and imaging speed.To offset the otherwise long imaging times typically associated with the mechanical process of moving filters in and out of the optical path of the emitted fluorescent light, the present disclosure provided herein may provide a detector with a numerical aperture that may comprise at least about 30% collection efficiency of the emitted fluorescent light emission.

[0052] In some cases, the detector may have a collection efficiency of about 10% to about 50%. In some cases, the detector may have a collection efficiency of about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 35%, about 10% to about 40%, about 10% to about 45%, about 10% to about 50%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 35%, about 15% to about 40%, about 15% to about 45%, about 15% to about 50%, about 20% to about 25%, about 20% to about 30%, about 20% to about 35%, about The detector may have a collection efficiency of about 20% to about 40%, about 20% to about 45%, about 20% to about 50%, about 25% to about 30%, about 25% to about 35%, about 25% to about 40%, about 25% to about 45%, about 25% to about 50%, about 30% to about 35%, about 30% to about 40%, about 30% to about 45%, about 30% to about 50%, about 35% to about 40%, about 35% to about 45%, about 35% to about 50%, about 40% to about 45%, about 40% to about 50%, or about 45% to about 50%. In some cases, the detector may have a collection efficiency of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. In some cases, the detector may have a collection efficiency of at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45%. In some cases, the detector may have a collection efficiency of up to about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

[0053] In some cases, collection efficiency may allow for short dwell times for one or more filters housed in a filter wheel that is in the emitted fluorescent beam optical path.

[0054] Aspects of the disclosure provided herein may include methods, systems, and devices configured to analyze a sample (e.g., a tissue sample). In some cases, the tissue sample may be tissue excised from a subject undergoing an operation to remove a suspected tumor from the subject. In some cases, the systems and devices disclosed herein may analyze tissue excised from a subject in an operating room.

[0055] The systems and devices of the present disclosure provided herein may analyze multiple tissue samples. The tissue samples may be solid or semi-solid tissue samples. The tissue samples may include tissue from the prostate, lung, kidney, brain, mucosa, skin, liver, colon, bladder, muscle, breast, eye, mouth, muscle, lymph node, ureter, urethra, esophagus, trachea, stomach, gallbladder, pancreas, intestine, heart, spleen, thymus, thyroid, ovary, uterus, lung, appendix, blood vessel, bone, rectum, testicle, or cervix, or any combination thereof. The tissue sample may be any tissue or organ that is accessible through non-surgical or surgical techniques. The tissue sample may be collected from a subject or patient and characterized during a surgical procedure to remove the tissue sample. For example, the tissue sample may be a biopsy that is analyzed in the operating room during surgery or in a pathology laboratory to provide a preliminary diagnosis prior to immunohistochemistry analysis.

[0056] In some cases, the system (300, 2300) may include an imaging system, a user interface, a processor, a non-transitory computer-readable storage medium including software, a dedicated power supply, or any combination thereof. In some cases, the system may be stored on a cart to allow the imaging system to be moved around the hospital and in the operating room. In some cases, the dedicated power supply may be plugged into a wall socket via cable 2320. The cable may provide operating power to the imaging system and / or charge the dedicated power supply of the imaging system. In some cases, the cable may be a retractable cable configured to retract flush against a surface upon actuation of a retraction mechanism of the imaging system. The systems and devices of the disclosure provided herein or components thereof may communicate between each other optically, electrically, mechanically, opto-mechanically, or any combination thereof. system Imaging System

[0057] In some cases, the system of the disclosure provided herein may include an imaging system 300, as seen in FIG. 3A, which may include an imaging engine 304, system electronics 305, a user interface 301, a processor, a non-transitory computer-readable storage medium containing software, a dedicated power supply 310, or any combination thereof. In some cases, the system may further include one or more user interaction devices 312, such as a mouse, a keyboard, a controller, a foot pedal, or any combination thereof. In some cases, the imaging system may be stored on a cart 302, which may allow the imaging system to be moved around in a hospital, a pathology lab, an operating room, or any combination thereof. In some cases, the non-transitory computer-readable storage medium containing software may include implementations of machine learning models that may analyze data generated by the imaging system.

[0058] FIG. 3B illustrates an example of an imaging system stored on a cart as described in the disclosure provided herein, which may include an imaging engine, a user interface, a processor, a non-transitory computer-readable storage medium containing software, and a dedicated power supply.

[0059] As seen in Figures 1 and 2, the imaging system may comprise an imaging system 100 capable of detecting one or more fluorescent or autofluorescent signals from a sample 114 (e.g., a tissue sample). In some cases, the devices and systems shown in Figures 1 and 2 may obtain one or more points of fluorescent or autofluorescent data across the entirety of the tissue sample. In some cases, the one or more points of fluorescent or autofluorescent data may comprise one or more fluorescent or multiple fluorescent lifetime data measurements. In some cases, the devices and systems described herein may process, classify, and pseudocolor one or more points of fluorescent or autofluorescent data to indicate to a user or operator of the device and / or system where the presence of the tissue or cell of interest may reside. In some cases, the tissue or cell of interest may include diseased tissue or cells. In some cases, the diseased tissue or cells may include cancerous tissue or cells.

[0060] The fluorescence imaging system may detect autofluorescence, intrinsic fluorescence, exogenous fluorescence, fluorescence lifetime, or any combination thereof signals from a tissue sample excited by the excitation light source 106. In some cases, the intrinsic fluorescence may be produced by one or more fluorophores. In some cases, the one or more intrinsic fluorophores may include flavin mononucleotide (FMN) riboflavin, flavin adenine dinucleotide (FAD) riboflavin, lipochromic pigments, endogenous porphyrins, free nicotinamide adenosine dinucleotide (NADH), bound NADH, pyridoxal phosphate-glutamic acid decarboxylase (PLP-GAD), or any combination thereof. In some cases, the exogenous fluorescence may be produced by an exogenous fluorophore. In some cases, the exogenous fluorophore may include ICG-labeled chlorotoxin, ICG-labeled knottin, Cy5-labeled knottin, Cy7-labeled knottin, fluorescently conjugated tumor-targeting antibody, fluorescently labeled tumor-targeting moiety, or any combination thereof. The imaging system may include a laser excitation delivery subsystem 104, a signal acquisition subsystem 102, analog and / or digital signal processing elements 124-128, a user interface 130, or any combination thereof.

[0061] In some cases, the imaging system may have an imaging acquisition rate of about 50 pixels / sec to about 200 pixels / sec. In some cases, the imaging system may have an imaging acquisition rate of about 50 pixels / sec to about 60 pixels / sec, about 50 pixels / sec to about 70 pixels / sec, about 50 pixels / sec to about 80 pixels / sec, about 50 pixels / sec to about 90 pixels / sec, about 50 pixels / sec to about 100 pixels / sec, about 50 pixels / sec to about 150 pixels / sec, about 50 pixels / sec to about 200 pixels / sec, about 60 pixels / sec to about 70 pixels / sec, about 60 pixels / sec to about 80 pixels / sec, about 60 pixels / sec to about 90 pixels / sec, about 60 pixels / sec to about 100 pixels / sec, about 60 pixels / sec to about 150 pixels / sec, about 60 pixels / sec to about 200 pixels / sec, about 70 pixels / sec to about 80 pixels / sec, about 70 The imaging rate may be from about 90 pixels / sec to about 90 pixels / sec, from about 70 pixels / sec to about 100 pixels / sec, from about 70 pixels / sec to about 150 pixels / sec, from about 70 pixels / sec to about 200 pixels / sec, from about 80 pixels / sec to about 90 pixels / sec, from about 80 pixels / sec to about 100 pixels / sec, from about 80 pixels / sec to about 150 pixels / sec, from about 80 pixels / sec to about 200 pixels / sec, from about 90 pixels / sec to about 100 pixels / sec, from about 90 pixels / sec to about 150 pixels / sec, from about 90 pixels / sec to about 200 pixels / sec, from about 100 pixels / sec to about 150 pixels / sec, from about 100 pixels / sec to about 200 pixels / sec, or from about 150 pixels / sec to about 200 pixels / sec. In some cases, the imaging system may have an imaging acquisition rate of about 50 pixels / sec, about 60 pixels / sec, about 70 pixels / sec, about 80 pixels / sec, about 90 pixels / sec, about 100 pixels / sec, about 150 pixels / sec, or about 200 pixels / sec. In some cases, the imaging system may have an imaging acquisition rate of at least about 50 pixels / sec, about 60 pixels / sec, about 70 pixels / sec, about 80 pixels / sec, about 90 pixels / sec, about 100 pixels / sec, or about 150 pixels / sec.In some cases, the imaging system may have an imaging acquisition rate of up to about 60 pixels / sec, about 70 pixels / sec, about 80 pixels / sec, about 90 pixels / sec, about 100 pixels / sec, about 150 pixels / sec, or about 200 pixels / sec.

[0062] In some cases, the laser excitation delivery subsystem 104 may comprise one or more excitation optics 110, a light source 106, or any combination thereof. In some cases, the one or more excitation optics elements may comprise mirrors, optical attenuators, optical isolators, filters, lenses, apertures, acousto-optic modulators (AOMs), or any combination thereof.

[0063] The light source 106 may be configured to generate excitation light 108 comprising a pulse or beam of continuous light at a predetermined excitation wavelength. The excitation light 108 generated by the light source 106 may comprise a pulse energy, a pulse frequency, a pulse width on the order of (nanoseconds), or any combination thereof.

[0064] In some cases, the excitation light is about 1 μJ / mm 2 ~about 60μJ / mm 2 In some cases, the excitation light may have a pulse energy of about 1 μJ / mm 2 ~about 2μJ / mm 2 , about 1μJ / mm 2 ~about 5μJ / mm 2 , about 1μJ / mm 2 ~about 10μJ / mm 2 , about 1μJ / mm 2 ~about 20μJ / mm 2 , about 1μJ / mm 2 ~about 30μJ / mm 2 , about 1μJ / mm 2 ~about 40μJ / mm 2 , about 1μJ / mm 2 ~about 50μJ / mm 2 , about 1μJ / mm 2 ~about 60μJ / mm 2 , about 2μJ / mm 2 ~about 5μJ / mm 2, about 2 μJ / mm 2 ~ about 10 μJ / mm 2 , about 2 μJ / mm 2 ~ about 20 μJ / mm 2 , about 2 μJ / mm 2 ~ about 30 μJ / mm 2 , about 2 μJ / mm 2 ~ about 40 μJ / mm 2 , about 2 μJ / mm 2 ~ about 50 μJ / mm 2 , about 2 μJ / mm 2 ~ about 60 μJ / mm 2 , about 5 μJ / mm 2 ~ about 10 μJ / mm 2 , about 5 μJ / mm 2 ~ about 20 μJ / mm 2 , about 5 μJ / mm 2 ~ about 30 μJ / mm 2 , about 5 μJ / mm 2 ~ about 40 μJ / mm 2 , about 5 μJ / mm 2 ~ about 50 μJ / mm 2 , about 5 μJ / mm 2 ~ about 60 μJ / mm 2 , about 10 μJ / mm 2 ~ about 20 μJ / mm 2 , about 10 μJ / mm 2 ~ about 30 μJ / mm 2 , about 10 μJ / mm 2 ~ about 40 μJ / mm 2 , about 10 μJ / mm 2 ~ about 50 μJ / mm 2 , about 10 μJ / mm 2 ~ about 60 μJ / mm 2 , about 20 μJ / mm 2 ~ about 30 μJ / mm 2 , about 20 μJ / mm 2 ~ about 40 μJ / mm 2 , about 20 μJ / mm 2 ~ about 50 μJ / mm 2 , about 20 μJ / mm 2 ~ about 60 μJ / mm 2 , about 30 μJ / mm 2 ~ about 40 μJ / mm 2 , about 30 μJ / mm 2 ~ about 50 μJ / mm 2, about 30μJ / mm 2 ~about 60μJ / mm 2 , about 40μJ / mm 2 ~about 50μJ / mm 2 , about 40μJ / mm 2 ~about 60μJ / mm 2 , or about 50 μJ / mm 2 ~about 60μJ / mm 2 In some embodiments, the excitation light may have a pulse energy of about 1 μJ / mm 2 , about 2μJ / mm 2 , about 5μJ / mm 2 , about 10μJ / mm 2 , about 20μJ / mm 2 , about 30μJ / mm 2 , about 40μJ / mm 2 , about 50μJ / mm 2 , or about 60 μJ / mm 2 In some cases, the excitation light may have a pulse energy of at least about 1 μJ / mm 2 , about 2μJ / mm 2 , about 5μJ / mm 2 , about 10μJ / mm 2 , about 20μJ / mm 2 , about 30μJ / mm 2 , about 40μJ / mm 2 , or about 50 μJ / mm 2 In some embodiments, the excitation light may have a pulse energy of up to about 2 μJ / mm 2 , about 5μJ / mm 2 , about 10μJ / mm 2 , about 20μJ / mm 2 , about 30μJ / mm 2 , about 40μJ / mm 2 , about 50μJ / mm 2 , or about 60 μJ / mm 2 The pulse energy may be

[0065] In some cases, the excitation light may have a pulse frequency of about 1 kilohertz (kHz) to about 10,000 kHz. In some cases, the excitation light may have a pulse frequency of about 1 kHz to about 5 kHz, about 1 kHz to about 10 kHz, about 1 kHz to about 20 kHz, about 1 kHz to about 50 kHz, about 1 kHz to about 100 kHz, about 1 kHz to about 500 kHz, about 1 kHz to about 1,000 kHz, about 1 kHz to about 5,000 kHz, about 1 kHz to about 10,000 kHz, about 5 kHz to about 10 kHz, about 5 kHz to about 20 kHz, about 5 kHz to about 50 kHz, about 5 kHz to about 1 00kHz, about 5kHz to about 500kHz, about 5kHz to about 1,000kHz, about 5kHz to about 5,000kHz, about 5kHz to about 10,000kHz, about 10kHz to about 20kHz, about 10kHz to about 50kHz, about 10kHz to about 100kHz, about 10kHz to about 500kHz, about 10kHz to about 1,000kHz, about 10kHz to about 5,000kHz, about 10kHz to about 10,000kHz, about 20kHz to about 50kHz, approx. 20kHz to approx. 100kHz, approx. 20kHz to approx. 500kHz, approx. 20kHz to approx. 1,000kHz, approx. 20kHz to approx. 5,000kHz, approx. 20kHz to approx. 10,000kHz, approx. 50kHz to approx. 100kHz, approx. 50kHz to approx. 500kHz, approx. 50kHz to approx. 1,000kHz, approx. 50kHz to approx. 5,000kHz, approx. 50kHz to approx. 10,000kHz, approx. 100kHz to approx. 500kHz, approx. The excitation light may have a pulse frequency of about 100 kHz to about 1,000 kHz, about 100 kHz to about 5,000 kHz, about 100 kHz to about 10,000 kHz, about 500 kHz to about 1,000 kHz, about 500 kHz to about 5,000 kHz, about 500 kHz to about 10,000 kHz, about 1,000 kHz to about 5,000 kHz, about 1,000 kHz to about 10,000 kHz, or about 5,000 kHz to about 10,000 kHz. In some cases, the excitation light may have a pulse frequency of about 1 kHz, about 5 kHz, about 10 kHz, about 20 kHz, about 50 kHz, about 100 kHz, about 500 kHz, about 1,000 kHz, about 5,000 kHz, or about 10,000 kHz.In some cases, the excitation light may have a pulse frequency of at least about 1 kHz, about 5 kHz, about 10 kHz, about 20 kHz, about 50 kHz, about 100 kHz, about 500 kHz, about 1,000 kHz, or about 5,000 kHz. In some cases, the excitation light may have a pulse frequency of up to about 5 kHz, about 10 kHz, about 20 kHz, about 50 kHz, about 100 kHz, about 500 kHz, about 1,000 kHz, about 5,000 kHz, or about 10,000 kHz.

[0066] In some cases, the excitation light may have a pulse width of about 1 picosecond (ps) to about 60,000 ps. In some cases, the excitation light may have a pulse width of about 1 ps to about 50 ps, ​​about 1 ps to about 100 ps, ​​about 1 ps to about 500 ps, ​​about 1 ps to about 1,000 ps, ​​about 1 ps to about 5,000 ps, ​​about 1 ps to about 10,000 ps, ​​about 1 ps to about 20,000 ps, ​​about 1 ps to about 40,000 ps, ​​about 1 ps to about 60,000 ps, ​​about 50 ps to about 100 ps, ​​about 50 ps to about 500 ps, ​​about 50 ps to about 1,000 ps, ​​about 50 ps to about 5,000 ps, ​​about 5 0 p seconds to approx. 10,000 p seconds, approx. 50 p seconds to approx. 20,000 p seconds, approx. 50 p seconds to approx. 40,000 p seconds, approx. 50 p seconds to approx. 60,000 p seconds, approx. 100 p seconds to approx. seconds ~ approx. 5,000 p seconds, approx. 100 p seconds ~ approx. 10,000 p seconds, approx. 100 p seconds ~ approx. 20,000 p seconds, approx. 100 p seconds ~ approx. 40,000 p seconds, approx. 00p seconds to approx. 5,000p seconds, approx. 500p seconds to approx. 10,000p seconds, approx. 500p seconds to approx. 20,000p seconds, approx. 500p seconds to approx. 40,000p seconds, approx. 500p seconds to approx. 60,000p seconds, approx. 1,000p seconds to approx. 0 p seconds, approximately 1,000 p seconds to approximately 10,000 p seconds, approximately 1,000 p seconds to approximately 20,000 p seconds, approximately 1,000 p seconds to approximately 40,000 p seconds, approximately 1,000 p seconds to approximately 60,000 p seconds, approximately 5,000 p seconds to approximately 10,000 p seconds The pulse width may be about 5,000 psec to about 20,000 psec, about 5,000 psec to about 40,000 psec, about 5,000 psec to about 60,000 psec, about 10,000 psec to about 20,000 psec, about 10,000 psec to about 40,000 psec, about 10,000 psec to about 60,000 psec, about 20,000 psec to about 40,000 psec, about 20,000 psec to about 60,000 psec, or about 40,000 psec to about 60,000 psec. In some cases, the excitation light may have a pulse width of about 1 psec, about 50 psec, about 100 psec, about 500 psec, about 1,000 psec, about 5,000 psec, about 10,000 psec, about 20,000 psec, about 40,000 psec, or about 60,000 psec.In some cases, the excitation light may have a pulse width of at least about 1 psec, about 50 psec, about 100 psec, about 500 psec, about 1,000 psec, about 5,000 psec, about 10,000 psec, about 20,000 psec, or about 40,000 psec. In some cases, the excitation light may have a pulse width of up to about 50 psec, about 100 psec, about 500 psec, about 1,000 psec, about 5,000 psec, about 10,000 psec, about 20,000 psec, about 40,000 psec, or about 60,000 psec.

[0067] Light source 106 may comprise any number of light sources, such as a pulsed laser, a continuous wave laser, a modulated laser, a tunable laser, an LED, or any combination thereof. The predetermined excitation wavelength of light source 106 may be in one or more of the ultraviolet spectrum, the visible spectrum, the near infrared spectrum, and / or the infrared spectrum, for example, within a range of about 300 nm to about 1,100 nm.

[0068] In some cases, the pulsed laser may be used as a master clock to time one or more other imaging system components, such as the stage, the scan controller 2426, the gain controller 221, the optical scanning element 112, data acquisition, or any combination thereof. In some cases, the pulsed laser clock signal may be generated internally and / or externally to the light source 106 by the pulse controller 2418 and / or the seed laser. In some cases, the pulse controller and / or the seed laser may provide a synchronous clock and / or trigger signal to the scan controller 2426. In some cases, the light source may comprise a circular or square ring LED light source, and the light emitted from one or more LEDs of the circular or square ring LED light source is in the visible spectrum. In some cases, the circular or square LED light source may be configured to illuminate the tissue sample to generate a diffuse visible light image that is detected by the camera and / or the visible light sensor 2428, as seen in FIG. 25. In some cases, the brightness of the circular or square LED may be current controlled or pulse width modulated controlled. The brightness of the LED light source may be adjusted to increase the signal-to-noise ratio of the visible light image captured by the camera and / or the visible light sensor. In some cases, the camera may generate a live image (e.g., video) of the tissue sample that may be used as an overlay and / or to correlate the spatial location of the fluorescent imaging data to a spatial location on the sample. In some cases, the camera may be equipped with a rolling shutter.

[0069] The predetermined excitation wavelength of the light source 106 may be within a range of about 330 nm to about 360 nm, about 420 nm to about 450 nm, about 660 nm to about 720 nm, or about 750 nm to about 780 nm. For example, the light source 106 may emit a light pulse at about 355 nm. The light source 106 may emit a light pulse at about 700 nm or about 710 nm. The wavelength of the light source 106 may be selected such that the sample 114 produces a responsive optical signal in response to excitation with the light pulse. The wavelength of the light source may be selected such that the sample 114 produces a responsive optical signal without being damaged.

[0070] In some cases, the pulsed laser may comprise a pulsed fiber laser. In some cases, the pulsed fiber laser may comprise a master oscillator power amplifier (MOPA) laser configuration. The master oscillator power amplifier laser configuration may comprise one or more laser subsystem components, such as a seed laser, an optical fiber amplifier, a harmonic module, or any combination thereof. In some cases, the MOPA laser configuration may provide a form factor to enable benchtop use of the imaging system.

[0071] In some cases, a pulsed fiber laser with a MOPA configuration may have a width of about 200 mm to about 500 mm.In some cases, the pulsed fiber laser with the MOPA configuration has a diameter of about 200 mm to about 220 mm, about 200 mm to about 240 mm, about 200 mm to about 260 mm, about 200 mm to about 280 mm, about 200 mm to about 300 mm, about 200 mm to about 320 mm, about 200 mm to about 340 mm, about 200 mm to about 360 mm, about 200 mm to about 380 mm, about 200 mm to about 400 mm, about 200 mm to about 500 mm, about 220 mm to about 240 mm, about 220 mm to about 260 mm, about 220 mm to about 280 mm, about 220 mm to about 300 mm, Approximately 220mm to approximately 320mm, approximately 220mm to approximately 340mm, approximately 220mm to approximately 360mm, approximately 220mm to approximately 380mm, approximately 220mm to approximately 400mm, approximately 220mm to approximately 500mm, approximately 240mm to approximately 260mm, approximately 240mm to approximately 280mm, approximately 240mm to approximately 3 00mm, about 240mm to about 320mm, about 240mm to about 340mm, about 240mm to about 360mm, about 240mm to about 380mm, about 240mm to about 400mm, about 240mm to about 500mm, about 260mm to about 280mm, about 260mm to about 300mm, about 260 mm ~ 320mm, 260mm ~ 340mm, 260mm ~ 360mm, 260mm ~ 380mm, 260mm ~ 400mm, 260mm ~ 500mm, 280mm ~ 300mm, 280mm ~ 320mm, 280mm ~ 340mm , about 280mm to about 360mm, about 280mm to about 380mm, about 280mm to about 400mm, about 280mm to about 500mm, about 300mm to about 320mm, about 300mm to about 340mm, about 300mm to about 360mm, about 300mm to about 380mm, about 300mm to about The width may be about 400 mm, about 300 mm to about 500 mm, about 320 mm to about 340 mm, about 320 mm to about 360 mm, about 320 mm to about 380 mm, about 320 mm to about 400 mm, about 320 mm to about 500 mm, about 340 mm to about 360 mm, about 340 mm to about 380 mm, about 340 mm to about 400 mm, about 340 mm to about 500 mm, about 360 mm to about 380 mm, about 360 mm to about 400 mm, about 360 mm to about 500 mm, about 380 mm to about 400 mm, about 380 mm to about 500 mm, or about 400 mm to about 500 mm.In some cases, a pulsed fiber laser with a MOPA configuration may have a width of about 200 mm, about 220 mm, about 240 mm, about 260 mm, about 280 mm, about 300 mm, about 320 mm, about 340 mm, about 360 mm, about 380 mm, about 400 mm, or about 500 mm. In some cases, a pulsed fiber laser with a MOPA configuration may have a width of at least about 200 mm, about 220 mm, about 240 mm, about 260 mm, about 280 mm, about 300 mm, about 320 mm, about 340 mm, about 360 mm, about 380 mm, or about 400 mm. In some cases, a pulsed fiber laser with a MOPA configuration may have a width of up to about 220 mm, about 240 mm, about 260 mm, about 280 mm, about 300 mm, about 320 mm, about 340 mm, about 360 mm, about 380 mm, about 400 mm, or about 500 mm.

[0072] In some cases, a pulsed fiber laser with a MOPA configuration may have a length of about 500 mm to about 800 mm.In some cases, the pulsed fiber laser with the MOPA configuration has a diameter of about 500 mm to about 520 mm, about 500 mm to about 540 mm, about 500 mm to about 560 mm, about 500 mm to about 580 mm, about 500 mm to about 600 mm, about 500 mm to about 620 mm, about 500 mm to about 640 mm, about 500 mm to about 660 mm, about 500 mm to about 680 mm, about 500 mm to about 700 mm, about 500 mm to about 800 mm, about 520 mm to about 540 mm, about 520 mm to about 560 mm, about 520 mm to about 580 mm, about 520 mm to about 600 mm, About 520mm to about 620mm, about 520mm to about 640mm, about 520mm to about 660mm, about 520mm to about 680mm, about 520mm to about 700mm, about 520mm to about 800mm, about 540mm to about 560mm, about 540mm to about 580mm, about 540mm to about 600mm, about 540mm to about 620mm, about 540mm to about 640mm, about 540mm to about 660mm, about 540mm to about 680mm, about 540mm to about 700mm, about 540mm to about 800mm, about 560mm to about 580mm, about 560mm to about 600mm, about 560 mm~620mm, 560mm~640mm, 560mm~680mm, 560mm~700mm, 560mm~800mm, 580mm~600mm, 580mm~620mm, 580mm~640mm , approx. 580mm ~ approx. 660mm, approx. 580mm ~ approx. 680mm, approx. 580mm ~ approx. 700mm, approx. 580mm ~ approx. 800mm, approx. 600mm ~ approx. The length may be about 700 mm, about 600 mm to about 800 mm, about 620 mm to about 640 mm, about 620 mm to about 660 mm, about 620 mm to about 680 mm, about 620 mm to about 700 mm, about 620 mm to about 800 mm, about 640 mm to about 660 mm, about 640 mm to about 680 mm, about 640 mm to about 700 mm, about 640 mm to about 800 mm, about 660 mm to about 680 mm, about 660 mm to about 700 mm, about 660 mm to about 800 mm, about 680 mm to about 700 mm, about 680 mm to about 800 mm, or about 700 mm to about 800 mm.In some cases, a pulsed fiber laser with a MOPA configuration may have a length of about 500 mm, about 520 mm, about 540 mm, about 560 mm, about 580 mm, about 600 mm, about 620 mm, about 640 mm, about 660 mm, about 680 mm, about 700 mm, or about 800 mm. In some cases, a pulsed fiber laser with a MOPA configuration may have a length of at least about 500 mm, about 520 mm, about 540 mm, about 560 mm, about 580 mm, about 600 mm, about 620 mm, about 640 mm, about 660 mm, about 680 mm, or about 700 mm. In some cases, a pulsed fiber laser with a MOPA configuration may have a length of up to about 520 mm, about 540 mm, about 560 mm, about 580 mm, about 600 mm, about 620 mm, about 640 mm, about 660 mm, about 680 mm, about 700 mm, or about 800 mm.

[0073] In some cases, a pulsed fiber laser with a MOPA configuration may have a height of about 50 mm to about 100 mm. In some cases, the pulsed fiber laser with the MOPA configuration has a diameter of about 50 mm to about 55 mm, about 50 mm to about 60 mm, about 50 mm to about 65 mm, about 50 mm to about 70 mm, about 50 mm to about 75 mm, about 50 mm to about 80 mm, about 50 mm to about 85 mm, about 50 mm to about 90 mm, about 50 mm to about 100 mm, about 55 mm to about 60 mm, about 55 mm to about 65 mm, about 55 mm to about 70 mm, about 55 mm to about 75 mm, about 55 mm to about 80 mm, about 55 mm to about 85 mm, about 55 mm to about 90 mm, about 55 mm to about 100 mm, about 60 mm to about 65 mm, about 60 mm to about 70 mm, about 60 mm to about 75 mm, about 60 mm to about 80 mm, about 60 mm to about 85 mm , about 60 mm to about 90 mm, about 60 mm to about 100 mm, about 65 mm to about 70 mm, about 65 mm to about 75 mm, about 65 mm to about 80 mm, about 65 mm to about 85 mm, about 65 mm to about 90 mm, about 65 mm to about 100 mm, about 70 mm to about 75 mm, about 70 mm to about 80 mm, about 70 mm to about 85 mm, about 70 mm to about 90 mm, about 70 mm to about 100 mm, about 75 mm to about 80 mm, about 75 mm to about 85 mm, about 75 mm to about 90 mm, about 75 mm to about 100 mm, about 80 mm to about 85 mm, about 80 mm to about 90 mm, about 80 mm to about 100 mm, about 85 mm to about 90 mm, about 85 mm to about 100 mm, or about 90 mm to about 100 mm. In some cases, the pulsed fiber laser with the MOPA configuration may have a height of about 50 mm, about 55 mm, about 60 mm, about 65 mm, about 70 mm, about 75 mm, about 80 mm, about 85 mm, about 90 mm, or about 100 mm. In some cases, the pulsed fiber laser with the MOPA configuration may have a height of at least about 50 mm, about 55 mm, about 60 mm, about 65 mm, about 70 mm, about 75 mm, about 80 mm, about 85 mm, or about 90 mm. In some cases, the pulsed fiber laser with the MOPA configuration may have a height of at most about 55 mm, about 60 mm, about 65 mm, about 70 mm, about 75 mm, about 80 mm, about 85 mm, about 90 mm, or about 100 mm.

[0074] In some cases, the MOPA laser configuration may provide a robust, long-life laser that can be continuously powered on without a warm-up period before imaging, which may reduce overall imaging time. In some cases, the MOPA laser configuration may reduce overall laser cost compared to the cost of a solid-state laser.

[0075] In some cases, the MOPA fiber laser may comprise a seed laser, which may comprise an infrared (IR) pulsed laser configured to continuously output pulses at a repetition rate of, for example, about 70 MHz to about 80 MHz, selected up to about 500 kHz repetition rate or pump light pulse frequency, as described elsewhere herein. In some cases, the infrared pulsed laser may comprise a pulse width or pump light pulse width of at least about 50 psec, as described elsewhere herein.

[0076] In some cases, the output wavelength of the infrared pulsed laser may comprise about 1,064 nanometers (nm). In some cases, the infrared pulsed laser may comprise an output wavelength of about 1,000 nm to about 1,600 nm. In some cases, the infrared pulsed laser may be from about 1,000 nm to about 1,020 nm, from about 1,000 nm to about 1,040 nm, from about 1,000 nm to about 1,060 nm, from about 1,000 nm to about 1,080 nm, from about 1,000 nm to about 1,100 nm, from about 1,000 nm to about 1,120 nm, from about 1,000 nm to about 1,140 nm, from about 1,000 nm to about 1,180 nm, from about 1,000 nm to about 1,200 nm, from about 1,000 nm to about 1,300 nm, from about 1,000 nm to about 1,600 nm, from about 1,020 nm to about 1,180 nm, from about 1,000 nm to about 1,200 nm, from about 1,000 nm to about 1,300 nm, from about 1,000 nm to about 1,400 nm, from about 1,020 nm to about 1,500 nm, from about 1,020 nm to about 1,600 nm, from about 1,020 nm to about 1,700 nm, from about 1,020 nm to about 1,800 nm, from about 1,020 nm to about 1,900 nm, from about 1,020 nm to about 1,100 nm, from about 1,020 nm to about 1,120 nm, from about 1,020 nm to about 1,140 nm, from about 1,000 nm to about 1,150 nm, from about 1,020 nm to about 1,160 nm, from about 1,020 nm to about 1,180 nm, from about 1,0 0nm~about 1,040nm, about 1,020nm~about 1,060nm, about 1,020nm~about 1,080nm, about 1,020nm~about 1,100nm, about 1,020nm~about 1,120nm, about 1,020nm~about 1,140nm, about 1,020nm~ Approximately 1,180nm, approximately 1,020nm ~ approximately 1,200nm, approximately 1,020nm ~ approximately 1,300nm, approximately 1,020nm ~ approximately 1,600nm, approximately 1,040nm ~ approximately 1,060nm, approximately 1,040nm ~ approximately 1,080nm, approximately 1,040nm ~ approximately 1,1 00nm, approximately 1,040nm to approximately 1,120nm, approximately 1,040nm to approximately 1,140nm, approximately 1,040nm to approximately 1,180nm, approximately 1,040nm to approximately 1,200nm, approximately 1,040nm to approximately 1,300nm, approximately 1,040nm to approximately 1,600nm , about 1,060 nm to about 1,080 nm, about 1,060 nm to about 1,100 nm, about 1,060 nm to about 1,120 nm, about 1,060 nm to about 1,140 nm, about 1,060 nm to about 1,180 nm, about 1,060 nm to about 1,200 nm, about 1, 060nm to approx. 1,300nm, approx. 1,060nm to approx. 1,600nm, approx. 1,080nm to approx. 1,100nm, approx. 1,080nm to approx. 1,120nm, approx. 1,080nm to approx. 1,140nm, approx. m ~ approx. 1,200 nm, approx. 1,080 nm ~ approx. 1,300 nm, approx. 1,080 nm ~ approx. 1,600 nm, approx. 1,100 nm ~ approx. 1,120 nm, approx.200nm, about 1,100nm to about 1,300nm, about 1,100nm to about 1,600nm, about 1,120nm to about 1,140nm, about 1,120nm to about 1,180nm, about 1, 120nm to approx. 1,200nm, approx. 1,120nm to approx. 1,300nm, approx. 1,120nm to approx. 1,600nm, approx. 1,140nm to approx. 1,180nm, approx. 1,140nm to approx. 1,2 The optical fiber may have an output wavelength of about 1,000 nm, about 1,140 nm to about 1,300 nm, about 1,140 nm to about 1,600 nm, about 1,180 nm to about 1,200 nm, about 1,180 nm to about 1,300 nm, about 1,180 nm to about 1,600 nm, about 1,200 nm to about 1,300 nm, about 1,200 nm to about 1,600 nm, or about 1,300 nm to about 1,600 nm. In some cases, the infrared pulsed laser may have an output wavelength of about 1,000 nm, about 1,020 nm, about 1,040 nm, about 1,060 nm, about 1,080 nm, about 1,100 nm, about 1,120 nm, about 1,140 nm, about 1,180 nm, about 1,200 nm, about 1,300 nm, or about 1,600 nm. In some cases, the infrared pulsed laser may have an output wavelength of at least about 1,000 nm, about 1,020 nm, about 1,040 nm, about 1,060 nm, about 1,080 nm, about 1,100 nm, about 1,120 nm, about 1,140 nm, about 1,180 nm, about 1,200 nm, or about 1,300 nm. In some cases, the infrared pulsed laser may have an output wavelength of up to about 1,020 nm, about 1,040 nm, about 1,060 nm, about 1,080 nm, about 1,100 nm, about 1,120 nm, about 1,140 nm, about 1,180 nm, about 1,200 nm, about 1,300 nm, or about 1,600 nm.

[0077] In some cases, the infrared pulsed laser may have an output power of about 1 W to about 20 W. In some cases, the infrared pulsed laser may have an output power of about 1 W to about 2 W, about 1 W to about 4 W, about 1 W to about 6 W, about 1 W to about 8 W, about 1 W to about 10 W, about 1 W to about 12 W, about 1 W to about 15 W, about 1 W to about 20 W, about 2 W to about 4 W, about 2 W to about 6 W, about 2 W to about 8 W, about 2 W to about 10 W, about 2 W to about 12 W, about 2 W to about 15 W, about 2 W to about 20 W, about 4 W to about 6 W, about 4 W to about 8 W, about 4 W to about 10 W, about 4 W to about The infrared pulsed laser may have an output power of about 12 W, about 4 W to about 15 W, about 4 W to about 20 W, about 6 W to about 8 W, about 6 W to about 10 W, about 6 W to about 12 W, about 6 W to about 15 W, about 6 W to about 20 W, about 8 W to about 10 W, about 8 W to about 12 W, about 8 W to about 15 W, about 8 W to about 20 W, about 10 W to about 12 W, about 10 W to about 15 W, about 10 W to about 20 W, about 12 W to about 15 W, about 12 W to about 20 W, or about 15 W to about 20 W. In some cases, the infrared pulsed laser may have an output power of about 1 W, about 2 W, about 4 W, about 6 W, about 8 W, about 10 W, about 12 W, about 15 W, or about 20 W. In some cases, the infrared pulsed laser may comprise an output power of at least about 1 W, about 2 W, about 4 W, about 6 W, about 8 W, about 10 W, about 12 W, or about 15 W. In some cases, the infrared pulsed laser may comprise an output power of up to about 2 W, about 4 W, about 6 W, about 8 W, about 10 W, about 12 W, about 15 W, or about 20 W.

[0078] In some cases, the harmonic module of the MOPA fiber laser may convert the pulsed IR seed laser into a pulsed ultraviolet (UV) laser with a spectral output (e.g., about 300 nanometers (nm) to about 365 nm). In some cases, the harmonic module may comprise a quartz crystal configured to convert the pulsed IR seed laser into UV pulses. In some cases, the quartz crystal may comprise a finite life of up to about 10,000 hours to output the UV pulses.

[0079] In some cases, the quartz may have a life span of about 1,000 hours to about 30,000 hours. In some cases, the quartz may have a life span of about 1,000 hours to about 2,000 hours, about 1,000 hours to about 5,000 hours, about 1,000 hours to about 10,000 hours, about 1,000 hours to about 15,000 hours, about 1,000 hours to about 20,000 hours, about 1,000 hours to about 25,000 hours, about 1,000 hours to about 30,000 hours, or about 1,000 hours to about 2,000 hours. 0 hours, about 2,000 hours to about 5,000 hours, about 2,000 hours to about 10,000 hours, about 2,000 hours to about 15,000 hours, about 2,000 hours to about 20,000 hours, about 2,000 hours to about 25,000 hours, about 2,000 hours to about 30,000 hours, about 5,000 hours to about 10,000 hours, about 5,000 hours to about 15,000 hours, about 5,000 hours to about 20,000 hours, about 5,000 hours to about 25,000 hours, about 5,000 hours to about 30,000 hours, about 10,000 hours to about 15,000 hours, about 10,000 hours to about 20,000 hours, about 10,000 hours to about 25,000 hours, about 10,000 hours to about The quartz crystal may have a lifespan of about 1,000 hours, about 2,000 hours, about 5,000 hours, about 10,000 hours, about 15,000 hours, about 20,000 hours, about 25,000 hours, about 30,000 hours, about 15,000 hours, about 20,000 hours, about 25,000 hours, about 20,000 hours, about 30,000 hours, or about 25,000 hours, about 30,000 hours. In some cases, the quartz crystal may have a lifespan of about 1,000 hours, about 2,000 hours, about 5,000 hours, about 10,000 hours, about 15,000 hours, about 20,000 hours, about 25,000 hours, or about 30,000 hours. In some cases, the quartz may have a lifespan of at least about 1,000 hours, about 2,000 hours, about 5,000 hours, about 10,000 hours, about 15,000 hours, about 20,000 hours, or about 25,000 hours. In some cases, the quartz may have a lifespan of up to about 2,000 hours, about 5,000 hours, about 10,000 hours, about 15,000 hours, about 20,000 hours, about 25,000 hours, or about 30,000 hours.

[0080] In some cases, the UV spectral output of the pulsed UV laser may comprise a bandwidth of at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, or at least about 10 nm. In some cases, the pulsed UV laser may comprise a pulse width, pulse frequency, and / or pulse energy of the excitation light as described elsewhere herein.

[0081] In some cases, the pulsed UV laser may have an output wavelength of about 300 nm to about 400 nm. In some cases, the pulsed UV laser may have an output wavelength of about 300 nm to about 310 nm, about 300 nm to about 320 nm, about 300 nm to about 330 nm, about 300 nm to about 340 nm, about 300 nm to about 350 nm, about 300 nm to about 360 nm, about 300 nm to about 370 nm, about 300 nm to about 380 nm, about 300 nm to about 390 nm, about 300 nm to about 400 nm, about 310 nm to about 320 nm, about 310 nm to about 330 nm, about 310 nm to about 340 nm, Approximately 310nm to approximately 350nm, approximately 310nm to approximately 360nm, approximately 310nm to approximately 370nm, approximately 310nm to approximately 380nm, approximately 310nm to approximately 390nm, approximately 310nm to approximately 400nm, approximately 320nm to approximately 330nm, approximately 3 20nm to about 340nm, about 320nm to about 350nm, about 320nm to about 360nm, about 320nm to about 370nm, about 320nm to about 380nm, about 320nm to about 390nm, about 320nm to about 400nm, about 330nm m ~ approx. 340 nm, approx. 330 nm ~ approx. 350 nm, approx. 330 nm ~ approx. 360 nm, approx. 330 nm ~ approx. 370 nm, approx. 330 nm ~ approx. 380 nm, approx. Approximately 350nm, approximately 340nm to approximately 360nm, approximately 340nm to approximately 370nm, approximately 340nm to approximately 380nm, approximately 340nm to approximately 390nm, approximately 340nm to approximately 400nm, approximately 350nm to approximately 360nm, approximately 350nm to approximately 37 In some cases, the pulsed UV laser may have an output wavelength of about 300 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm, about 350 nm, about 360 nm, about 370 nm, about 360 nm, about 380 nm, about 360 nm, about 390 nm, about 360 nm, about 400 nm, about 370 nm, about 380 nm, about 390 nm, about 370 nm, about 400 nm, about 380 nm, about 390 nm, or about 400 nm.In some cases, the pulsed UV laser may have an output wavelength of at least about 300 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm, about 350 nm, about 360 nm, about 370 nm, about 380 nm, or about 390 nm. In some cases, the pulsed UV laser may have an output wavelength of at most about 310 nm, about 320 nm, about 330 nm, about 340 nm, about 350 nm, about 360 nm, about 370 nm, about 380 nm, about 390 nm, or about 400 nm.

[0082] Excitation by the light pulse may cause the sample 114 to generate a responsive optical signal that may be collected by the signal collection subsystem 102. In this manner, a single excitation light pulse may be used to gather both time-resolved (fluorescence decay) as well as wavelength-resolved (fluorescence intensity) information from the responsive optical signal that is damaged by the light pulse in real time or near real time. For example, ultraviolet light may be selected to excite a wide range of fluorophores in a tissue sample and may be used to excite multiple fluorophores simultaneously. However, prolonged exposure to ultraviolet light may cause cell damage, at least in some cases. Thus, in cases where exposure to ultraviolet light is a concern, near-infrared or infrared light may be a safer alternative. The infrared light source may be configured to excite a range of fluorophores similar to that of ultraviolet light by using two-photon (or multi-photon) techniques. For example, the infrared light source may be configured to emit multiple light pulses in very rapid succession such that two photons of the light pulse simultaneously illuminate the sample 114. When two or more photons simultaneously illuminate the sample 114, their energies can be added together and the sample can produce a responsive optical signal similar to that which can be produced in response to illumination with ultraviolet light, but with reduced potential safety risks.

[0083] In some cases, the excitation light 108 of the light source 106 may be directed towards the sample 114 by one or more excitation optics (110) and an optical scanning element 112, such as an angled partially reflective mirror, a dichroic mirror, a hot mirror, a cold mirror, one or more galvanic scanning mirrors, or any combination thereof. In some cases, the optical scanning element 112 may comprise a filter in the optical path of the optical scanning element 112 before the objective lens and / or the scan lens, configured to transmit a pulsed UV laser source, for example, as described elsewhere herein, and to remove and / or reflect any autofluorescence generated by the interaction of the pulsed UV light source with any of the optical components of the imaging system disposed between the light source and the filter. In some cases, the optical signal transmission element 112 may direct the excitation beam 108 to the tissue sample and direct an emitted beam 117, which may result from the interaction of the tissue sample and the excitation beam, to the signal collection subsystem 102. In some cases, the optical signal transmission element 112 may comprise a slotted mirror beam splitter, a dichroic mirror, a beam splitter, or any combination thereof. In some cases, the emitted beam 117 may comprise an emission beam of autofluorescence, phosphorescence, fluorescence lifetime, intrinsic fluorescence, exogenous fluorescence, or any combination thereof.

[0084] In some cases, the optical signal transmission element 112 may include a retroreflector optically coupled to one or more excitation optics (110). The retroreflector may be mechanically coupled to the chassis of the optical signal transmission element 112. In some cases, the retroreflector may extend the optical path length for the imaging system to achieve a depth of focus of at least about 5 mm with a beam spot size of at least about 75 micrometers (μm) using a long focal length lens (effective focal length of at least about 10 mm). A depth of focus of at least about 5 mm with a beam spot size of at least about 75 μm may provide an optimal spot size and correspondingly increased signal-to-noise of the detected emitted fluorescent signal for, for example, tissue samples with various heights spatially across the tissue sample.

[0085] In some cases, the one or more excitation optics 110 provide the imaging system with a depth of focus of about 0.1 mm to about 100 mm. In some cases, the one or more excitation optics 110 provide the imaging system with a depth of focus of about 0.1 mm to about 0.5 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 20 mm, about 0.1 mm to about 30 mm, about 0.1 mm to about 40 mm, about 0.1 mm to about 50 mm, about 0.1 mm to about 70 mm, about 0.1 mm to about 80 mm, about 0.1 mm to about 100 mm, about 0.5 mm to about 1 mm, about 0.5 mm to about 5 mm, about 0.5 mm to about 10 mm, about 0. 5mm to about 20mm, about 0.5mm to about 30mm, about 0.5mm to about 40mm, about 0.5mm to about 50mm, about 0.5mm to about 70mm, about 0.5mm to about 80mm, about 0.5mm to about 100mm, about 1mm to about 5mm, about 1mm to about 1 0mm, about 1mm to about 20mm, about 1mm to about 30mm, about 1mm to about 40mm, about 1mm to about 50mm, about 1mm to about 70mm, about 1mm to about 80mm, about 1mm to about 100mm, about 5mm to about 10mm, about 5mm to about 20mm, about 5 mm~30mm, 5mm~40mm, 5mm~50mm, 5mm~70mm, 5mm~80mm, 5mm~100mm, 10mm~20mm, 10mm~30mm, 10mm~40mm, 10m m ~ approx. 50 mm, approx. 10 mm ~ approx. 70 mm, approx. 10 mm ~ approx. 80 mm, approx. 10 mm ~ approx. 100 mm, approx. 20 mm ~ approx. 30 mm, approx. The focal depth may be from 20 mm to about 100 mm, from about 30 mm to about 40 mm, from about 30 mm to about 50 mm, from about 30 mm to about 70 mm, from about 30 mm to about 80 mm, from about 30 mm to about 100 mm, from about 40 mm to about 50 mm, from about 40 mm to about 70 mm, from about 40 mm to about 80 mm, from about 40 mm to about 100 mm, from about 50 mm to about 70 mm, from about 50 mm to about 80 mm, from about 50 mm to about 100 mm, from about 70 mm to about 80 mm, from about 70 mm to about 100 mm, or from about 80 mm to about 100 mm.In some cases, the one or more excitation optics 110 may have a focal depth of about 0.1 mm, about 0.5 mm, about 1 mm, about 5 mm, about 10 mm, about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 70 mm, about 80 mm, or about 100 mm. In some cases, the one or more excitation optics 110 may have a focal depth of at least about 0.1 mm, about 0.5 mm, about 1 mm, about 5 mm, about 10 mm, about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 70 mm, or about 80 mm. In some cases, the one or more excitation optics 110 may have a focal depth of at most about 0.5 mm, about 1 mm, about 5 mm, about 10 mm, about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 70 mm, about 80 mm, or about 100 mm.

[0086] In some cases, the retroreflector and / or one or more excitation optics (110) may provide the imaging system with a beam spot size of about 60 μm to about 450 μm.In some cases, the retroreflector and / or one or more excitation optics (110) may provide a focal distance of about 60 μm to about 75 μm, about 60 μm to about 80 μm, about 60 μm to about 120 μm, about 60 μm to about 140 μm, about 60 μm to about 180 μm, about 60 μm to about 200 μm, about 60 μm to about 250 μm, about 60 μm to about 300 μm, about 60 μm to about 350 μm, about 60 μm to about 400 μm, about 60 μm to about 450 μm, about 75 μm to about 80 μm, about 75 μm to about 120 μm, about 75 μm to about 140 μm, about 75 μm to about 180 μm, , about 75 μm to about 200 μm, about 75 μm to about 250 μm, about 75 μm to about 300 μm, about 75 μm to about 350 μm, about 75 μm to about 400μm, about 75μm to about 450μm, about 80μm to about 120μm, about 80μm to about 140μm, about 80μm to about 180μm, about 8 0μm to about 200μm, about 80μm to about 250μm, about 80μm to about 300μm, about 80μm to about 350μm, about 80μm to about 400 μm, approximately 80 μm to approximately 450 μm, approximately 120 μm to approximately 140 μm, approximately 120 μm to approximately 180 μm, approximately 120 μm to approximately 200 μm, approximately 12 0μm~about 250μm, about 120μm~about 300μm, about 120μm~about 350μm, about 120μm~about 400μm, about 120μm~ Approximately 450μm, approximately 140μm to approximately 180μm, approximately 140μm to approximately 200μm, approximately 140μm to approximately 250μm, approximately 140μm to approximately 300 μm, approximately 140 μm to approximately 350 μm, approximately 140 μm to approximately 400 μm, approximately 140 μm to approximately 450 μm, approximately 180 μm to approximately 200 μm, approximately 180μm~about 250μm, about 180μm~about 300μm, about 180μm~about 350μm, about 180μm~about 400μm, about 180μm In one embodiment, a beam spot size of about 200 μm to about 450 μm, about 200 μm to about 250 μm, about 200 μm to about 300 μm, about 200 μm to about 350 μm, about 200 μm to about 400 μm, about 200 μm to about 450 μm, about 250 μm to about 300 μm, about 250 μm to about 350 μm, about 250 μm to about 400 μm, about 250 μm to about 450 μm, about 300 μm to about 350 μm, about 300 μm to about 400 μm, about 300 μm to about 450 μm, about 350 μm to about 400 μm, about 350 μm to about 450 μm, or about 400 μm to about 450 μm may be provided.In some cases, the retroreflector and / or one or more excitation optics (110) may provide a beam spot size of about 60 μm, about 75 μm, about 80 μm, about 120 μm, about 140 μm, about 180 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, or about 450 μm to the imaging system. In some cases, the retroreflector and / or one or more excitation optics (110) may provide a beam spot size of at least about 60 μm, about 75 μm, about 80 μm, about 120 μm, about 140 μm, about 180 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, or about 400 μm to the imaging system. In some cases, the retroreflector and / or one or more excitation optics (110) may provide the imaging system with a beam spot size of up to about 75 μm, about 80 μm, about 120 μm, about 140 μm, about 180 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, or about 450 μm.

[0087] In some cases, the long focal length lens may have a focal length of about 10 mm to about 1,000 mm. In some cases, the long focal length lens may have a focal length of about 10 mm to about 50 mm, about 10 mm to about 100 mm, about 10 mm to about 150 mm, about 10 mm to about 200 mm, about 10 mm to about 250 mm, about 10 mm to about 300 mm, about 10 mm to about 400 mm, about 10 mm to about 500 mm, about 10 mm to about 700 mm, about 10 mm to about 800 mm, about 10 mm to about 1,000 mm, about 50 mm to about 100 mm, about 50 mm to about 150 mm, about 50 mm to about 200 mm, about 50 mm to about 250 mm, about 50 mm to about 300 mm, about 50 mm to about 400 mm, about 50mm to about 500mm, about 50mm to about 700mm, about 50mm to about 800mm, about 50mm to about 1,000mm, about 100mm to about 150mm, about 100mm to about 200mm, about 100mm to about 250mm, about 100mm to about 300mm, about 100mm to about 400mm, about 100mm to about 500mm, about 100mm to about 700mm, about 100mm to about 800mm, about 100mm to about 1,000mm, about 150mm to about 200mm, about 150mm to about 250mm, about 150mm to about 300mm, about 150mm to about 400mm, about 50mm~500mm, 150mm~700mm, 150mm~800mm, 150mm~1,000mm, 200mm~250mm, 200mm~300mm, 200mm~400mm, 200mm~500mm, 200mm~ Approx. 700mm, Approx. 200mm~Approx. 800mm, Approx. 200mm~Approx. 1,000mm, Approx. 250mm~Approx. 300mm, Approx. 250mm~Approx. 400mm, Approx. 250mm~Approx. 500mm, Approx. The focal length may be about 0 mm, about 300 mm to about 400 mm, about 300 mm to about 500 mm, about 300 mm to about 700 mm, about 300 mm to about 800 mm, about 300 mm to about 1,000 mm, about 400 mm to about 500 mm, about 400 mm to about 700 mm, about 400 mm to about 800 mm, about 400 mm to about 1,000 mm, about 500 mm to about 700 mm, about 500 mm to about 800 mm, about 500 mm to about 1,000 mm, about 700 mm to about 800 mm, about 700 mm to about 1,000 mm, or about 800 mm to about 1,000 mm.In some cases, the long focal length lens may have a focal length of about 10 mm, about 50 mm, about 100 mm, about 150 mm, about 200 mm, about 250 mm, about 300 mm, about 400 mm, about 500 mm, about 700 mm, about 800 mm, or about 1,000 mm. In some cases, the long focal length lens may have a focal length of at least about 10 mm, about 50 mm, about 100 mm, about 150 mm, about 200 mm, about 250 mm, about 300 mm, about 400 mm, about 500 mm, about 700 mm, or about 800 mm. In some cases, the long focal length lens may have a focal length of up to about 50 mm, about 100 mm, about 150 mm, about 200 mm, about 250 mm, about 300 mm, about 400 mm, about 500 mm, about 700 mm, about 800 mm, or about 1,000 mm.

[0088] In some cases, the sample 114 may be placed on a stage 116 that may translate the tissue sample so that the imaging system may obtain imaging data for multiple locations on the tissue sample. The stage may include a removable and disposable tray on which the sample 114 may be placed for analysis. In some cases, the disposable tray (i.e., carrier) may be made of nylon 6,6 (polyamide) polymer, acrylonitrile butadiene styrene (ABS), natural off-white ABS, high impact ABS, black nylon, Celcon TM , Acetal Copolymer, Hylex TM , Polycarbonate, Lexan TM , black high density polyethylene (HDPE), blue HDPE, green HDPE, orange HDPE, red HDPE, yellow HDPE, black HDPE, green HDPE, nitrile plastic, blue vinyl, brown vinyl, green vinyl, orange vinyl, pink vinyl, red vinyl, purple vinyl, white vinyl, ultra high molecular weight (UHMW) polyethylene, blue UHMW polyethylene, black UHMW polyethylene, white UHMW polyethylene, off-white nylon, abrasion resistant nylon, black abrasion resistant nylon, or polylactic acid.

[0089] In some cases, the stage may be configured to translate the sample 114 in one, two, or three dimensions while the optical signal transmission element 112 remains stationary. In some cases, the optical signal transmission element 112, one or more excitation optics 110, the optical scanning element 112, the wavelength splitting element 120, the photomultiplier tube 122, one or more collection optics (204, 208), or any combination thereof may be mounted on a stage and / or fixture that may be scanned across the tissue sample to obtain imaging data for multiple locations on the sample 114 while the sample 114 remains stationary. In some cases, both the sample 114 and the optical signal transmission element 112 may move independently of each other. In some cases, the optical scanning element 112 may move independently of the excitation light 108. In some cases, the excitation light 108 may be in mechanical communication with, e.g., directly mounted to, the optical scanning element 112, such that a beam of the excitation light 108 may be incident on the sample 114. In some cases, the stage may be configured to translate the optical signal transmission element 112 in one, two, or three dimensions.

[0090] In some cases, the imaging system may include an imaging probe configured to couple to an opto-mechanical surface of the imaging system, which is optically and / or electrically coupled to the imaging system components described elsewhere herein. In some cases, the imaging system may couple the light source 106 to an imaging probe with one or more excitation optics 110 (e.g., one or more lenses, collimators, cylindrical lenses, mirrors, acousto-optic modulators, etc.). In some cases, the imaging system optical scanning element 112 may translate to a position where the output of the optical scanning element 112 may couple the light source output (e.g., via a folding mirror, one or more stationary mirrors and / or lenses) into the imaging probe mounted to the surface of the imaging system. In some cases, the probe may include a handle-held probe. The probe may include a fiber optic probe, which may include one or more fibers and / or fiber bundles. The probe may include a window and / or lens at the tip of the probe configured to deliver light source excitation to the sample and / or collect the sample's emitted fluorescent light. In some cases, the probe may direct the collected autofluorescence light emitted from the sample to collection optics 118, a wavelength splitting element 120, and / or a detector 122 (e.g., a PMT) to detect the collected autofluorescence signal of the sample.

[0091] In some cases, the imaging system may include a drawer 2226 as seen in FIGS. 22A-22B, 23, 24A and 24C, and 25, in which the sample 114 may be placed for imaging by a user, a physician, operating room medical personnel, or any combination of such individuals. The drawer may include a mounting (e.g., cut out of the drawer) feature 2230 for receiving the barrier 2206. In some cases, the movement of the drawer 2226, e.g., opening and / or closing of the drawer, may be controlled by a drawer controller 2422, electrically and / or operably coupled to a motor 2229 configured to open and / or close the drawer. In some cases, a user may manually override control of the movement of the drawer by the drawer controller, e.g., in situations where the drawer needs to be opened immediately. In some cases, the drawer controller 2422 may be configured to receive input from one or more drawer controls and / or drawer control interfaces 2420. In some cases, the drawer control device and / or the drawer control interface may include actuating and / or pressing a pedal, a button 2306, a switch, or any combination thereof. The pedal may comprise a foot pedal, which is a pedal separate from the imaging system that is operably connected to the imaging system via a cable and / or wireless interface. In some cases, the button 2306 and / or switch may be located on the imaging system and / or provided in a button and / or switch box separate from the imaging system that is operably coupled to the imaging system via a cable and / or wireless interface. In some cases, the drawer 2226 may be opened and / or closed by voice commands detected by a microphone of the imaging system that is electrically coupled to a processor of the imaging system. In some cases, other functionality of the imaging system, such as starting and / or stopping imaging and / or scanning of a sample, may also be actuated by voice commands.Voice commands may be used to initiate processing and / or analysis of the fluorescent imaging data, for example, to indicate the last scan of a sample, or to perform a particular processing or analysis method on the fluorescent imaging data, as described elsewhere herein. In some cases, the drawer 2226 may include features (e.g., a recessed edge 2319 when the drawer is closed) that may enable a user of the imaging system to manually interact with the drawer and open and / or close the drawer 2226.

[0092] In some cases, the drawer controller 2422 may receive sample height information from a sample height sensor 2235, as seen in Figure 23, described elsewhere herein. Information from the sample height sensor may be taken into account when the drawer controller determines whether the drawer can be safely opened without damaging the sample and / or other imaging system components.

[0093] In some cases, the drawer may be mechanically coupled to a motor 2229 configured to open and / or close the drawer 2226 upon a user inputting a command to the imaging system, as described elsewhere herein, to open and / or close the drawer.

[0094] In some cases, the drawer 2226 may include a lock 2231 configured to lock the position of the drawer in place when the linear actuator 2228 described elsewhere herein is raised and / or extended. The lock may prevent a user from inadvertently opening the drawer while the light source is imaging the sample. The lock may be mechanically coupled to a bottom surface of the linear actuator coupling interface 2232 such that when the linear actuator coupling interface is extended, the lock 2231 may pivot to a latched, locked position, thereby constraining the movement of the drawer 2226. In some cases, the linear actuator coupling interface may include one or more kinematic features (2241, 2238) configured to mechanically couple the linear actuator coupling interface and the barrier kinematic features 2218A-2218C. In some cases, the linear actuator kinematic features (2241, 2238) may comprise one or more recesses 2238 and / or one or more protruding features 240, such as holes, slots, circular features, cylindrical features, button features, and / or other polygonal structural features. In some cases, the linear actuator kinematic features (2241, 2238) may comprise one or more chamfered surfaces configured to facilitate coupling between the linear actuator coupling interface and the barrier. In some cases, the linear actuator kinematic features may compensate for manufacturing errors in one or more barrier-to-linear actuator kinematic features 2218A-2218C by neither over-constraining nor under-constraining the coupling between the linear actuator kinematic features and the barrier-to-linear actuator kinematic features.

[0095] As seen in FIGS. 21C-21D and 22A-22B, the barrier 2206 may comprise geometric features (2210, 2212) configured to prevent tissue sample liquid from flowing into the compartment below the mounting feature 2230 of the drawer 2226. In some cases, the compartment below the mounting feature 2230 of the drawer 2226 may comprise imaging optics, system electronics, a power supply, or any combination thereof, as described elsewhere herein, that may be damaged by the sample liquid. In some cases, the barrier 2206 may comprise flanged and / or edge features 2216, the surface of which is configured to mate with and seal the interface between the barrier 2206 and the surface 2234 of the drawer, and prevent sample liquid from flowing into the compartment below the mounting feature 2230 of the drawer 2226. In some cases, the geometric features (2210, 2212) of the barrier 2206 may comprise, e.g., mounds, edges, protrusions, moats, etc., disposed between the drawer surface 2234 and the barrier 2206 to prevent sample liquid from flowing from the barrier 2206 to the drawer surface 2234 and / or from the barrier 2206 into a compartment below the mounting features 2230 of the drawer 2226. In some cases, the geometric features (2210, 2212) may comprise recessed features, e.g., moats, disposed around the periphery of the barrier to prevent sample liquid from flowing to the drawer surface 2234 and / or to prevent sample liquid from flowing into a compartment below the mounting features 2230 of the drawer. In some cases, the geometric features (2210, 2212) may comprise protruding features 2212 (e.g., mounds, projections, raised lips, etc.) that, depending on the height and / or shape of the protrusions, prevent the flow of tissue liquid from the barrier 2206 to the surface 2234 of the drawer and / or from the barrier to a compartment below the mounting feature 2230 of the drawer 2226.The barrier 2206 may comprise a carrier coupling surface 2214 comprising one or more barrier kinematic features 2208 shown in FIG. 21C configured to couple to one or more carrier kinematic features 2204A-2204C of the carrier shown in FIG. 21B disposed on the carrier coupling surface 2203. In some cases, the one or more carrier kinematic features 2204A-2204C may compensate for manufacturing errors of the one or more barrier kinematic features 2208 by neither over-constraining nor under-constraining the coupling between the one or more carrier kinematic features and the one or more barrier kinematic features. In some cases, the one or more barrier kinematic features 2208 may be positioned along the circumference of a circle, each spaced 120 degrees apart from one another. The one or more barrier kinematic features may comprise one or more recesses and / or one or more protruding features, e.g., holes, slots, circular features, and / or other polygonal structural features. In some cases, one or more barrier kinematic features. The combination of one or more barrier kinematic features and one or more carrier kinematic features may isolate one or more degrees of freedom of the carrier 2200. In some cases, a single barrier may be used when imaging one or more samples (e.g., 5-10 samples) from a single patient. In some cases, the barrier 2206 may comprise one or more features 2209, e.g., one or more raised and / or protruding structures displayed in an array or discrete objects, e.g., a protruding shape of a circle with a cross and / or a line through the circle, configured to inhibit or prevent placement of samples on the carrier binding surface 2214.

[0096] In some cases, the barrier 2206 may comprise structural features 2217, such as edges, margins, protruding edges, and / or flanges, that may provide an interface for a user to interact with the barrier 2206 when the barrier is installed within the mounting feature 2230 of the drawer 2226. In some cases, the barrier may comprise directional and / or topological structural features 2215 that limit, restrict, and / or constrain the orientation of the barrier relative to the mounting feature 2230 of the drawer 2226.

[0097] In some cases, the barrier 2206 may comprise a barrier-to-linear actuator coupling interface 2222, as seen in the bottom perspective view of the barrier shown in FIG. 21D. In some instances, the barrier-to-linear actuator coupling interface 2222 may comprise one or more barrier-to-linear actuator kinematic features 2218A-2218C configured to couple and / or mate with a coupling interface 2232 of a linear actuator 2228, motor, and / or piston configured to raise the barrier 2206, carrier 2200, and sample 114 up to the optical scanning element 112 of the imaging system for imaging the sample. The one or more barrier-to-linear actuator kinematic features 2218 may comprise one or more recesses and / or one or more protruding features, such as holes, slots, circular features, and / or other polygonal structural features. In some cases, one or more barrier-to-linear actuator kinematic features may comprise constraining shapes, such as a circle 2218A, an oval 2218B, and / or a slot 2218C, where the circle 2218A may be configured to constrain the translation of the barrier, the oval 2218B may be configured to constrain the rotation of the barrier, and the slot 2218C is configured to constrain the angle of the barrier linear actuator coupling surface 2222 relative to the planar surface of the linear actuator coupling interface 2232.

[0098] In some cases, the barrier, carrier, and sample are raised, lifted, and / or extended into the depth of field of the optical scanning element 112. In some cases, when the barrier 2206 and carrier 2200 are raised and / or elevated normal to the surface 2234 of the drawer, the drawer 2226 may lock in place with interference between the linear actuator 2228, motor, and / or piston in its engaged and elevated state and the mounting feature 2230 of the drawer 2226. In some cases, the linear actuator 2228, motor, and / or piston in the extended, elevated, and / or elevated state during a power outage of the system may collapse and / or retract to a home state where the carrier 2200 and barrier 2206 contact the mounting feature 2230 of the drawer 2226 under the weight of the sample 114, carrier 2200, and / or barrier 2206. In the home state, the drawer of the system may be opened and the sample may be removed.

[0099] 21A-21B and 22A-22B, the carrier 2200 may include one or more structural features 2201, e.g., one or more recessed and / or protruding structures, configured to align and secure the sample during transport, e.g., from the surgical field to the imaging system. In some cases, the one or more structural features may include a lip and / or flange protrusion 2202 extending outward from a central axis of the carrier 2200. In some cases, the lip and / or flange 2202 protrusion may provide a handle and / or grip for a user, physician, medical operating room personnel, nurse, or any combination of such individuals to transport the carrier 2200 and the sample 114 disposed on the surface of the carrier to the imaging system without contaminating the sterility of the sample. In some cases, the one or more structural features may comprise one or more sample alignment features 2201 (e.g., one or more concentric rings arranged as alignment targets) that center the sample on the carrier. The centering of the sample may provide better imaging system resolution (e.g., consistent optical scanning element spot size) than expected across the sample. In some cases, the one or more structural features of the carrier may comprise a raised edge 2205 that protrudes normal to the surface of the carrier, and the raised edge may be configured to contain the liquid of the sample so that it does not flow over the outer edge surface of the carrier. The one or more structural features of the carrier may comprise one or more protruding and / or recessed features on the top surface of the carrier configured to prevent movement of the sample disposed on the surface of the carrier. The carrier 2200 may comprise a carrier-to-barrier mating surface 2203 configured to mate with a carrier mating surface 2214 of the barrier 2206. The carrier-to-barrier mating surface 2203 may comprise one or more carrier kinematic features 2204A-2240C configured to mate with one or more barrier kinematic features 2208.In some cases, one or more carrier kinematic features may be positioned along the circumference of a circle, each spaced 120 degrees apart from one another. One or more carrier kinematic features 2204A-2240C may comprise one or more recessed and / or one or more protruding features, such as holes, slots, circular features, and / or other polygonal structural features. In some cases, one or more carrier kinematic features 2204A-2240C may be configured to constrain one or more degrees of freedom of carrier 2200 relative to barrier 2206. In some cases, one or more carrier kinematic features may comprise constraining shapes, such as a circle 2204A, an ellipse 2204B, and / or a slot 2204C, where the circle 2204 may be configured to constrain the translation of the barrier, the ellipse 2204 may be configured to constrain the rotation of the barrier, and the slot 2204C is configured to constrain the angle of the barrier linear actuator coupling surface 2222 relative to the planar surface of the linear actuator coupling interface 2232. Isolating one or more degrees of freedom of the carrier 2200 may stabilize the carrier 2200 and prevent unwanted motion artifacts generated by sample movement during imaging. By preventing motion artifacts, imaging performance may be improved, for example, by maintaining uniform image resolution across the sample and / or improving co-registration of one or more scan areas and / or sections of fluorescent imaging data of the sample.

[0100] In some cases, the carrier may include a material that, when excited with a light source as described elsewhere herein, emits a fluorescence lifetime with an intensity and fluorescence lifetime range similar to that of the sample, in some cases, the fluorescence lifetime range is within at least about 5%, at least about 10%, at least about 20%, at least about 50%, or at least about 100% of the fluorescence lifetime range of the tissue.

[0101] In some cases, the imaging system 2300 may include a compartment 2314 in which the carrier 2200 and / or barrier 2206 may be stored prior to use when imaging a sample, as seen in FIG. 24B. In some cases, the compartment 2314 may include shelves (e.g., vertical and / or horizontal) and / or subcompartments in which the carrier 2200 and / or barrier 2206 may be stored. In some cases, the compartment may include a cover 2310, which may include a material that is optically transparent to visible light such that a user, physician, medical operating room personnel, and / or nurse may visualize the presence of one or more carriers and / or one or more barriers prior to using the imaging system. The cover 2310 may maintain the atmosphere and / or temperature of the local environment surrounding the carrier 2200 and / or barrier 2206 to maintain the sterility and material properties of the stored carrier and / or barrier.

[0102] In some cases, the carrier and / or barrier may be labeled with, for example, a barcode, QR code, symbol, or feature distinguishable by a visible light sensor (e.g., one or more photodiodes in a one-dimensional or two-dimensional sensor array, a single detector). In some cases, the carrier and / or barrier may include a material with multiple fluorescence lifetimes and / or fluorescence intensities for authentication, calibration, and system self-test procedures. In some cases, the spatial location of the material with multiple fluorescence lifetimes and / or fluorescence intensities may be sensed and / or detected with respect to the location of a visible feature that may be imaged by a visible light camera of the imaging system. In some cases, the labeled carrier and / or barrier may be scanned and interpreted by a sensor of the imaging system, which is operably connected to one or more processors. In some cases, the labeling of the carrier and / or barrier may provide information about the particular carrier and / or barrier (e.g., material, calibration information for a given carrier and / or barrier, etc.). In some cases, the information may be stored in a cloud database and provided to the system when cross-referenced with the characteristics of the label, fluorescence lifetime, fluorescence intensity, spatial geometric features, visible image, or any combination thereof, of the carrier and / or barrier when scanned. In some cases, the characteristics of the label, fluorescence lifetime, fluorescence intensity, spatial geometric features, visible image, or any combination thereof, of the carrier and / or barrier may be used to determine validity and / or authenticate the carrier and / or barrier to prevent unsafe use of the imaging system and / or damage to the sample being imaged.

[0103] In some cases, the carrier and / or barrier may comprise one or more features configured to calibrate and / or test the performance of an imaging system, as described elsewhere herein. In some cases, the carrier and / or barrier may comprise spatially varying material properties that provide different fluorescence lifetime imaging data when excited by a light source of an imaging system, as described elsewhere herein.

[0104] In some cases, the fluorescence imaging system 2300 may comprise an extendable work surface 2308 mechanically coupled to an exterior surface of the fluorescence imaging system, as seen in FIGS. 24A-24C. The work surface 2308 may comprise structural features 2318, cutouts and / or protrusions configured to provide a surface that a user may grasp and / or handle to extend the extendable work surface 2308 away from the imaging system body. In some cases, the imaging system may comprise a recessed feature 2317 configured to provide access to the structural features 2318 of the extendable work surface 2308. The work surface may comprise a hinge coupled to the exterior surface of the fluorescence imaging system, the hinge configured to pivot and fasten the work surface from a collapsed and / or folded state (FIG. 24A) to an unfolded and / or extended state (FIGS. 24B and 24C). In some cases, the work surface may comprise a sterilizable material (e.g., a biocompatible inert plastic and / or polymer).

[0105] In some cases, the fluorescence imaging system may include a sample retrieval hatch configured to provide access to the sample when a system failure occurs (e.g., a drawer does not open to remove the sample). In some cases, the sample retrieval hatch may be disposed on a surface of the imaging system enclosure. In some cases, the sample retrieval hatch may include a door and / or surface that can be manually operated by an individual, such as a user, a physician, a scrub medical personnel, a nurse, or any combination thereof, to access the sample. In some cases, the sample retrieval hatch may include a locking feature (e.g., a latch) configured to secure the sample retrieval hatch in a closed state when not operated by an individual, such as a user, a physician, a scrub medical personnel, a nurse, or any combination thereof.

[0106] In some cases, the imaging system (300, 2300) may include a sample height sensor 2235 as seen in FIG. 25 configured to repeatedly translate along a first planar axis of a surface on which the sample is disposed and / or a second axis normal to a planar surface containing the first axis to determine the presence of a sample within the field of view of the optical scanning element 112. The height of the sample determined by the sample height sensor may be used in determining the position of the optical scanning element 112 prior to scanning and / or imaging the sample. The position of the optical scanning element may be positioned such that the nominal depth of field of the optical scanning element is aligned with the highest point across the sample as determined by the sample height sensor. In some cases, the nominal depth of field of the optical scanning element may comprise a distance of up to about 8.5 mm from the surface of the optical scanning element 112. In some cases, the sample height sensor may comprise a light source 2236 and a detector 2234, as seen in FIG. 23, and the presence of the object and / or sample is determined when the detector 2239 does not detect light emitted from the light source 2236 (i.e., the light source is obstructed or blocked by the object and / or sample). In some cases, the light source 2236 may comprise an optical fiber. In some cases, the light source 2236 may comprise an infrared light source, a visible light source, or any combination thereof. In some cases, the light source may comprise a laser or a light emitting diode. The light source 2236 may comprise a collimated parallel beam light source. The detector 2239 may comprise one or more photodiode, CMOS, CCD, or any combination thereof sensors. In some cases, the sample height sensor may comprise a controller 2242 configured to be electrically and / or optically associated with the light source 2236, the detector 2234, the device controller 222, and / or the computer system 804. In some cases, the controller 2242 may couple light from a light source within the controller to the light source 2236 via a fiber.

[0107] The sample height sensor may be positioned at an offset distance 2237 from the surface of the optical scanning element 112, as seen in Figure 23. The offset distance 2237 of the sample height sensor from the surface of the optical scanning element 112 may allow the sample height sensor to translate in approximately 10 mm step increments to determine the height of the sample (e.g., a tissue sample) without damaging the sample and / or the optical scanning element 112. The offset distance may allow for gross movement of the sample along the second axis since there is a fixed clearance between the detection planes of the optics.

[0108] In some cases, the sample height sensor may translate in step increments of about 0.1 mm to about 14 mm. In some cases, the sample height sensor may translate in step increments of about 0.1 mm to about 0.5 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 1.5 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 2.5 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 5.5 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 14 mm, about 0.5 mm to about 1 mm, about 0.5 mm to about 1.5 mm, about 0.5 mm to about 2 mm, about 0.5 mm to about 2.5 mm. m, about 0.5mm to about 5mm, about 0.5mm to about 5.5mm, about 0.5mm to about 8mm, about 0.5mm to about 10mm, about 0.5mm to about 12mm, about 0.5mm to about 14mm, about 1mm to about 1.5mm, about 1mm to about 2mm, about 1mm ~2.5mm, 1mm~5mm, 1mm~5.5mm, 1mm~8mm, 1mm~10mm, 1mm~12mm, 1mm~14mm, 1.5mm~2mm, 1.5mm~2.5mm, 1.5mm~ Approximately 5mm, approximately 1.5mm to approximately 5.5mm, approximately 1.5mm to approximately 8mm, approximately 1.5mm to approximately 10mm, approximately 1.5mm to approximately 12mm, approximately 1.5mm to approximately 14mm, approximately 2mm to approximately 2.5mm, approximately 2mm to approximately 5mm, approximately 2mm to approximately 5.5mm, approximately 2mm to about 8mm, about 2mm to about 10mm, about 2mm to about 12mm, about 2mm to about 14mm, about 2.5mm to about 5mm, about 2.5mm to about 5.5mm, about 2.5mm to about 8mm, about 2.5mm to about 10mm, about 2.5mm to about 12mm, It may translate in step increments of about 2.5 mm to about 14 mm, about 5 mm to about 5.5 mm, about 5 mm to about 8 mm, about 5 mm to about 10 mm, about 5 mm to about 12 mm, about 5 mm to about 14 mm, about 5.5 mm to about 8 mm, about 5.5 mm to about 10 mm, about 5.5 mm to about 12 mm, about 5.5 mm to about 14 mm, about 8 mm to about 10 mm, about 8 mm to about 12 mm, about 8 mm to about 14 mm, about 10 mm to about 12 mm, about 10 mm to about 14 mm, or about 12 mm to about 14 mm.In some cases, the sample height sensor may translate in step increments of about 0.1 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm, about 5 mm, about 5.5 mm, about 8 mm, about 10 mm, about 12 mm, or about 14 mm. In some cases, the sample height sensor may translate in step increments of at least about 0.1 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm, about 5 mm, about 5.5 mm, about 8 mm, about 10 mm, or about 12 mm. In some cases, the sample height sensor may translate in step increments of up to about 0.5 mm, about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm, about 5 mm, about 5.5 mm, about 8 mm, about 10 mm, about 12 mm, or about 14 mm.

[0109] In some embodiments, the disclosure describes a method for determining the height of a sample. In some cases, the method may include (a) providing a sample on a surface; (b) translating a sample height sensor along a first axis parallel to the surface; and (c) translating the sample along a second axis normal to the surface when the sample height sensor detects a tissue blockage or its absence in a path between the sample height light source and the detector. In some cases, prior to (b), the sample may be translated away from or toward the sample height sensor along the second axis by at least about 1 mm, at least about 5 mm, at least about 10 mm, at least about 20 mm, at least about 30 mm, or at least about 40 mm. In some cases, steps (a)-(c) may be repeated one or more times. In some cases, (b)-(c) may be repeated one or more times. In some cases, while repeating steps (b)-(c), the sample is translated along the second axis by at least about 1 mm, at least about 5 mm, at least about 10 mm, at least about 20 mm, at least about 30 mm, or at least about 40 mm. In some cases, the translation of the sample may comprise translation of the sample in a first direction along the second axis and in a second direction along the second axis, the first direction and the second direction being opposite to each other. In some cases, the translation of the sample when repeating steps (b)-(c) of the method may alternate between the first direction and the second direction. In some cases, the translation of the sample when alternating between the first and second directions may comprise a first translated distance with respect to the first direction and a second translated distance with respect to the second direction, the first translated distance being greater than the second translated distance. In some cases, the method may include (d) determining a height of the sample when the difference between the first translated distance and the second translated distance of the sample is less than about 0.1 mm, less than about 1 mm, less than about 2 mm, or less than about 5 mm.In some cases, the method may further include (e) positioning the sample along a second axis, the height of the sample corresponding to a working distance of the optical scanning element. The working distance may comprise a plane and / or a point within the depth of field closest to the optical scanning element.

[0110] In some cases, the emitted beam 117 may be collected for further analysis by the signal collection subsystem 102. The signal collection subsystem may include collection optics 118, wavelength splitting element 120, detector 122, or any combination thereof. As shown in FIG. 1B and FIG. 25, the collection optics 118 may include one or more lenses and / or lens arrays (208, 204), optical fibers 206, multiple relay optics 2430, or any combination thereof. In some cases, the arrays configured to collect and / or relay the emitted fluorescent light by the lenses and / or lens arrays (208, 204) to the detector may include a double achromatic doublet pair, an objective lens, a scan lens, or any combination thereof. In some cases, as seen in FIG. 25, the multiple relay optics 2430 may include one or more optical elements configured to transmit and / or relay the autofluorescence light emitted from the sample collected by the collection optics 118 to the wavelength splitting element 120. In some cases, the collection. In some cases, the core size of the optical fiber 206 may allow image photons to be captured at various depths of field.

[0111] In some cases, the doublet achromatic doublet pair may have an outer diameter of about 1 inch to about 10 inches. In some cases, the doublet achromatic doublet pair may have an outer diameter of about 1 inch to about 1.5 inches, about 1 inch to about 2 inches, about 1 inch to about 2.5 inches, about 1 inch to about 3 inches, about 1 inch to about 3.5 inches, about 1 inch to about 4 inches, about 1 inch to about 5 inches, about 1 inch to about 6 inches, about 1 inch to about 8 inches, about 1 inch to about 9 inches, about 1 inch to about 10 inches, about 1.5 inches to about 2 inches, about 1.5 inches to about 2.5 inches, about 1.5 inches to about 3 inches, about 1.5 inches to about 3.5 inches, about 1.5 inches ... 2 inches to about 4 inches, about 1.5 inches to about 5 inches, about 1.5 inches to about 6 inches, about 1.5 inches to about 8 inches, about 1.5 inches to about 9 inches, about 1.5 inches to about 10 inches, about 2 inches to about 2.5 inches, about 2 inches to about 3 inches, about 2 inches to about 3.5 inches, about 2 inches to about 4 inches, about 2 inches to about 5 inches, about 2 inches to about 6 inches, about 2 inches to about 8 inches, about 2 inches to about 9 inches, about 2 inches to about 10 inches, about 2.5 inches to about 3 inches, about 2.5 inches to about 3.5 inches, about 2.5 inches to about 2.5 inches inch to about 4 inches, about 2.5 inches to about 5 inches, about 2.5 inches to about 6 inches, about 2.5 inches to about 8 inches, about 2.5 inches to about 9 inches, about 2.5 inches to about 10 inches, about 3 inches to about 3.5 inches, about 3 inches to about 4 inches, about 3 inches to about 5 inches, about 3 inches to about 6 inches, about 3 inches to about 8 inches, about 3 inches to about 9 inches, about 3 inches to about 10 inches, about 3.5 inches to about 4 inches, about 3.5 inches to about 5 inches, about 3.5 inches to about 6 inches, about 3.5 inches to about 8 inches, about It may have an outer diameter of 3.5 inches to about 9 inches, about 3.5 inches to about 10 inches, about 4 inches to about 5 inches, about 4 inches to about 6 inches, about 4 inches to about 8 inches, about 4 inches to about 9 inches, about 4 inches to about 10 inches, about 5 inches to about 6 inches, about 5 inches to about 8 inches, about 5 inches to about 9 inches, about 5 inches to about 10 inches, about 6 inches to about 8 inches, about 6 inches to about 9 inches, about 6 inches to about 10 inches, about 8 inches to about 9 inches, about 8 inches to about 10 inches, or about 9 inches to about 10 inches.In some cases, the double achromatic doublet pair may have an outer diameter of about 1 inch, about 1.5 inches, about 2 inches, about 2.5 inches, about 3 inches, about 3.5 inches, about 4 inches, about 5 inches, about 6 inches, about 8 inches, about 9 inches, or about 10 inches. In some cases, the double achromatic doublet pair may have an outer diameter of at least about 1 inch, about 1.5 inches, about 2 inches, about 2.5 inches, about 3 inches, about 3.5 inches, about 4 inches, about 5 inches, about 6 inches, about 8 inches, or about 9 inches. In some cases, the double achromatic doublet pair may have an outer diameter of at most about 1.5 inches, about 2 inches, about 2.5 inches, about 3 inches, about 3.5 inches, about 4 inches, about 5 inches, about 6 inches, about 8 inches, about 9 inches, or about 10 inches.

[0112] In some cases, the double achromatic doublet pair may have an outer diameter of about 1 inch (in) to about 10 inches. In some cases, the double achromatic doublet pair may have an outer diameter of about 1 inch to about 2 inches, about 1 inch to about 3 inches, about 1 inch to about 4 inches, about 1 inch to about 5 inches, about 1 inch to about 6 inches, about 1 inch to about 7 inches, about 1 inch to about 8 inches, about 1 inch to about 9 inches, about 1 inch to about 10 inches, about 2 inches to about 3 inches, about 2 inches to about 4 inches, about 2 inches to about 5 inches, about 2 inches to about 6 inches, about 2 inches to about 7 inches, about 2 inches to about 8 inches, about 2 inches to about 9 inches, about 2 inches to about 10 inches, about 3 inches to about 4 inches, about 3 inches to about 5 inches, about 3 inches to about 6 inches, about 3 inches to about 7 inches, about 3 inches to about 8 inches, about 3 inches to about 8 inches, about 3 inches to about 9 inches, about 3 inches to about 10 inches, about 3 inches to about 4 inches, about 3 inches to about 5 inches, about 3 inches to about 6 inches, about 3 inches to about 7 ...5 inches, about 3 inches to about 6 inches, about 3 inches to about 7 inches, about 3 inches to about 8 inches, about 3 inches to about 9 inches, about 3 inches to about The outer diameter may be about 1 to about 9 inches, about 3 inches to about 10 inches, about 4 inches to about 5 inches, about 4 inches to about 6 inches, about 4 inches to about 7 inches, about 4 inches to about 8 inches, about 4 inches to about 9 inches, about 4 inches to about 10 inches, about 5 inches to about 6 inches, about 5 inches to about 7 inches, about 5 inches to about 8 inches, about 5 inches to about 9 inches, about 5 inches to about 10 inches, about 6 inches to about 7 inches, about 6 inches to about 8 inches, about 6 inches to about 9 inches, about 6 inches to about 10 inches, about 7 inches to about 8 inches, about 7 inches to about 9 inches, about 7 inches to about 10 inches, about 8 inches to about 9 inches, about 8 inches to about 10 inches, or about 9 inches to about 10 inches. In some cases, the double achromatic doublet pair may have an outer diameter of about 1 inch, about 2 inches, about 3 inches, about 4 inches, about 5 inches, about 6 inches, about 7 inches, about 8 inches, about 9 inches, or about 10 inches. In some cases, the double achromatic doublet pair may have an outer diameter of at least about 1 inch, about 2 inches, about 3 inches, about 4 inches, about 5 inches, about 6 inches, about 7 inches, about 8 inches, or about 9 inches. In some cases, the double achromatic doublet pair may have an outer diameter of at most about 2 inches, about 3 inches, about 4 inches, about 5 inches, about 6 inches, about 7 inches, about 8 inches, about 9 inches, or about 10 inches.

[0113] In some cases, the collecting optics may have an F-number of about 1 to about 12. In some cases, the collecting optics may have an F-number of about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1 to about 6, about 1 to about 7, about 1 to about 8, about 1 to about 9, about 1 to about 10, about 1 to about 11, about 1 to about 12, about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2 to about 6, about 2 to about 7, about 2 to about 8, about 2 to about 9, about 2 to about 10, about 2 to about 11, about 2 to about 12, about 3 to about 4, about 3 to about 5, about 3 to about 6, about 3 to about 7, about 3 to about 8, about 3 to about 9, about 3 to about 10, about 3 to about 11, about 3 to about 12, about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about The F number may be 4 to about 9, about 4 to about 10, about 4 to about 11, about 4 to about 12, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 5 to about 11, about 5 to about 12, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 6 to about 11, about 6 to about 12, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 7 to about 11, about 7 to about 12, about 8 to about 9, about 8 to about 10, about 8 to about 11, about 8 to about 12, about 9 to about 10, about 9 to about 11, about 9 to about 12, about 10 to about 11, about 10 to about 12, or about 11 to about 12. In some cases, the collection optics may have an F-number of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12. In some cases, the collection optics may have an F-number of at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or about 11. In some cases, the collection optics may have an F-number of up to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12.

[0114] In some cases, the collection optics may have an F-number of about 1 to about 10. In some cases, the collection optics may be about 1 to about 1.5, about 1 to about 2, about 1 to about 2.5, about 1 to about 3, about 1 to about 3.5, about 1 to about 4, about 1 to about 5, about 1 to about 6, about 1 to about 8, about 1 to about 9, about 1 to about 10, about 1.5 to about 2, about 1.5 to about 2.5, about 1.5 to about 3, about 1.5 to about 3.5, about 1.5 to about 4, about 1.5 to about 5, about 1.5 to about 6, about 1.5 to about 8, about 1.5 to about 9, about 1.5 to about 10, about 2 to about 2.5, about 2 to about 3, about 2 to about 3.5, about 2 to about 4, about 2 to about 5, about 2 to about 6, about 2 to about 8, about 2 to about 9, about 2 to about 10, about 2.5 to about 3, about 2.5 to about 3.5, about It may have an F number of 2.5 to about 4, about 2.5 to about 5, about 2.5 to about 6, about 2.5 to about 8, about 2.5 to about 9, about 2.5 to about 10, about 3 to about 3.5, about 3 to about 4, about 3 to about 5, about 3 to about 6, about 3 to about 8, about 3 to about 9, about 3 to about 10, about 3.5 to about 4, about 3.5 to about 5, about 3.5 to about 6, about 3.5 to about 8, about 3.5 to about 9, about 3.5 to about 10, about 4 to about 5, about 4 to about 6, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 5 to about 6, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 8 to about 9, about 8 to about 10, or about 9 to about 10. In some cases, the collection optics may have an F-number of about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 5, about 6, about 8, about 9, or about 10. In some cases, the collection optics may have an F-number of at least about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 5, about 6, about 8, or about 9. In some cases, the collection optics may have an F-number of up to about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 5, about 6, about 8, about 9, or about 10.

[0115] In some cases, one or more lenses and / or lens arrays (204, 208) and / or one or more lenses of the relay optics 2430 may have an outer diameter of about 10 mm to about 220 mm.In some cases, the one or more lenses and / or lens arrays (204, 208) and / or one or more lenses of the plurality of relay optics 2430 may have a diameter of about 10 mm to about 20 mm, about 10 mm to about 30 mm, about 10 mm to about 40 mm, about 10 mm to about 50 mm, about 10 mm to about 100 mm, about 10 mm to about 120 mm, about 10 mm to about 140 mm, about 10 mm to about 160 mm, about 10 mm to about 180 mm, about 10 mm to about 200 mm, about 10 mm to about 220 mm, about 20 mm to about 30 mm, about 20 mm to about 40 mm, about 2 ...20 mm to about 60 mm, about 20 mm to about 60 mm, about 20 mm to about 70 mm, about 20 mm to about 80 mm, about 20 mm to about 90 mm, about 20 mm to about 100 mm, about 20 mm to about 120 mm, about 20 mm to about 140 mm, about 20 mm to about 160 mm, about 20 mm to about 180 mm, about 20 mm to about 180 mm, about 20 mm to about 190 mm, about 20 mm to about 0mm, approximately 20mm to approximately 50mm, approximately 20mm to approximately 100mm, approximately 20mm to approximately 120mm, approximately 20mm to approximately 140mm, approximately 20mm to approximately 160mm, approximately 20mm to approximately 180mm, approximately 20mm to approximately 200mm, approximately 20mm to approximately 220mm, approximately 30mm to approximately 40mm, approximately 30mm to about 50mm, about 30mm to about 100mm, about 30mm to about 120mm, about 30mm to about 140mm, about 30mm to about 160mm, about 30mm to about 180mm, about 30mm to about 200mm, about 30mm to about 220mm, about 40mm to about 50mm, about 40mm to about 100mm, about 40mm to about 120mm, about 40mm to about 140mm, about 40mm to about 160mm, about 40mm to about 180mm, about 40mm to about 200mm, about 40mm to about 220mm, about 50mm to about 100mm, about 50mm to about 120mm, about 50mm to about 140mm, about 50mm to about 160mm, about 50mm to about 180mm, about 50mm to about 200mm, about 50mm to about 220mm, about 100mm to about 120mm, about 100mm to about 140mm, about 100mm to about 160mm, about 100mm to about 180mm, about 100mm to about 20 The outer diameter may be about 100 mm, about 100 mm to about 220 mm, about 120 mm to about 140 mm, about 120 mm to about 160 mm, about 120 mm to about 180 mm, about 120 mm to about 200 mm, about 120 mm to about 220 mm, about 140 mm to about 160 mm, about 140 mm to about 180 mm, about 140 mm to about 200 mm, about 140 mm to about 220 mm, about 160 mm to about 180 mm, about 160 mm to about 200 mm, about 160 mm to about 220 mm, about 180 mm to about 200 mm, about 180 mm to about 220 mm, or about 200 mm to about 220 mm.In some cases, the one or more lenses and / or lens arrays (204, 208) and / or one or more lenses of the plurality of relay optics 2430 may have an outer diameter of about 10 mm, about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 100 mm, about 120 mm, about 140 mm, about 160 mm, about 180 mm, about 200 mm, or about 220 mm. In some cases, the one or more lenses and / or lens arrays (204, 208) and / or one or more lenses of the plurality of relay optics 2430 may have an outer diameter of at least about 10 mm, about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 100 mm, about 120 mm, about 140 mm, about 160 mm, about 180 mm, or about 200 mm. In some cases, the one or more lenses and / or lens arrays (204, 208) and / or one or more lenses of the plurality of relay optics 2430 may have an outer diameter of up to about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 100 mm, about 120 mm, about 140 mm, about 160 mm, about 180 mm, about 200 mm, or about 220 mm. In some cases, the collection optics 118 may collect and transmit to the wavelength splitting element 120 the autofluorescence light emitted from the tissue sample with an angular spread of about 2 degrees to about 16 degrees. In some cases, the collection optics 118 may be between about 2 degrees and about 4 degrees, between about 2 degrees and about 6 degrees, between about 2 degrees and about 8 degrees, between about 2 degrees and about 10 degrees, between about 2 degrees and about 12 degrees, between about 2 degrees and about 14 degrees, between about 2 degrees and about 16 degrees, between about 4 degrees and about 6 degrees, between about 4 degrees and about 8 degrees, between about 4 degrees and about 10 degrees, between about 4 degrees and about 12 degrees, between about 4 degrees and about 14 degrees, between about 4 degrees and about 16 degrees, between about 6 degrees and about 8 degrees, between about 6 degrees and about 10 degrees, between about 6 degrees and about 12 degrees, between about 6 degrees and about 16 degrees, Autofluorescent light emitted from the tissue sample with an angular spread of about 14 degrees, about 6 degrees to about 16 degrees, about 8 degrees to about 10 degrees, about 8 degrees to about 12 degrees, about 8 degrees to about 14 degrees, about 8 degrees to about 16 degrees, about 10 degrees to about 12 degrees, about 10 degrees to about 14 degrees, about 10 degrees to about 16 degrees, about 12 degrees to about 14 degrees, about 12 degrees to about 16 degrees, or about 14 degrees to about 16 degrees may be collected and transmitted to the wavelength division element 120.In some cases, the collection optics 118 may collect and transmit to the wavelength splitting element 120 the autofluorescence light emitted from the tissue sample with an angular spread of about 2 degrees, about 4 degrees, about 6 degrees, about 8 degrees, about 10 degrees, about 12 degrees, about 14 degrees, or about 16 degrees. In some cases, the collection optics 118 may collect and transmit to the wavelength splitting element 120 the autofluorescence light emitted from the tissue sample with an angular spread of at least about 2 degrees, about 4 degrees, about 6 degrees, about 8 degrees, about 10 degrees, about 12 degrees, or about 14 degrees. In some cases, the collection optics 118 may collect and transmit to the wavelength splitting element 120 the autofluorescence light emitted from the tissue sample with an angular spread of at most about 4 degrees, about 6 degrees, about 8 degrees, about 10 degrees, about 12 degrees, about 14 degrees, or about 16 degrees.

[0116] In some cases, the collection optics 118 configured to capture fluorescent light emitted from the tissue sample may have a numerical aperture of about 0.1 to about 0.4. In some cases, the collection optics 118 configured to capture fluorescent light emitted from the tissue sample may have a numerical aperture of about 0.1 to about 0.12, about 0.1 to about 0.14, about 0.1 to about 0.18, about 0.1 to about 0.2, about 0.1 to about 0.22, about 0.1 to about 0.26, about 0.1 to about 0.28, about 0.1 to about 0.3, about 0.1 to about 0.34, about 0.1 to about 0.36, about 0.1 to about 0.4, about 0.12 to about 0.14, about 0.12 to about 0.18, about 0.12 to about 0.2 ...2, about 0.1 to about 0.26, about 0.1 to about 0.28, about 0.1 to about 0.3, about 0.1 to about 0.34, about 0.1 to about 0.36, about 0.1 to about 0.4, about 0. 0.12 to about 0.22, about 0.12 to about 0.26, about 0.12 to about 0.28, about 0.12 to about 0.3, about 0.12 to about 0.34, about 0.12 to about 0.36, about 0.12 to about 0.4, about 0.14 to about 0.18, about 0.14 to about 0.2, about 0.14 to about 0.22, about 0.14 to about 0.26, about 0.14 to about 0.28, about 0.14 to about 0.3, about 0.14 to about 0.34, about 0.14 to about 0.36, about 0.14 to about 0.4, about 0.18 to about 0.2, about 0.18 to about about 0.22, about 0.18 to about 0.26, about 0.18 to about 0.28, about 0.18 to about 0.3, about 0.18 to about 0.34, about 0.18 to about 0.36, about 0.18 to about 0.4, about 0.2 to about 0.22, about 0.2 to about 0.26, about 0.2 to about 0.28, about 0.2 to about 0.3, about 0.2 to about 0.34, about 0.2 to about 0.36, about 0.2 to about 0.4, about 0.22 to about 0.26, about 0.22 to about 0.28, about 0.22 to about 0.3, about 0.22 to about 0.34, about 0.2 The aperture may have a numerical aperture of 2 to about 0.36, about 0.22 to about 0.4, about 0.26 to about 0.28, about 0.26 to about 0.3, about 0.26 to about 0.34, about 0.26 to about 0.36, about 0.26 to about 0.4, about 0.28 to about 0.3, about 0.28 to about 0.34, about 0.28 to about 0.36, about 0.28 to about 0.4, about 0.3 to about 0.34, about 0.3 to about 0.36, about 0.3 to about 0.4, about 0.34 to about 0.36, about 0.34 to about 0.4, or about 0.36 to about 0.4.In some cases, the collection optics 118 configured to capture fluorescent light emitted from the tissue sample may comprise a numerical aperture of about 0.1, about 0.12, about 0.14, about 0.18, about 0.2, about 0.22, about 0.26, about 0.28, about 0.3, about 0.34, about 0.36, or about 0.4. In some cases, the collection optics 118 configured to capture fluorescent light emitted from the tissue sample may comprise a numerical aperture of at least about 0.1, about 0.12, about 0.14, about 0.18, about 0.2, about 0.22, about 0.26, about 0.28, about 0.3, about 0.34, or about 0.36. In some cases, the collection optics 118, configured to capture fluorescent light emitted from the tissue sample, may comprise a numerical aperture of up to about 0.12, about 0.14, about 0.18, about 0.2, about 0.22, about 0.26, about 0.28, about 0.3, about 0.34, about 0.36, or about 0.4.

[0117] In some cases, the lens and / or lens array (204, 208) and / or relay optics 2430 may be configured to collect and / or relay fluorescent light emitted by the sample at a beam spot of about 2 mm to about 14 mm to the PMT 122. In some cases, the lens and / or lens array (204, 208) and / or relay optics 2430 may be configured to collect and / or relay fluorescent light emitted by the sample at a beam spot of about 2 mm to about 3 mm, about 2 mm to about 4 mm, about 2 mm to about 5 mm, about 2 mm to about 6 mm, about 2 mm to about 7 mm, about 2 mm to about 8 mm, about 2 mm to about 9 mm, about 2 mm to about 10 mm, about 2 mm to about 11 mm, about 2 mm to about 12 mm, about 2 mm to about 14 mm, about 3 mm to about 4 mm, about 3 mm to about 5 mm, about 3 mm to about 6 mm, about 3 mm to about 7 mm, about 3 mm to about 8 mm, about 3 mm to about 9 mm, about 3 ...14 mm, about 3 mm to about 16 mm, about 3 mm to about 18 mm, about 3 mm to about 19 mm, about 3 mm to about 20 mm, about 3 mm to about 21 mm, about 3mm to about 8mm, about 3mm to about 9mm, about 3mm to about 10mm, about 3mm to about 11mm, about 3mm to about 12mm, about 3mm to about 14mm, about 4mm to about 5mm, about 4mm to about 6mm, about 4mm to about 7mm, about 4mm Approximately 8mm, approximately 4mm to approximately 9mm, approximately 4mm to approximately 10mm, approximately 4mm to approximately 11mm, approximately 4mm to approximately 12mm, approximately 4mm to approximately 14mm, approximately 5mm to approximately 6mm, approximately 5mm to approximately 7mm, approximately 5mm to approximately 8mm, approximately 5mm to approximately 9mm, approximately 5m m to about 10mm, about 5mm to about 11mm, about 5mm to about 12mm, about 5mm to about 14mm, about 6mm to about 7mm, about 6mm to about 8mm, about 6mm to about 9mm, about 6mm to about 10mm, about 6mm to about 11mm, about 6mm to about 12mm, about 6mm to about 14mm, about 7mm to about 8mm, about 7mm to about 9mm, about 7mm to about 10mm, about 7mm to about 11mm, about 7mm to about 12mm, about 7mm to about 14mm, about 8mm to about 9mm, about 8mm to about 10mm, about 8mm The PMT may be configured to collect and / or relay to the PMT fluorescent light emitted by the sample in a beam spot of about 11 mm, about 8 mm to about 12 mm, about 8 mm to about 14 mm, about 9 mm to about 10 mm, about 9 mm to about 11 mm, about 9 mm to about 12 mm, about 9 mm to about 14 mm, about 10 mm to about 11 mm, about 10 mm to about 12 mm, about 10 mm to about 14 mm, about 11 mm to about 12 mm, about 11 mm to about 14 mm, or about 12 mm to about 14 mm.In some cases, the lens and / or lens array (204, 208) and / or relay optics 2430 may be configured to collect and / or relay to the PMT fluorescent light emitted by the sample at a beam spot of about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, or about 14 mm. In some cases, the lens and / or lens array (204, 208) and / or relay optics 2430 may be configured to collect and / or relay to the PMT fluorescent light emitted by the sample at a beam spot of at least about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, or about 12 mm. In some cases, the lens and / or lens array (204, 208) and / or multiple relay optics 2430 may be configured to collect and / or relay to the PMT fluorescent light emitted by the sample in a beam spot of up to about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, or about 14 mm.

[0118] In some cases, the optical fiber 206 may have a length of about 0.3 meters (m) to about 10 m. In some cases, the optical fiber 206 may have a length of about 0.3 m to about 0.5 m, about 0.3 m to about 0.7 m, about 0.3 m to about 1 m, about 0.3 m to about 2 m, about 0.3 m to about 3 m, about 0.3 m to about 4 m, about 0.3 m to about 5 m, about 0.3 m to about 6 m, about 0.3 m to about 7 m, about 0.3 m to about 8 m, about 0.3 m to about 10 m, about 0.5 m to about 0.7 m, about 0.5 m to about 1 m, about 0.5m~2m, 0.5m~3m, 0.5m~4m, 0.5m~5m, 0.5m~6m, 0.5m~7m, 0.5m~8m, 0.5m~10m, Approximately 0.7m to approximately 1m, approximately 0.7m to approximately 2m, approximately 0.7m to approximately 3m, approximately 0.7m to approximately 4m, approximately 0.7m to approximately 5m, approximately 0.7m to approximately 6m, approximately 0.7m to approximately 7m, approximately 0.7m to approximately 8m, Approximately 0.7m to approximately 10m, approximately 1m to approximately 2m, approximately 1m to approximately 3m, approximately 1m to approximately 4m, approximately 1m to approximately 5m, approximately 1m to approximately 6m, approximately 1m to approximately 7m, approximately 1m to approximately 8m, approximately 1m to approximately 10m, approximately 2m Approximately 3m, approximately 2m to approximately 4m, approximately 2m to approximately 5m, approximately 2m to approximately 6m, approximately 2m to approximately 7m, approximately 2m to approximately 8m, approximately 2m to approximately 10m, approximately 3m to approximately 4m, approximately 3m to approximately 5m, approximately 3m to approximately 6m, approximately 3m The optical fiber 206 may have a length of about 0.3 m, about 0.5 m, about 0.7 m, about 1 m, about 2 m, about 3 m, about 4 m, about 5 m, about 6 m, about 7 m, about 8 m, about 10 m, about 4 m, about 5 m, about 6 m, about 7 m, about 8 m, about 10 m, about 7 m, about 8 m, about 10 m, about 10 m, about 7 m, about 8 m, about 10 m, or about 10 m. In some cases, the optical fiber 206 may have a length of about 0.3 m, about 0.5 m, about 0.7 m, about 1 m, about 2 m, about 3 m, about 4 m, about 5 m, about 6 m, about 7 m, about 8 m, or about 10 m. In some cases, the optical fiber 206 may comprise a length of at least about 0.3 m, about 0.5 m, about 0.7 m, about 1 m, about 2 m, about 3 m, about 4 m, about 5 m, about 6 m, about 7 m, or about 8 m. In some cases, the optical fiber 206 may comprise a length of up to about 0.5 m, about 0.7 m, about 1 m, about 2 m, about 3 m, about 4 m, about 5 m, about 6 m, about 7 m, about 8 m, or about 10 m.

[0119] In some cases, the optical fiber 206 may have a core size of about 10 micrometers (μm) to about 10,000 μm. In some cases, the optical fiber 206 may have a thickness of about 10 μm to about 20 μm, about 10 μm to about 50 μm, about 10 μm to about 100 μm, about 10 μm to about 500 μm, about 10 μm to about 1,000 μm, about 10 μm to about 2,000 μm, about 10 μm to about 4,000 μm, about 10 μm to about 6,000 μm, about 10 μm to about 8,000 μm, about 10 μm to about 10,000 μm, about 20 μm to about 50 μm, about 20 μm to about 100 μm, about 20 μm to about 500 μm, about 20 μm to about 1,000 μm, about 20 μm to about 2,000 μm. , about 20μm to about 4,000μm, about 20μm to about 6,000μm, about 20μm to about 8,000μm, about 20μm to about 10,000μm, about 50μm to about 100μm, about 50μm to about 500μm, about 50μm to about 1,000μm, about 50μm to about 2,0 00μm, approx. 50μm to approx. 4,000μm, approx. 50μm to approx. 6,000μm, approx. 50μm to approx. 8,000μm, approx. 50μm to approx. 10,000μm, approx. 100μm to approx. 4,000μm, approx. 100μm to approx. 6,000μm, approx. 100μm to approx. 8,000μm, approx. 100μm to approx. 10,000μm, approx. 500μm to approx. 1,000μm, approx. 500μm to about 6,000μm, about 500μm to about 8,000μm, about 500μm to about 10,000μm, about 1,000μm to about 2,000μm, about 1,000μm to about 4,000μm, about 1,000μm to about 6,000μm, about 1,000μm to about 8 The core size may be about 1,000 μm, about 1,000 μm to about 10,000 μm, about 2,000 μm to about 4,000 μm, about 2,000 μm to about 6,000 μm, about 2,000 μm to about 8,000 μm, about 2,000 μm to about 10,000 μm, about 4,000 μm to about 6,000 μm, about 4,000 μm to about 8,000 μm, about 4,000 μm to about 10,000 μm, about 6,000 μm to about 8,000 μm, about 6,000 μm to about 10,000 μm, or about 8,000 μm to about 10,000 μm.In some cases, the optical fiber 206 may comprise a core size of about 10 μm, about 20 μm, about 50 μm, about 100 μm, about 500 μm, about 1,000 μm, about 2,000 μm, about 4,000 μm, about 6,000 μm, about 8,000 μm, or about 10,000 μm. In some cases, the optical fiber 206 may comprise a core size of at least about 10 μm, about 20 μm, about 50 μm, about 100 μm, about 500 μm, about 1,000 μm, about 2,000 μm, about 4,000 μm, about 6,000 μm, or about 8,000 μm. In some cases, the optical fiber 206 may comprise a core size of up to about 20 μm, about 50 μm, about 100 μm, about 500 μm, about 1,000 μm, about 2,000 μm, about 4,000 μm, about 6,000 μm, about 8,000 μm, or about 10,000 μm.

[0120] In some cases, the optical fiber 206 may provide a depth of field of about 0.01 mm to about 20 mm. In some cases, the optical fiber 206 may provide a depth of field of about 0.01 mm to about 0.1 mm, about 0.01 mm to about 1 mm, about 0.01 mm to about 5 mm, about 0.01 mm to about 7 mm, about 0.01 mm to about 9 mm, about 0.01 mm to about 12 mm, about 0.01 mm to about 14 mm, about 0.01 mm to about 16 mm, about 0.01 mm to about 18 mm, about 0.01 mm to about 20 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 20 mm, about 1 mm to about 5 mm, about 1 mm to about 7 mm, about 1 mm to about 9 mm, about 1 mm to about 12 mm, about 1 mm to about 14 mm, about 1 mm to about 16 mm, about 1 mm to about 18 mm, About 1mm to about 20mm, about 5mm to about 7mm, about 5mm to about 9mm, about 5mm to about 12mm, about 5mm to about 14mm, about 5mm to about 16mm, about 5mm to about 18mm, about 5mm to about 20mm, about 7mm to about 9mm, about 7mm to about 12mm, about 7mm to about 14mm, about 7mm to about 16mm, about 7mm to about 18mm, about 7mm to about 20mm, about 9mm to about 12mm, about 9mm to about 14mm m, about 9 mm to about 16 mm, about 9 mm to about 18 mm, about 9 mm to about 20 mm, about 12 mm to about 14 mm, about 12 mm to about 16 mm, about 12 mm to about 18 mm, about 12 mm to about 20 mm, about 14 mm to about 16 mm, about 14 mm to about 18 mm, about 14 mm to about 20 mm, about 16 mm to about 18 mm, about 16 mm to about 20 mm, or about 18 mm to about 20 mm. In some cases, optical fiber 206 may provide a depth of field of about 0.01 mm, about 0.1 mm, about 1 mm, about 5 mm, about 7 mm, about 9 mm, about 12 mm, about 14 mm, about 16 mm, about 18 mm, or about 20 mm. In some cases, the optical fiber 206 may provide a depth of field of at least about 0.01 mm, about 0.1 mm, about 1 mm, about 5 mm, about 7 mm, about 9 mm, about 12 mm, about 14 mm, about 16 mm, or about 18 mm.In some cases, the optical fiber 206 may provide a depth of field of up to about 0.1 mm, about 1 mm, about 5 mm, about 7 mm, about 9 mm, about 12 mm, about 14 mm, about 16 mm, about 18 mm, or about 20 mm.

[0121] In some cases, the optical fiber 206 may have a numerical aperture of about 0.12 to about 0.5. In some cases, the optical fiber 206 may have a numerical aperture of about 0.12 to about 0.2, about 0.12 to about 0.25, about 0.12 to about 0.3, about 0.12 to about 0.35, about 0.12 to about 0.4, about 0.12 to about 0.45, about 0.12 to about 0.5, about 0.2 to about 0.25, about 0.2 to about 0.3, about 0.2 to about 0.35, about 0.2 to about 0.4, about 0.2 to about 0.45, about 0.2 to about 0.5, about 0.25 to about 0. In some cases, the optical fiber 206 may have a numerical aperture of about 0.12, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.25, about 0.45, about 0.25, about 0.5, about 0.3, about 0.3, about 0.4, about 0.3, about 0.45, about 0.3, about 0.5, about 0.35, about 0.4, about 0.35, about 0.45, about 0.35, about 0.5, about 0.4, about 0.4, about 0.5, or about 0.45 to about 0.5. In some cases, the optical fiber 206 may have a numerical aperture of at least about 0.12, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, or about 0.45. In some cases, the optical fiber 206 may have a numerical aperture of at most about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, or about 0.5.

[0122] In some cases, the optical fiber 206 may comprise a single mode, polarization maintaining, photonic crystal, multimode, or any combination thereof fiber. In some cases, the collection optics may comprise one or more plano-convex, bi-convex, bi-concave, or plano-concave lenses. In some cases, the optical fiber 206 may comprise one or more fibers, e.g., a fiber bundle. In some cases, the fiber bundle may comprise at least one fiber.

[0123] In some cases, the signal collection subsystem 102 may include a wavelength splitting element 120 that may split the emitted beam 117 into multiple beams in different wavelength ranges of interest. The wavelength splitting element 120 may include a filter wheel, such as a rotatable wheel of optical filters to allow only certain wavelength ranges to pass therethrough at a given time, or a demultiplexer, for example, including an array of filters and mirrors to split the emitted beam 117 into wavelength ranges. In some cases, the filter wheel may be rotated continuously or at a specific rate. In some cases, the filter wheel may be rotated at least one full and / or partial rotation of the filter wheel in at least about 1 second, at least about 2 seconds, at least about 3 seconds, at least about 4 seconds. In some cases, the filter wheel may be rotated such that each filter is placed in the path of the emitted fluorescent light of the sample for about 2 seconds. The wavelength splitting element 120 may include one or more filters with one or more emission cutoff wavelengths. The wavelength splitting element 120 may comprise one or more filters that may filter the emitted beam 117 into up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or more emission channels. The emission channels may comprise wavelength ranges from about 365 nm to about 410 nm, about 410 nm to about 450 nm, about 450 nm to about 480 nm, about 500 nm to about 560 nm, about 560 nm to about 600 nm, and about 600 nm or greater. In some cases, the splitting element 120 may comprise a filter wheel that may rotate multiple filters as the imaging system is imaging the tissue sample to generate a signal for each emission channel.

[0124] In some cases, the filters of one or more filters may provide upper and lower wavelength cutoffs for a wavelength transmission band for the filter.

[0125] In some cases, the one or more filters may have a wavelength of at most about 400 nm, at most about 402 nm, at most about 404 nm, at most about 408 nm, at most about 410 nm, at most about 412 nm, at most about 414 nm, at most about 418 nm, at most about 420 nm, at most about 422 nm, at most about 424 nm, at most about 426 nm, at most about 428 nm, at most about 430 nm, at most about 432 nm, at most about 434 nm, at most about 436 nm, at most about 438 nm, at most about 440 nm, at most about 444 nm, at most about 446 nm, at most about 448 nm, at most about 450 nm, at most about 452 nm, at most about 454 nm, at most about 456 nm, at most about 458 nm, at most about 459 nm, at most about 460 nm, at most about 461 nm, at most about 462 nm, at most about 463 nm, at most about 464 nm, at most about 465 nm, at most about 466 nm, at most about 467 nm, at most about 468 nm, at most about 469 nm, at most about 470 nm, at most about 471 nm, at most about 472 nm, at most about 473 nm, at most about 474 nm, at most about 475 nm, at most about 476 nm, at most about 477 nm, at most about 478 nm, at most about 479 nm, at most about 480 nm, at most about 481 nm, at most about 482 nm, at most about 483 nm, at most about 484 nm, at most about 485 nm, at most about 486 nm, at most 48nm, up to about 450nm, up to about 452nm, up to about 454nm, up to about 456nm, up to about 458nm, up to about 460nm, up to about 462nm, up to about 464nm, up to about 466, up to about 468nm, up to about 470nm, up to about 472nm, up to about 474nm, up to about 476nm, up to about 478nm, up to about 480nm, up to about 482nm, up to about 484nm, up to about 486nm, up to about 488nm, up to about 490nm, up to about 492nm, up to about 494nm, up to about 496nm, up to 498nm at maximum, 500nm at maximum, 502nm at maximum, 504nm at maximum, 506nm at maximum, 508nm at maximum, 510nm at maximum, 512nm at maximum, 514nm at maximum, 516nm at maximum, 518nm at maximum, 520nm at maximum, 522nm at maximum, 524nm at maximum, 526nm at maximum, 528nm at maximum, 530nm at maximum, 532nm at maximum, 534nm at maximum, 536nm at maximum, 538nm at maximum, 540nm at maximum, 542nm at maximum, 544nm at maximum, 546nm, up to about 548nm, up to about 550nm, up to about 552nm, up to about 554nm, up to about 580nm, up to about 582nm, up to about 584nm, up to about 586nm, up to about 588nm, up to about 590nm, up to about 592nm, up to about 594nm, up to about 596nm, up to about 598nm, up to about 600nm, up to 602nm, up to 604nm, up to about 606nm, up to about 608nm, up to about 610nm, up to about 612nm, up to about 614nm, up to about 616nm, up to about 618nm,Or up to about 620 nm upper wavelength cutoff. In some cases, one or more filters of the wavelength division element may have different upper wavelength cutoff values, as described elsewhere herein.

[0126] In some cases, the filter of one or more filters is at least about 358 nm, at least about 360 nm, at least about 362 nm, at least about 364 nm, at least about 366 nm, at least about 368 nm, at least about 370 nm, at least about 372 nm, at least about 374 nm, at least about 376 nm, at least about 378 nm, at least about 380 nm, at least about 382 nm, at least about 384 nm, at least about 386 nm, at least about 388 nm, at least about 390 nm, at least about 392 nm, at least about 396 nm, at least about 398 nm, at least about 398 nm, at least about 399 nm, at least about 400 nm, at least about 401 nm, at least about 402 nm, at least about 403 nm, at least about 404 nm, at least about 405 nm, at least about 406 nm, at least about 407 nm, at least about 408 nm, at least about 409 nm, at least about 410 nm, at least about 411 nm, at least about 412 nm, at least about 413 nm, at least about 414 nm, at least about 415 nm, at least about 416 nm, at least about 417 nm, at least about 418 nm, at least about 419 nm, at least about 420 nm, at least about 425 nm, at least about 426 nm, at least about 427 nm, at least about 428 nm, at least about 429 nm, at least about 430 nm, at least about 431 nm, at least about 432 nm, at least about 433 nm, at least about 434 nm, at least about 435 nm, at least about 436 nm, at least about at least about 394 nm, at least about 396 nm, at least about 398 nm, at least about 400 nm, at least about 402 nm, at least about 404 nm, at least about 408 nm, at least about 410 nm, at least about 412 nm, at least about 414 nm, at least about 418 nm, at least about 420 nm, at least about 422 nm, at least about 424 nm, at least about 426 nm, at least about 428 nm, at least about 430 nm, at least about 432 nm, at least about 434 nm, at least about 436 nm, at least about 438 nm, at least about 440 nm, at least about 444 nm, at least about 446 nm, at least about 448 nm, at least about 450 nm, at least about 452 nm, at least about 454 nm, at least about 456 nm, at least about 458 nm, at least about 460 nm, at least about 462 nm, at least about 464 nm, at least about 466, at least about 468 nm, at least about 470 nm, at least about 472 nm, at least about 474 nm, at least about 476 nm, at least about 478 nm, at least about 480 nm, at least about 482 nm, at least about 484 nm, at least about 486 nm, at least about 488 nm, at least about 490 nm, at least about 492 nm, at least about 494 nm, at least about 496 nm, at least about 498 nm, at least about 500 nm, at least about 502 nm, at least about 504 nm, at least about 506 nm, at least about 508 nm, at least about 510 nm, at least about 512 nm, at least about 514 nm, at least about 516 nm, at least about 518 nm, at least about 520 nm, at least about 522 nm, at least about 524 nm,at least about 526 nm, at least about 528 nm, at least about 530 nm, at least about 532 nm, at least about 534 nm, at least about 536 nm, at least about 538 nm, at least about 540 nm, at least about 542 nm, at least about 544 nm, at least about 546 nm, at least about 548 nm, at least about 550 nm, at least about 552 nm, at least about 554 nm, at least about 580 nm, at least about 582 nm, at least about 584 nm, at least about 586 nm, at least about 588 nm, at least about 590 nm, at least about 592 nm, at least about 594 nm, at least about 596 nm, at least about 598 nm, at least about 600 nm, at least 602 nm, at least 604 nm, at least about 606 nm, at least about 608 nm, at least about 610 nm, at least about 612 nm, at least about 614 nm, at least about 616 nm, at least about 618 nm, or at least about 620 nm. In some cases, one or more filters of the wavelength division element may each have a different lower wavelength cutoff value, as described elsewhere herein.

[0127] In some cases, the filter wheel may comprise multiple spectral filters. Passing the emitted beam 117 sequentially through the spectral filters of the filter wheel to generate the spectral bands may impart a predetermined time delay between the spectral bands generated by the different spectral filters. The filter wheel may comprise multiple encoders, with each spectral filter associated with at least one encoder. The filter wheel comprises a rotating filter wheel. The optical assembly may further comprise a mirror galvanometer for selectively focusing the responsive optical signal onto at least one spectral filter of the filter wheel.

[0128] In some cases, the spectral band resulting from the emitted beam 117 traversing through one or more filters of the filter wheel may be in the range of about 370 nm to about 900 nm. The spectral band may be in the range of about 365 nm or less, about 365 nm to about 410 nm, about 410 nm to about 450 nm, about 450 nm to about 480 nm, about 500 nm to about 560 nm, about 560 nm to about 600 nm, and about 600 nm or more. The spectral band may be in the range of about 400 nm or less, about 415 nm to about 450 nm, about 455 nm to about 480 nm, and about 500 nm or more.

[0129] In some cases, the emitted beam 117 may comprise one or more of a fluorescent spectrum, a Raman spectrum, a UV-visible spectrum, or an infrared spectrum.

[0130] In some cases, the light source 106 may emit pulses of light in the ultraviolet, visible, near-infrared, or infrared spectrum.

[0131] In some cases, light source 106 may emit light in a wavelength band within the range of about 300 nm to about 1,100 nm. Light source 106 may emit light in a wavelength band within the range of about 330 nm to about 360 nm, about 420 nm to about 450 nm, about 660 nm to about 720 nm, or about 750 nm to about 780 nm.

[0132] In some cases, the signal collection subsystem 102 may include a detector, which may include a photomultiplier tube (PMT) 122, a PIN detector, an avalanche photodiode, or any combination thereof. The photomultiplier tube 122 may detect and convert the optical light energy of the emitted beam 117 into an electrical signal. The gain of the PMT may be adjusted by a voltage power supply 220 capable of providing a modular voltage output.

[0133] In some cases, the active area of ​​the detector is pi × (d2) / 4, where d may comprise the diameter of the active area of ​​the detector. In some cases, the diameter of the active area of ​​the detector, d, may be from about 50 μm to about 50,000 μm. In some cases, the diameter of the detector's active area, d, is from about 50 μm to about 125 μm, from about 50 μm to about 400 μm, from about 50 μm to about 1,000 μm, from about 50 μm to about 2,000 μm, from about 50 μm to about 10,000 μm, from about 50 μm to about 12,000 μm, from about 50 μm to about 20,000 μm, from about 50 μm to about 30,000 μm, from about 50 μm to about 45,000 μm, from about 50 μm to about 50,000 μm, from about 125 μm to about 400 μm, from about 125 μm to about 1,000 μm, from about 125 μm to about 2,000 μm, from about 125 μm to about 30,000 μm, Approximately 10,000μm, approximately 125μm to approximately 12,000μm, approximately 125μm to approximately 20,000μm, approximately 125μm to approximately 30,000μm, approximately 125μm to approximately 45,000μm, approximately 125μm to approximately 50,000μm, approximately 400μm to approximately 1,000μm, approximately 400μm μm ~ approx. 2,000 μm, approx. 400 μm ~ approx. 10,000 μm, approx. 400 μm ~ approx. 12,000 μm, approx. 400 μm ~ approx. 20,000 μm, approx. 1,000μm to about 2,000μm, about 1,000μm to about 10,000μm, about 1,000μm to about 12,000μm, about 1,000μm to about 20,000μm, about 1,000μm to about 30,000μm, about 1,000μm to about 45,000μm, about 1 ,000μm~about 50,000μm, about 2,000μm~about 10,000μm, about 2,000μm~about 12,000μm, about 2,000μm~about 20,000μm, about 2,000μm~about 30,000μm, about 2,000μm~about 45,000μm, about 2, 000μm~about 50,000μm, about 10,000μm~about 12,000μm, about 10,000μm~about 20,000μm, about 10,000μm~about 30,000μm, about 10,000μm~about 45,000μm, about 10,000μm~about 50,000μm , about 12,000 μm to about 20,000 μm, about 12,000 μm to about 30,000 μm, about 12,000 μm to about 45,000 μm, about 12,000 μm to about 50,000 μm, about 20,000 μm to about 30,000 μm, about 20,000 μm to about 45In some cases, the diameter d of the detector active area may be about 50 μm, about 125 μm, about 400 μm, about 1,000 μm, about 2,000 μm, about 10,000 μm, about 12,000 μm, about 20,000 μm, about 30,000 μm, about 45,000 μm, about 30,000 μm, about 50,000 μm, or about 45,000 μm to about 50,000 μm. In some cases, the diameter of the detector active area, d, may be at least about 50 μm, about 125 μm, about 400 μm, about 1,000 μm, about 2,000 μm, about 10,000 μm, about 12,000 μm, about 20,000 μm, about 30,000 μm, or about 45,000 μm. In some cases, the diameter of the detector active area, d, may be at least about 125 μm, about 400 μm, about 1,000 μm, about 2,000 μm, about 10,000 μm, about 12,000 μm, about 20,000 μm, about 30,000 μm, about 45,000 μm, or about 50,000 μm.

[0134] In some cases, an acceptable cone angle for the detector may be from about -70 degrees to about 0 degrees. In some cases, an acceptable cone angle for the detector may be from about 0 degrees to about -10 degrees, from about 0 degrees to about -15 degrees, from about 0 degrees to about -20 degrees, from about 0 degrees to about -25 degrees, from about 0 degrees to about -30 degrees, from about 0 degrees to about -35 degrees, from about 0 degrees to about -40 degrees, from about 0 degrees to about -45 degrees, from about 0 degrees to about -50 degrees, from about 0 degrees to about -60 degrees, from about 0 degrees to about -70 degrees, from about -10 degrees to about -15 degrees, from about -10 degrees to about -20 degrees, from about -10 degrees to about -25 degrees, from about -10 degrees to about -30 degrees, from about -10 degrees to about -35 degrees, from about -10 degrees to about -40 degrees, from about -1 ... -10 degrees to about -50 degrees, from about -10 degrees to about -60 degrees, from about -10 degrees to about -70 degrees, from about -10 degrees to about -15 degrees, from about -10 degrees to about -20 degrees, from about -10 degrees to about -25 degrees, from about -10 degrees to about -30 degrees, from about -10 degrees to about -35 degrees, from about -10 degrees to about -40 degrees, from about -1 0 degrees, approx. -10 degrees to approx. -45 degrees, approx. -10 degrees to approx. -50 degrees, approx. -10 degrees to approx. -60 degrees, approx. -10 degrees to approx. -70 degrees, approx. -15 degrees to approx. -20 degrees, approx. -15 degrees to approx. -25 degrees, approx. -15 degrees to approx. -30 degrees, approx. -15 degrees to approx. -35 degrees, approx. -15 degrees to approx. -40 degrees, approx. -15 degrees to approx. -45 degrees, approx. -15 degrees to approx. -50 degrees, approx. -15 degrees to approx. -60 degrees, approx. -15 degrees to approx. -70 degrees, approx. -20 degrees to approx. -25 degrees, approx. -20 degrees to approx. -30 degrees, approx. -20 degrees to approx. -35 degrees, approx. -20 degrees ~-40 degrees, ~-20 degrees to ~-45 degrees, ~-20 degrees to ~-50 degrees, ~-20 degrees to ~-60 degrees, ~-20 degrees to ~-70 degrees, ~-25 degrees to ~-30 degrees, ~-25 degrees to ~-35 degrees, ~-25 degrees to ~-40 degrees, ~-25 degrees to ~-45 degrees, ~-25 degrees to ~-50 degrees, ~-25 degrees to ~-60 degrees, ~-25 degrees to ~-70 degrees, ~-30 degrees to ~-35 degrees, ~-30 degrees to ~-40 degrees, ~-30 degrees to ~-45 degrees, ~-30 degrees to ~-50 degrees, ~-30 degrees to ~-60 degrees, The temperature may be from -30 degrees to about -70 degrees, from about -35 degrees to about -40 degrees, from about -35 degrees to about -45 degrees, from about -35 degrees to about -50 degrees, from about -35 degrees to about -60 degrees, from about -35 degrees to about -70 degrees, from about -40 degrees to about -45 degrees, from about -40 degrees to about -50 degrees, from about -40 degrees to about -60 degrees, from about -40 degrees to about -70 degrees, from about -45 degrees to about -50 degrees, from about -45 degrees to about -60 degrees, from about -45 degrees to about -70 degrees, from about -50 degrees to about -60 degrees, from about -50 degrees to about -70 degrees, or from about -60 degrees to about -70 degrees. In some cases, an acceptable cone angle for a detector may be about 0 degrees, about -10 degrees, about -15 degrees, about -20 degrees, about -25 degrees, about -30 degrees, about -35 degrees, about -40 degrees, about -45 degrees, about -50 degrees, about -60 degrees, or about -70 degrees.In some cases, the acceptable cone angle of the detector may be at least about 0 degrees, about -10 degrees, about -15 degrees, about -20 degrees, about -25 degrees, about -30 degrees, about -35 degrees, about -40 degrees, about -45 degrees, about -50 degrees, or about -60 degrees. In some cases, the acceptable cone angle of the detector may be at least about -10 degrees, about -15 degrees, about -20 degrees, about -25 degrees, about -30 degrees, about -35 degrees, about -40 degrees, about -45 degrees, about -50 degrees, about -60 degrees, or about -70 degrees.

[0135] In some cases, the acceptable cone angle of the detector may be from about 0 degrees to about 70 degrees. In some cases, the acceptable cone angle of the detector may be from about 0 degrees to about 10 degrees, from about 0 degrees to about 15 degrees, from about 0 degrees to about 20 degrees, from about 0 degrees to about 25 degrees, from about 0 degrees to about 30 degrees, from about 0 degrees to about 35 degrees, from about 0 degrees to about 40 degrees, from about 0 degrees to about 45 degrees, from about 0 degrees to about 50 degrees, from about 0 degrees to about 60 degrees, from about 0 degrees to about 70 degrees, from about 10 degrees to about 15 degrees, from about 10 degrees to about 20 degrees, from about 10 degrees to about 25 degrees, from about 10 degrees to about 30 degrees, from about 10 degrees to about 35 degrees, About 10 degrees to about 40 degrees, about 10 degrees to about 45 degrees, about 10 degrees to about 50 degrees, about 10 degrees to about 60 degrees, about 10 degrees to about 70 degrees, about 15 degrees to about 20 degrees, about 15 degrees to about 25 degrees, about 15 degrees to about 30 degrees, about 15 degrees to about 35 degrees, about 15 degrees to about 40 degrees, about 15 degrees to about 45 degrees, about 15 degrees to about 50 degrees, about 15 degrees to about 60 degrees, about 15 degrees to about 70 degrees, about 20 degrees to about 25 degrees, about 20 degrees to about 30 degrees, about 20 degrees to about 35 degrees, About 20 degrees to about 40 degrees, about 20 degrees to about 45 degrees, about 20 degrees to about 50 degrees, about 20 degrees to about 60 degrees, about 20 degrees to about 70 degrees, about 25 degrees to about 30 degrees, about 25 degrees to about 35 degrees, about 25 degrees to about 40 degrees, about 25 degrees to about 45 degrees, about 25 degrees to about 50 degrees, about 25 degrees to about 60 degrees, about 25 degrees to about 70 degrees, about 30 degrees to about 35 degrees, about 30 degrees to about 40 degrees, about 30 degrees to about 45 degrees, about 30 degrees to about 50 degrees, about 30 degrees to about 60 degrees, The cone angle may be about 30 degrees to about 70 degrees, about 35 degrees to about 40 degrees, about 35 degrees to about 45 degrees, about 35 degrees to about 50 degrees, about 35 degrees to about 60 degrees, about 35 degrees to about 70 degrees, about 40 degrees to about 45 degrees, about 40 degrees to about 50 degrees, about 40 degrees to about 60 degrees, about 40 degrees to about 70 degrees, about 45 degrees to about 50 degrees, about 45 degrees to about 60 degrees, about 45 degrees to about 70 degrees, about 50 degrees to about 60 degrees, about 50 degrees to about 70 degrees, or about 60 degrees to about 70 degrees. In some cases, the acceptable cone angle of the detector may be about 0 degrees, about 10 degrees, about 15 degrees, about 20 degrees, about 25 degrees, about 30 degrees, about 35 degrees, about 40 degrees, about 45 degrees, about 50 degrees, about 60 degrees, or about 70 degrees. In some cases, the acceptable cone angle of the detector may be at least about 0 degrees, about 10 degrees, about 15 degrees, about 20 degrees, about 25 degrees, about 30 degrees, about 35 degrees, about 40 degrees, about 45 degrees, about 50 degrees, or about 60 degrees. In some cases, the acceptable cone angle of the detector may be at least about 10 degrees, about 15 degrees, about 20 degrees, about 25 degrees, about 30 degrees, about 35 degrees, about 40 degrees, about 45 degrees, about 50 degrees, about 60 degrees, or about 70 degrees.

[0136] In some cases, the photomultiplier electrical signals may be processed and / or analyzed by digital and / or analog signal processing elements 124-128. The digital and / or analog signal processing elements may comprise attenuation-amplification electronics 124, a digitizer (126, 234), system control electronics (128, 221, 222), or any combination thereof. In some cases, the attenuation-amplification electronics 124 may comprise at least two attenuators (226, 230), at least two preamplifiers (228, 232), a programmable attenuator 2600, a fixed attenuator 2604, an amplifier 2602, or any combination thereof. In some cases, the attenuation-amplification electronics 124 may comprise a programmable attenuator 2600, an amplifier 2602, a fixed attenuator 2604, or any combination thereof, electrically coupled to each other and / or to the digitizer 234. In some cases, the electrical connectors between the attenuation-amplification electronics 124 may include connectors configured to reduce a connection distance between a first component and / or connector and a second component and / or connector and / or to reduce radio frequency electrical signal reflections.

[0137] The programmable attenuator 2600 may provide an attenuation of about 1 dB to about 100 dB. The programmable attenuator 2600 may provide an attenuation of about 1 dB to about 5 dB, about 1 dB to about 10 dB, about 1 dB to about 15 dB, about 1 dB to about 20 dB, about 1 dB to about 30 dB, about 1 dB to about 50 dB, about 1 dB to about 60 dB, about 1 dB to about 70 dB, about 1 dB to about 80 dB, about 1 dB to about 90 dB, about 1 dB to about 100 dB, about 5 dB to about 10 dB, about 5 dB to about 15 dB, about 5 dB to about 20 dB, about 5 dB to about 30 dB, about 5 dB to about 50 dB, about 5 dB to about 60 dB. , about 5dB to about 70dB, about 5dB to about 80dB, about 5dB to about 90dB, about 5dB to about 100dB, about 10dB to about 15dB, about 10dB to about 20dB, about 10dB to about 30dB, about 10dB to about 50dB, about 10dB to about 60dB, about 10dB to about 70dB, about 10dB to about 80dB, about 10dB to about 90dB, about 10dB to about 100dB, about 15dB to about 20dB, about 15dB to about 30dB, about 15dB to about 50dB, about 15dB to about 60dB, 15dB to 70dB, 15dB to 80dB, 15dB to 90dB, 15dB to 100dB, 20dB to 30dB, 20dB to 50dB, 20dB to 60dB, 20dB to 70dB, 20dB to 80dB, 20dB to 90dB, 20dB to 100dB, 30dB to 50dB, 30dB to 60dB, 30dB to 70dB, 30dB to 80dB, 30dB to 90dB, 30dB to 1 may have an attenuation of about 00 dB, about 50 dB to about 60 dB, about 50 dB to about 70 dB, about 50 dB to about 80 dB, about 50 dB to about 90 dB, about 50 dB to about 100 dB, about 60 dB to about 70 dB, about 60 dB to about 80 dB, about 60 dB to about 90 dB, about 60 dB to about 100 dB, about 70 dB to about 80 dB, about 70 dB to about 90 dB, about 70 dB to about 100 dB, about 80 dB to about 90 dB, about 80 dB to about 100 dB, or about 90 dB to about 100 dB. The programmable attenuator 2600 may provide an attenuation of about 1 dB, about 5 dB, about 10 dB, about 15 dB, about 20 dB, about 30 dB, about 50 dB, about 60 dB, about 70 dB, about 80 dB, about 90 dB, or about 100 dB.The programmable attenuator 2600 may provide at least about 1 dB, about 5 dB, about 10 dB, about 15 dB, about 20 dB, about 30 dB, about 50 dB, about 60 dB, about 70 dB, about 80 dB, or about 90 dB of attenuation. The programmable attenuator 2600 may provide up to about 5 dB, about 10 dB, about 15 dB, about 20 dB, about 30 dB, about 50 dB, about 60 dB, about 70 dB, about 80 dB, about 90 dB, or about 100 dB of attenuation.

[0138] The programmable attenuator 2600 may have an attenuation resolution of about 0.1 dB to about 30 dB. The programmable attenuator 2600 may have an attenuation resolution of about 0.1 dB to about 0.25 dB, about 0.1 dB to about 0.3 dB, about 0.1 dB to about 0.5 dB, about 0.1 dB to about 1 dB, about 0.1 dB to about 1.5 dB, about 0.1 dB to about 2 dB, about 0.1 dB to about 3 dB, about 0.1 dB to about 5 dB, about 0.1 dB to about 10 dB, about 0.1 dB to about 20 dB, about 0.1 dB to about 30 dB, about 0.25 dB to about 0.3 dB, about 0.25 dB to about 0.5 dB, about 0.25 dB to about 1 ... Approx. 1.5dB, Approx. 0.25dB~Approx. 2dB, Approx. 0.25dB~Approx. 3dB, Approx. 0.25dB~Approx. 5dB, Approx. 0.25dB~Approx. 10dB, Approx. 0.25dB~Approx. 20dB, Approx. 0.25dB~Approx. 30dB, Approx. 0.3dB~Approx. 0.5dB, Approx. dB ~ approx. 1 dB, approx. 0.3 dB ~ approx. 1.5 dB, approx. 0.3 dB ~ approx. 2 dB, approx. 0.3 dB ~ approx. 3 dB, approx. 0.3 dB ~ approx. 5 dB, approx. 0.3 dB ~ approx. 10 dB, approx. dB, about 0.5dB to about 1.5dB, about 0.5dB to about 2dB, about 0.5dB to about 3dB, about 0.5dB to about 5dB, about 0.5dB to about 10dB, about 0.5dB to about 20dB, about 0.5dB to about 30dB, about 1dB to about 1.5dB, about 1dB to about 2dB, about 1dB to about 3dB, about 1dB to about 5dB, about 1dB to about 10dB, about 1dB to about 20dB, about 1dB to about 30dB, about 1.5dB to about 2dB, about 1.5dB to about 3dB, about 1.5dB to about 5dB, about 1.5dB to about The attenuation resolution may be 10 dB, about 1.5 dB to about 20 dB, about 1.5 dB to about 30 dB, about 2 dB to about 3 dB, about 2 dB to about 5 dB, about 2 dB to about 10 dB, about 2 dB to about 20 dB, about 2 dB to about 30 dB, about 3 dB to about 5 dB, about 3 dB to about 10 dB, about 3 dB to about 20 dB, about 3 dB to about 30 dB, about 5 dB to about 10 dB, about 5 dB to about 20 dB, about 5 dB to about 30 dB, about 10 dB to about 20 dB, about 10 dB to about 30 dB, or about 20 dB to about 30 dB. The programmable attenuator 2600 may have an attenuation resolution of about 0.1 dB, about 0.25 dB, about 0.3 dB, about 0.5 dB, about 1 dB, about 1.5 dB, about 2 dB, about 3 dB, about 5 dB, about 10 dB, about 20 dB, or about 30 dB.The programmable attenuator 2600 may provide an attenuation resolution of at least about 0.1 dB, about 0.25 dB, about 0.3 dB, about 0.5 dB, about 1 dB, about 1.5 dB, about 2 dB, about 3 dB, about 5 dB, about 10 dB, or about 20 dB. The programmable attenuator 2600 may provide an attenuation resolution of up to about 0.25 dB, about 0.3 dB, about 0.5 dB, about 1 dB, about 1.5 dB, about 2 dB, about 3 dB, about 5 dB, about 10 dB, about 20 dB, or about 30 dB.

[0139] The fixed attenuator 2604 may have an attenuation of about 0.1 dB to about 30 dB. The fixed attenuator 2604 may have an attenuation of about 0.1 dB to about 0.25 dB, about 0.1 dB to about 0.3 dB, about 0.1 dB to about 0.5 dB, about 0.1 dB to about 1 dB, about 0.1 dB to about 1.5 dB, about 0.1 dB to about 2 dB, about 0.1 dB to about 3 dB, about 0.1 dB to about 6 dB, about 0.1 dB to about 10 dB, about 0.1 dB to about 20 dB, about 0.1 dB to about 30 dB, about 0.25 dB to about 0.3 dB, about 0.25 dB to about 0.5 dB, about 0.25 dB to about 1 dB, about 0.25 dB to about 1. 5dB, approximately 0.25dB to approximately 2dB, approximately 0.25dB to approximately 3dB, approximately 0.25dB to approximately 6dB, approximately 0.25dB to approximately 10dB, approximately 0.25dB to approximately 20dB, approximately 0.25dB to approximately 30dB, approximately 0.3dB to approximately 0.5dB, approximately 0.3dB ~1dB, approx. 0.3dB ~ approx. 1.5dB, approx. 0.3dB ~ approx. 2dB, approx. 0.3dB ~ approx. 3dB, approx. 0.3dB ~ approx. 6dB, approx. 0.3dB ~ approx. 10dB, approx. 0.3dB ~ approx. 20dB, approx. 0.3dB ~ approx. 30dB, approx. B, about 0.5dB to about 1.5dB, about 0.5dB to about 2dB, about 0.5dB to about 3dB, about 0.5dB to about 6dB, about 0.5dB to about 10dB, about 0.5dB to about 20dB, about 0.5dB to about 30dB, about 1dB to about 1.5dB, about 1dB to about 2dB, about 1dB to about 3dB, about 1dB to about 6dB, about 1dB to about 10dB, about 1dB to about 20dB, about 1dB to about 30dB, about 1.5dB to about 2dB, about 1.5dB to about 3dB, about 1.5dB to about 6dB, about 1.5dB The attenuation may be about 10 dB, about 1.5 dB to about 20 dB, about 1.5 dB to about 30 dB, about 2 dB to about 3 dB, about 2 dB to about 6 dB, about 2 dB to about 10 dB, about 2 dB to about 20 dB, about 2 dB to about 30 dB, about 3 dB to about 6 dB, about 3 dB to about 10 dB, about 3 dB to about 20 dB, about 3 dB to about 30 dB, about 6 dB to about 10 dB, about 6 dB to about 20 dB, about 6 dB to about 30 dB, about 10 dB to about 20 dB, about 10 dB to about 30 dB, or about 20 dB to about 30 dB. The fixed attenuator 2604 may provide an attenuation of about 0.1 dB, about 0.25 dB, about 0.3 dB, about 0.5 dB, about 1 dB, about 1.5 dB, about 2 dB, about 3 dB, about 6 dB, about 10 dB, about 20 dB, or about 30 dB.The fixed attenuator 2604 may provide an attenuation of at least about 0.1 dB, about 0.25 dB, about 0.3 dB, about 0.5 dB, about 1 dB, about 1.5 dB, about 2 dB, about 3 dB, about 6 dB, about 10 dB, or about 20 dB. The fixed attenuator 2604 may provide an attenuation of up to about 0.25 dB, about 0.3 dB, about 0.5 dB, about 1 dB, about 1.5 dB, about 2 dB, about 3 dB, about 6 dB, about 10 dB, about 20 dB, or about 30 dB.

[0140] In some cases, the digitizer (126, 234) may include an analog-to-digital circuit (i.e., DAC) configured to sample the photomultiplier tube analog electrical signal after amplification and attenuation, as described elsewhere herein. In some cases, the digitizer may include an input signal detection range of positive 1 volt to negative 1 volt. In some cases, the digitizer may include an input signal damage voltage threshold of positive 3 volts to negative 3 volts.

[0141] In some cases, the digitizer (126, 234) may be electrically coupled to an electrical component of the imaging system, such as a field programmable gate array (FPGA), a graphics processing unit (GPU), a solid-state memory of the system, or any combination thereof. In some cases, the digitizer may transfer data directly to the FPGA or GPU without sending the digitized data to a processor before sending the data to the FPGA or GPU. In some cases, the FPGA and / or GPU may pre-process (2450) the output signal from the attenuation-amplification electronics 124 prior to sending, forwarding, and / or transmitting the fluorescent imaging data to the predictive model pipeline 2452, as shown in FIG. 25. In some cases, the predictive model pipeline 2452 may perform one or more processing methods on the fluorescent imaging data, such as dimensionality reduction, feature engineering, classification, image processing, further signal pre-processing, or any combination thereof. In some cases, the predictive model pipeline may perform one or more processing methods on the fluorescent imaging data on the computer system 804, offline in the cloud computing architecture 816, or a combination thereof. In some cases, the digitizer may convert the analog electrical signal of the photomultiplier tube to a digital signal and then transmit the digitized signal to the GPU for further signal processing (e.g., determining the fluorescence lifetime of the pulsed electrical signal of the photomultiplier tube). In some cases, the digitizer may convert the analog pulsed electrical signal provided by the photomultiplier tube to a digital signal and then transmit the digitized signal to the FPGA. The FPGA may be configured to detect the total optical energy detected by the photomultiplier tube. In some cases, the FPGA may be configured to measure the peak amplitude and / or area under each pulse of the pulsed signal.

[0142] The signal processing performed by the GPU and / or FPGA may include signal processing steps of aligning the detected pulsed signal of the electrical signal provided by the photomultiplier tube, filtering the aligned pulsed signal, averaging the pulsed signal, extracting decay and / or peak values ​​from the averaged pulses, or any combination thereof.

[0143] In some cases, the digitizer may have an analog bandwidth of about 50 megahertz (MHz) to about 20,000 MHz. In some cases, the digitizer may have an analog bandwidth of about 50 MHz to about 100 MHz, about 50 MHz to about 500 MHz, about 50 MHz to about 700 MHz, about 50 MHz to about 1,000 MHz, about 50 MHz to about 2,000 MHz, about 50 MHz to about 4,000 MHz, about 50 MHz to about 6,000 MHz, about 50 MHz to about 8,000 MHz, about 50 MHz to about 9,000 MHz, about 50 MHz to about 10,000 MHz, about 50 MHz to about 20,000 MHz, about 100 MHz to about 500 MHz, about 100 MHz to about 700 MHz, about 10 0MHz~Approx. 1,000MHz, Approx. 100MHz~Approx. 2,000MHz, Approx. 100MHz~Approx. 4,000MHz, Approx. 100MHz~Approx. 6,000MHz, Approx. 100MHz~Approx. 8,000MHz, Approx. 100MHz~Approx. 9,000MHz, Approx. 100MHz~Approx. 10,000MHz, Approx. 100MHz~Approx. 20,000MHz, Approx. 500MHz~Approx. 700MHz, Approx. 500MHz~Approx. 1,000MHz, Approx. 500MHz~Approx. 2,000MHz, Approx. 500MHz~Approx. 4,000MHz, Approx. 500MHz~Approx. 6,000MHz, Approx. 500MHz Hz ~ approximately 8,000MHz, approximately 500MHz ~ approximately 9,000MHz, approximately 500MHz ~ approximately 10,000MHz, approximately 500MHz ~ approximately 20,000MHz, approximately 700MHz ~ approximately 1,000MHz, approximately 700MHz ~ approximately 2,000MHz, approximately 700MHz ~ approximately 4,00 0MHz, approximately 700MHz to approximately 6,000MHz, approximately 700MHz to approximately 8,000MHz, approximately 700MHz to approximately 9,000MHz, approximately 700MHz to approximately 10,000MHz, approximately 700MHz to approximately 20,000MHz, approximately 1,000MHz to approximately 2,000MHz, approximately 1,000MHz to about 4,000MHz, about 1,000MHz to about 6,000MHz, about 1,000MHz to about 8,000MHz, about 1,000MHz to about 9,000MHz, about 1,000MHz to about 10,000MHz, about 1,000MHz to about 20,000MHz, about 2,000MHz to about 4,000MHz, about 2,000MHz to about 6,000MHz, about 2,000MHz to about 8,000MHz, about 2,000MHz to about 9,000MHz, about 2,000MHz to about 10,000MHz, about 2,000MHz to about 20,000MHz, approx. 4,000MHz to approx. 6,000MHz, approx. 4,000MHz to approx. 8,000MHz, approx. 4,000MHz to approx. 9,000MHz, approx. 4,000MHz to approx. 10,000MHz, approx. 4,000MHz to approx. 20,000MHz, approx. 6,000MHz to approx. 8,000MHz, approx. 6,000MHz to approx. 9,000MHz, approx. 6,000MHz to approx. 10,000MHz Hz, about 6,000 MHz to about 20,000 MHz, about 8,000 MHz to about 9,000 MHz, about 8,000 MHz to about 10,000 MHz, about 8,000 MHz to about 20,000 MHz, about 9,000 MHz to about 10,000 MHz, about 9,000 MHz to about 20,000 MHz, or about 10,000 MHz to about 20,000 MHz. In some cases, the digitizer may have an analog bandwidth of about 50 MHz, about 100 MHz, about 500 MHz, about 700 MHz, about 1,000 MHz, about 2,000 MHz, about 4,000 MHz, about 6,000 MHz, about 8,000 MHz, about 9,000 MHz, about 10,000 MHz, or about 20,000 MHz. In some cases, the digitizer may have an analog bandwidth of at least about 50 MHz, about 100 MHz, about 500 MHz, about 700 MHz, about 1,000 MHz, about 2,000 MHz, about 4,000 MHz, about 6,000 MHz, about 8,000 MHz, about 9,000 MHz, or about 10,000 MHz. In some cases, the digitizer may have an analog bandwidth of at least about 100 MHz, about 500 MHz, about 700 MHz, about 1,000 MHz, about 2,000 MHz, about 4,000 MHz, about 6,000 MHz, about 8,000 MHz, about 9,000 MHz, about 10,000 MHz, or about 20,000 MHz.

[0144] In some cases, the digitizer may have a sampling rate of about 50 megasamples per second (Ms / s) to about 20,000 Ms / s. In some cases, the digitizer may be configured to operate at a speed between about 50 Ms / sec and about 100 Ms / sec, between about 50 Ms / sec and about 500 Ms / sec, between about 50 Ms / sec and about 700 Ms / sec, between about 50 Ms / sec and about 1,000 Ms / sec, between about 50 Ms / sec and about 2,000 Ms / sec, between about 50 Ms / sec and about 4,000 Ms / sec, between about 50 Ms / sec and about 6,000 Ms / sec, between about 50 Ms / sec and about 8,000 Ms / sec, between about 50 Ms / sec and about 9,000 Ms / sec, between about 50 Ms / sec and about 10,000 Ms / sec, between about 50 Ms / sec and about 20,000 Ms / sec, between about 100 Ms / sec and about 5 00Ms / sec, approx. 100Ms / sec ~ approx. 700Ms / sec, approx. 100Ms / sec ~ approx. 1,000Ms / sec, approx. 100Ms / sec ~ approx. 2,0 00Ms / sec, approx. 100Ms / sec ~ approx. 4,000Ms / sec, approx. 100Ms / sec ~ approx. 6,000Ms / sec, approx. 100Ms / sec ~ approx. 8 ,000Ms / sec, approx. 100Ms / sec ~ approx. 9,000Ms / sec, approx. 100Ms / sec ~ approx. 10,000Ms / sec, approx. 100Ms / sec ~20,000Ms / sec, approx. 500Ms / sec ~700Ms / sec, approx. 500Ms / sec ~ approx. 1,000Ms / sec, approx. 500Ms / sec ~2,000Ms / sec, 500Ms / sec~4,000Ms / sec, 500Ms / sec~6,000Ms / sec, 500Ms / sec~8,000Ms / sec, 500Ms / sec~9,000Ms / sec, 500Ms / sec~10,000Ms / sec, approx. 50 0Ms / sec ~ approx. 20,000Ms / sec, approx. 700Ms / sec ~ approx. 1,000Ms / sec, approx. 700Ms / sec ~ approx. 2,000Ms / sec, Approx. 700Ms / sec ~ approx. 4,000Ms / sec, approx. 700Ms / sec ~ approx. 6,000Ms / sec, approx. 700Ms / sec ~ approx. 8,000Ms / sec , about 700Ms / sec to about 9,000Ms / sec, about 700Ms / sec to about 10,000Ms / sec, about 700Ms / sec to about 20,000 Ms / sec, approx. 1,000Ms / sec ~ approx. 2,000Ms / sec, approx. 1,000Ms / sec ~ approx. 4,000Ms / sec, approx. 1,000Ms / seconds ~ approx. 6,000Ms / sec, approx. 1,000Ms / sec ~ approx. 8,000Ms / sec, approx. 1,000Ms / sec ~ approx. 9,000Ms / sec, approx. 1,000Ms / sec ~ approx. 10,000Ms / sec, approx. 1,000Ms / sec ~ approx. 20,000Ms / sec, approx. 2,000Ms / sec ~ approx. 4,000Ms / sec, approx. 2,000Ms / sec ~ approx. 6,000Ms / sec, approx. 2,000Ms / sec ~ approx. 8,000Ms / sec, approx. 2,000Ms / sec ~ approx. 9,000Ms / sec, approx. 2,000Ms / sec ~ approx. 10,000Ms / sec, approx. 2,000Ms / sec ~ approx. 20,000Ms / sec, Approx. 4,000Ms / sec ~ approx. 6,000Ms / sec, approx. 4,000Ms / sec ~ approx. 8,000Ms / sec, approx. 4,000Ms / sec ~ approx. 9,00 0Ms / sec, approx. 4,000Ms / sec ~ approx. 10,000Ms / sec, approx. 4,000Ms / sec ~ approx. 20,000Ms / sec, approx. 6,000Ms The sampling rate may be from about 10,000 Ms / sec to about 20,000 Ms / sec, from about 8,000 Ms / sec to about 9,000 Ms / sec, from about 6,000 Ms / sec to about 10,000 Ms / sec, from about 6,000 Ms / sec to about 20,000 Ms / sec, from about 8,000 Ms / sec to about 9,000 Ms / sec, from about 8,000 Ms / sec to about 10,000 Ms / sec, from about 8,000 Ms / sec to about 20,000 Ms / sec, from about 9,000 Ms / sec to about 10,000 Ms / sec, from about 9,000 Ms / sec to about 20,000 Ms / sec, or from about 10,000 Ms / sec to about 20,000 Ms / sec. In some cases, the digitizer may have a sampling rate of about 50 Ms / sec, about 100 Ms / sec, about 500 Ms / sec, about 700 Ms / sec, about 1,000 Ms / sec, about 2,000 Ms / sec, about 4,000 Ms / sec, about 6,000 Ms / sec, about 8,000 Ms / sec, about 9,000 Ms / sec, about 10,000 Ms / sec, or about 20,000 Ms / sec. In some cases, the digitizer may have a sampling rate of at least about 50 Ms / sec, about 100 Ms / sec, about 500 Ms / sec, about 700 Ms / sec, about 1,000 Ms / sec, about 2,000 Ms / sec, about 4,000 Ms / sec, about 6,000 Ms / sec, about 8,000 Ms / sec, about 9,000 Ms / sec, or about 10,000 Ms / sec. In some cases, the digitizer may have a sampling rate of at least about 100 Ms / sec, about 500 Ms / sec, about 700 Ms / sec, about 1,000 Ms / sec, about 2,000 Ms / sec, about 4,000 Ms / sec, about 6,000 Ms / sec, about 8,000 Ms / sec, about 9,000 Ms / sec, about 10,000 Ms / sec, or about 20,000 Ms / sec.

[0145] In some cases, the at least two preamplifiers (228, 232) and / or amplifier 2602 may have a frequency response of about 8 kilohertz (kHz) to about 3,000,000 kHz. In some cases, the at least two preamplifiers (228, 232) and / or amplifier 2602 may have a frequency response of about 8 kHz to about 100 kHz, about 8 kHz to about 1,000 kHz, about 8 kHz to about 10,000 kHz, about 8 kHz to about 50,000 kHz, about 8 kHz to about 100,000 kHz, about 8 kHz to about 150,000 kHz, about 8 kHz to about 250,000 kHz, about 8 kHz to about 500,000 kHz, about 8 kHz to about 1,000,000 kHz, about 8 kHz to about 2,000,000 kHz, about 8 kHz to about 3,000,000 kHz, about 8 kHz to about 4,000,000 kHz, about 8 kHz to about 5,000,000 kHz, about 8 kHz to about 6,000,000 kHz, about 8 kHz to about 7,000,000 kHz, about 8 kHz to about 8,000,000 kHz, about 8 kHz to about 9,000,000 kHz, about 8 kHz to about 10,000,000 kHz, about 8 kHz to about 2,000,000 kHz, about 8 kHz to about 3,000,000 kHz, about 8 kHz to about 4,000,000 kHz, about 8 kHz to about 5,000, ~3,000,000kHz, ~100kHz~1,000kHz, ~100kHz~10,000kHz, ~100kHz~50,000kHz, ~100kHz~100,000kHz, ~100kHz~150,000kHz, ~100kHz~250,000kHz, ~100kHz~500,000kHz, ~100kHz~1,000,000kHz, ~100kHz~2,000,000kHz, ~100kHz~3,000,000kHz, ~1,000kHz~ Approximately 10,000kHz, approximately 1,000kHz to approximately 50,000kHz, approximately 1,000kHz to approximately 100,000kHz, approximately 1,000kHz to approximately 150,000kHz, approximately 1,000kHz to approximately 250,000kHz, approximately 1,000kHz to approximately 500,000kHz, approximately 1,000kHz to approximately 1,000,000kHz, approximately 1,000kHz to approximately 2,000,000kHz, approximately 1,000kHz to approximately 3,000,000kHz, approximately 10,000kHz to approximately 50,000kHz, approximately 10,000kHz to approximately 1 00,000kHz, about 10,000kHz to about 150,000kHz, about 10,000kHz to about 250,000kHz, about 10,000kHz to about 500,000kHz, about 10,000kHz to about 1,000,000kHz, about 10,000kHz to about 2,000,000kHz, about 10,000kHz to about 3,000,000kHz, about 50,000kHz to about 100,000kHz, about 50,000kHz to about 150,000kHz, about 50,000kHz to about 250,000kHz, about 50,000kHz to approximately 500,000kHz, approximately 50,000kHz to approximately 1,000,000kHz, approximately 50,000kHz to approximately 2,000,000kHz, approximately 50,000kHz to approximately 3,000,000kHz, approximately 100,000kHz to approximately 150,000kHz, approximately 100,000kHz to approximately 250,000kHz, approximately 100,000kHz to approximately 500,000 kHz, about 100,000kHz to about 1,000,000kHz, about 100,000kHz to about 2,000,000kHz, about 100,000kHz to about 3,000,000kHz, about 150,000kHz to about 250,000kHz, about 150,000kHz to about 500,000kHz, about 150,000kHz to about 1,000,000kHz, about 150,00 0kHz to about 2,000,000kHz, about 150,000kHz to about 3,000,000kHz, about 250,000kHz to about 500,000kHz, about 250,000kHz to about 1,000,000kHz, about 250,000kHz to about 2,000,000kHz, about 250,000kHz to about 3,000,000kHz, about 500,000kHz to about 1, The optical fiber may have a frequency response of about 1,000,000 kHz, about 500,000 kHz to about 2,000,000 kHz, about 500,000 kHz to about 3,000,000 kHz, about 1,000,000 kHz to about 2,000,000 kHz, about 1,000,000 kHz to about 3,000,000 kHz, or about 2,000,000 kHz to about 3,000,000 kHz. In some cases, the at least two preamplifiers (228, 232) and / or the amplifier 2602 may have a frequency response of about 8 kHz, about 100 kHz, about 1,000 kHz, about 10,000 kHz, about 50,000 kHz, about 100,000 kHz, about 150,000 kHz, about 250,000 kHz, about 500,000 kHz, about 1,000,000 kHz, about 2,000,000 kHz, or about 3,000,000 kHz. In some cases, the at least two preamplifiers (228, 232) and / or amplifier 2602 may be configured to provide at least about 8 kHz, about 100 kHz, about 1,000 kHz, about 10,000 kHz, about 50,000 kHz, about 100,000 kHz, about 150,000 kHz, about 250,000 kHz, about 500,000 kHz, about 1,000,In some cases, the at least two preamplifiers (228, 232) and / or amplifier 2602 may have a frequency response of at least about 100 kHz, about 1,000 kHz, about 10,000 kHz, about 50,000 kHz, about 100,000 kHz, about 150,000 kHz, about 250,000 kHz, about 500,000 kHz, about 1,000,000 kHz, about 2,000,000 kHz, or about 3,000,000 kHz.

[0146] In some cases, the at least two preamplifiers (228, 232) and / or amplifier 2602 may have a gain of about 2 dB to about 60 dB. In some cases, the at least two preamplifiers (228, 232) and / or amplifier 2602 may have a gain of about 2 dB to about 4 dB, about 2 dB to about 6 dB, about 2 dB to about 8 dB, about 2 dB to about 10 dB, about 2 dB to about 12 dB, about 2 dB to about 15 dB, about 2 dB to about 20 dB, about 2 dB to about 30 dB, about 2 dB to about 40 dB, about 2 dB to about 50 dB, about 2 dB to about 60 dB, about 4 dB to about 6 dB, about 4 dB to about 8 dB, about 4 dB to about 10 ... 2dB, approx. 4dB to approx. 15dB, approx. 4dB to approx. 20dB, approx. 4dB to approx. 30dB, approx. 4dB to approx. 40dB, approx. 4dB to approx. 50dB, approx. 4dB to approx. 60dB, approx. 6dB to approx. 8dB, approx. 6dB to approx. 10dB, approx. 6dB to approx. 12dB, approx. 6dB to approx. 15dB, approx. 6dB to approx. 20dB, approx. 6dB to approx. 30dB, approx. 6dB to approx. 40dB, approx. 6dB to approx. 50dB, approx. 6dB to approx. 60dB, approx. 8dB to approx. 10dB, approx. 8dB to approx. 12dB, approx. 8dB to approx. 15dB, approx. 8dB to 20dB, 8dB to 30dB, 8dB to 40dB, 8dB to 50dB, 8dB to 60dB, 10dB to 12dB, 10dB to 15dB, 10dB to 20dB, 10dB to 30dB, 10dB to 40dB, 10dB to 50dB, 10dB to 60dB, 12dB to 15dB, 12dB to 20dB, 12dB to 30dB, 12dB to 40dB, 12dB to 50dB, 12dB to 60dB, The optical fiber may have a gain of about 15 dB to about 60 dB, about 15 dB to about 20 dB, about 15 dB to about 30 dB, about 15 dB to about 40 dB, about 15 dB to about 50 dB, about 15 dB to about 60 dB, about 20 dB to about 30 dB, about 20 dB to about 40 dB, about 20 dB to about 50 dB, about 20 dB to about 60 dB, about 30 dB to about 40 dB, about 30 dB to about 50 dB, about 30 dB to about 60 dB, about 40 dB to about 50 dB, about 40 dB to about 60 dB, or about 50 dB to about 60 dB. In some cases, the at least two preamplifiers (228, 232) and / or the amplifier 2602 may have a gain of about 2 dB, about 4 dB, about 6 dB, about 8 dB, about 10 dB, about 12 dB, about 15 dB, about 20 dB, about 30 dB, about 40 dB, about 50 dB, or about 60 dB.In some cases, the at least two preamplifiers (228, 232) and / or amplifier 2602 may have a gain of at least about 2 dB, about 4 dB, about 6 dB, about 8 dB, about 10 dB, about 12 dB, about 15 dB, about 20 dB, about 30 dB, about 40 dB, or about 50 dB. In some cases, the at least two preamplifiers (228, 232) and / or amplifier 2602 may have a gain of up to about 4 dB, about 6 dB, about 8 dB, about 10 dB, about 12 dB, about 15 dB, about 20 dB, about 30 dB, about 40 dB, about 50 dB, or about 60 dB.

[0147] In some cases, the thermal noise of the at least two preamplifiers (228, 232) and / or the amplifier 2602 may comprise a thermal noise of about 0.01 dB to about 6 dB. In some cases, the thermal noise of the at least two preamplifiers (228, 232) and / or amplifier 2602 is between about 0.01 dB and about 0.05 dB, between about 0.01 dB and about 0.07 dB, between about 0.01 dB and about 0.1 dB, between about 0.01 dB and about 0.25 dB, between about 0.01 dB and about 0.5 dB, between about 0.01 dB and about 1 dB, between about 0.01 dB and about 2 dB, between about 0.01 dB and about 3 dB, between about 0.01 dB and about 4 dB, between about 0.01 dB and about 5 dB, between about 0.01 dB and about 6 dB, between about 0.05 dB and about 0.07 dB, between about 0. 0.05dB to 0.1dB, 0.05dB to 0.25dB, 0.05dB to 0.5dB, 0.05dB to 1dB, 0.05dB to 2dB, 0.05dB to 3dB, 0.05dB to 4dB, 0.05dB to 5dB, 0.05dB to 6dB, 0.07dB to 0.1dB, 0.07dB to 0.25dB, 0.07dB to 0.5dB, 0.07dB to 1dB, 0.07dB to 2dB, 0.07dB to 3dB, 0.07dB to 4dB, 0.07dB to 5dB, 0.05dB to 6dB, B ~ approx. 5 dB, approx. 0.07 dB ~ approx. 6 dB, approx. 0.1 dB ~ approx. 0.25 dB, approx. 0.1 dB ~ approx. 0.5 dB, approx. 0.1 dB ~ approx. 1 dB, approx. 0.1 dB ~ approx. 2 dB, approx. 1dB to about 6dB, about 0.25dB to about 0.5dB, about 0.25dB to about 1dB, about 0.25dB to about 2dB, about 0.25dB to about 3dB, about 0.25dB to about 4dB, about 0.25dB to about 5dB, about 0.25dB to about 6dB, about 0.5dB to about 1dB , about 0.5 dB to about 2 dB, about 0.5 dB to about 3 dB, about 0.5 dB to about 4 dB, about 0.5 dB to about 5 dB, about 0.5 dB to about 6 dB, about 1 dB to about 2 dB, about 1 dB to about 3 dB, about 1 dB to about 4 dB, about 1 dB to about 5 dB, about 1 dB to about 6 dB, about 2 dB to about 3 dB, about 2 dB to about 4 dB, about 2 dB to about 5 dB, about 2 dB to about 6 dB, about 3 dB to about 4 dB, about 3 dB to about 5 dB, about 3 dB to about 6 dB, about 4 dB to about 5 dB, about 4 dB to about 6 dB, or about 5 dB to about 6 dB.In some cases, the thermal noise of the at least two preamplifiers (228, 232) and / or amplifier 2602 may comprise a thermal noise of about 0.01 dB, about 0.05 dB, about 0.07 dB, about 0.1 dB, about 0.25 dB, about 0.5 dB, about 1 dB, about 2 dB, about 3 dB, about 4 dB, about 5 dB, or about 6 dB. In some cases, the thermal noise of the at least two preamplifiers (228, 232) and / or amplifier 2602 may comprise a thermal noise of at least about 0.01 dB, about 0.05 dB, about 0.07 dB, about 0.1 dB, about 0.25 dB, about 0.5 dB, about 1 dB, about 2 dB, about 3 dB, about 4 dB, or about 5 dB. In some cases, the thermal noise of the at least two preamplifiers (228, 232) and / or the amplifier 2602 may comprise a thermal noise of up to about 0.05 dB, about 0.07 dB, about 0.1 dB, about 0.25 dB, about 0.5 dB, about 1 dB, about 2 dB, about 3 dB, about 4 dB, about 5 dB, or about 6 dB.

[0148] In some cases, the detected optical signal from the tissue sample may vary depending on the molecule of interest that is excited. The detected optical signal may saturate the detectable range of the optical signal of the PMT, for example, for highly responsive or highly fluorescent molecules in the tissue sample, or may not be detectable relative to the noise floor of the PMT for less responsive or less fluorescent molecules in the tissue sample. For example, a fluorophore emits a fluorescence spectrum with an intensity based on the quantum efficiency and / or absorption of the excitation light used to excite it. Depending on the conditions in which the fluorophore is present, the intensity of the fluorophore may differ. For example, a fluorophore in a tissue sample may have a different intensity than the same fluorophore in a blood sample or when isolated due to differences in its surroundings. To properly record the fluorescence spectrum, the gain of the detector (e.g., PMT) may be adjusted so that high fluorescence emissions do not saturate the signal and low fluorescence emissions do not reduce the signal-to-noise ratio. This may be achieved by rapidly changing the voltage (i.e., slew rate) of the voltage power supply 220 of the PMT 122.

[0149] In some cases, the slew rate of the voltage power supply may be from about 1 V / μs to about 1,000 V / μs. In some cases, the slew rate of the voltage power supply may be from about 1 V / μs to about 5 V / μs, from about 1 V / μs to about 10 V / μs, from about 1 V / μs to about 25 V / μs, from about 1 V / μs to about 50 V / μs, from about 1 V / μs to about 100 V / μs, from about 1 V / μs to about 200 V / μs, from about 1 V / μs to about 400 V / μs, from about 1 V / μs to about 800 V / μs, from about 1 V / μs to about 1,000 V / μs, from about 5 V / μs to about 10 V / μs, from about 5 V / μs to about 25 V / μs, from about 5 V / μs to about 50 V / μs, from about 5 V / μs to about 100 V / μs, from about 5 V / μs to about 200 V / μs, from about 5 V / μs to about 400 V / μs, from about 5 V / μs to about 800 V / μs, from about 5 V / μs to about 1,000 V / μs, from about 10 V / μs to about 25 V / μs, from about 10 V / μs to about 50 V / μs, from about 10 V / μs to about 100 V / μs, from about 10 V / μs to about 200 V / μs, from about 10 V / μs to about 400 V / μs, from about 10 V / μs to about 800 V / μs, from about 10 V / μs to about 1,000 V / μs, from about 25 V / μs to about 50 V / μs, from about 25 V / μs to about 100 V / μs, from about 25 V / μs to about 200 V / μs, from about 25 V / μs to about 400 V / μs, from about 25 V / μs to about 800 V / μs, from about 25 V / μs to about 1,000 V / μs, from about 50 V / μs to about 100 V / μs, from about 50 V / μs to about 200 V / μs, from about 50 V / μs to about 400 V / μs, from about 50 V / μs to about 800 V / μs, from about 50 V / μs to about 1,000 V / μs, from about 100 V / μs to about 200 V / μs, from about 100 V / μs to about 400 V / μs, from about 100 V / μs to about 800 V / μs, from about 100 V / μs to about 1,000 V / μs, from about 200 V / μs to about 400 V / μs, from about 200 V / μs to about 800 V / μs, from about 200 V / μs to about 1,000 V / μs, from about 400 V / μs to about 800 V / μs, from about 400 V / μs to about 1,000 V / μs, or from about 800 V / μs to about 1,000 V / μs. In some cases, the slew rate of the voltage power supply may be about 1 V / μs, about 5 V / μs, about 10 V / μs, about 25 V / μs, about 50 V / μs, about 100 V / μs, about 200 V / μs, about 400 V / μs, about 800 V / μs, or about 1,000 V / μs.In some cases, the slew rate of the voltage power supply may be at least about 1 V / μsec, about 5 V / μsec, about 10 V / μsec, about 25 V / μsec, about 50 V / μsec, about 100 V / μsec, about 200 V / μsec, about 400 V / μsec, or about 800 V / μsec. In some cases, the slew rate of the voltage power supply may be at least about 5 V / μsec, about 10 V / μsec, about 25 V / μsec, about 50 V / μsec, about 100 V / μsec, about 200 V / μsec, about 400 V / μsec, about 800 V / μsec, or about 1,000 V / μsec.

[0150] In some cases, the frequency response of the voltage power supply may comprise between about 1 kHz and about 1,000 kHz. In some cases, the frequency response of the voltage power supply may comprise between about 1 kHz and about 5 kHz, between about 1 kHz and about 10 kHz, between about 1 kHz and about 25 kHz, between about 1 kHz and about 50 kHz, between about 1 kHz and about 100 kHz, between about 1 kHz and about 200 kHz, between about 1 kHz and about 400 kHz, between about 1 kHz and about 800 kHz, between about 1 kHz and about 1,000 kHz, between about 5 kHz and about 10 kHz, between about 5 kHz and about 25 kHz, between about 5 ... Hz ~ about 50kHz, about 5kHz - about 100kHz, about 5kHz - about 200kHz, about 5kHz - about 400kHz, about 5kHz - about 800kHz, about 5kHz - about 1,000kHz, about 10kHz Approximately 25kHz, approximately 10kHz to approximately 50kHz, approximately 10kHz to approximately 100kHz, approximately 10kHz to approximately 200kHz, approximately 10kHz to approximately 400kHz, approximately 10kHz to approximately 800kHz, approximately 10kHz to approximately 1,000kHz, approx. 25kHz to approx. 50kHz, approx. 25kHz to approx. 100kHz, approx. 25kHz to approx. 200kHz, approx. 25kHz to approx. 400kHz, approx. 25kHz to approx. 800kHz, approx. 25kHz to approx. 1,000kHz, approx. 50kHz to approx. 100kHz, approx. 50kHz to approx. 200kHz, approx. 50kHz to approx. 400kHz, approx. 50kHz to approx. 800kHz, approx. 50kHz to approx. 1,000kHz, The frequency response of the voltage power supply may comprise about 100 kHz to about 200 kHz, about 100 kHz to about 400 kHz, about 100 kHz to about 800 kHz, about 100 kHz to about 1,000 kHz, about 200 kHz to about 400 kHz, about 200 kHz to about 800 kHz, about 200 kHz to about 1,000 kHz, about 400 kHz to about 800 kHz, about 400 kHz to about 1,000 kHz, or about 800 kHz to about 1,000 kHz. In some cases, the frequency response of the voltage power supply may comprise about 1 kHz, about 5 kHz, about 10 kHz, about 25 kHz, about 50 kHz, about 100 kHz, about 200 kHz, about 400 kHz, about 800 kHz, or about 1,000 kHz. In some cases, the frequency response of the voltage power supply may comprise at least about 1 kHz, about 5 kHz, about 10 kHz, about 25 kHz, about 50 kHz, about 100 kHz, about 200 kHz, about 400 kHz, or about 800 kHz.In some cases, the frequency response of the voltage power supply may comprise at least about 5 kHz, about 10 kHz, about 25 kHz, about 50 kHz, about 100 kHz, about 200 kHz, about 400 kHz, about 800 kHz, or about 1,000 kHz.

[0151] In some cases, the voltage of the voltage power supply may be adjusted at a rate to achieve an imaging scan duration of up to about 1 minute with at least about a 2-fold, at least 3-fold, or at least 4-fold increase in the imaging resolution of the fluorescence imaging system.

[0152] In some cases, the voltage power supply may output a voltage between about -5,000 volts (V) and about 3,000V.In some cases, the voltage power supply may be between about -5,000V and about -3,000V, between about -5,000V and about -1,000V, between about -5,000V and about -500V, between about -5,000V and about 0V, between about -5,000V and about 100V, between about -5,000V and about 200V, between about -5,000V and about 400V, between about -5,000V and about 800V, between about -5,000V and about 1,000V, between about -5,000V and about 2,000V, between about -5,000V and about 3,000V, between about -3,000V and about -1,000V, between about -3,000V and about -500V, between about -3,000V and about 0V, between about - 3,000V to approx. 100V, approx. -3,000V to approx. 200V, approx. -3,000V to approx. 400V, approx. -3,000V to approx. 800V, approx. -3,000V to approx. 1,000V, approx. -3,000V to approx. 2,000V, approx. -3,000V to approx. 3,000V, approx. -1,000V to approx. -50 0V, approx. -1,000V to approx. 0V, approx. -1,000V to approx. 100V, approx. -1,000V to approx. 200V, approx. -1,000V to approx. 400V, approx. -1,000V to approx. 800V, approx. -1,000V to approx. 1,000V, approx. -1,000V to approx. 2,000V, approx. -1,000V to approx. 3,0 00V, about -500V to about 0V, about -500V to about 100V, about -500V to about 200V, about -500V to about 400V, about -500V to about 800V, about -500V to about 1,000V, about -500V to about 2,000V, about -500V to about 3,000V, about 0V to about 100V, About 0V to about 200V, about 0V to about 400V, about 0V to about 800V, about 0V to about 1,000V, about 0V to about 2,000V, about 0V to about 3,000V, about 100V to about 200V, about 100V to about 400V, about 100V to about 800V, about 100V to about 1,000V, about 100V to about A voltage of about 2,000V, about 100V to about 3,000V, about 200V to about 400V, about 200V to about 800V, about 200V to about 1,000V, about 200V to about 2,000V, about 200V to about 3,000V, about 400V to about 800V, about 400V to about 1,000V, about 400V to about 2,000V, about 400V to about 3,000V, about 800V to about 1,000V, about 800V to about 2,000V, about 800V to about 3,000V, about 1,000V to about 2,000V, about 1,000V to about 3,000V, or about 2,000V to about 3,000V may be output.In some cases, the voltage power source may output a voltage of about -5,000 V, about -3,000 V, about -1,000 V, about -500 V, about 0 V, about 100 V, about 200 V, about 400 V, about 800 V, about 1,000 V, about 2,000 V, or about 3,000 V. In some cases, the voltage power source may output a voltage of at least about -5,000 V, about -3,000 V, about -1,000 V, about -500 V, about 0 V, about 100 V, about 200 V, about 400 V, about 800 V, about 1,000 V, or about 2,000 V. In some cases, the voltage power supply may output a voltage of at least about -3,000V, about -1,000V, about -500V, about 0V, about 100V, about 200V, about 400V, about 800V, about 1,000V, about 2,000V, or about 3,000V. In some cases, the voltage of the voltage power supply 220 may be controlled by a gain controller 221 or by an FPGA. In some cases, the gain controller may comprise an STM32 chipset. The gain controller 221 may control at least two attenuators (226, 230) through transistor-transistor-logic (TTL). The gain controller 221 may reduce or increase the voltage of the PMT 122 detected and recorded by the digitizer (126, 234) by controlling the at least two attenuators (226, 230). In some cases, the gain controller 221 may receive input from the digitizer (126, 234) via a universal serial bus (USB) interface. In some cases, the gain controller 221 may provide an input signal to the digitizer (126, 234). In some cases, the gain controller 221 may control the gain of the programmable attenuator 2600. In some cases, the gain controller 221 may provide control inputs to and / or receive control signals from one or more acousto-optic modulators of the excitation optics 110. In some cases, the gain controller may receive input signals from and / or provide signals to the computer system 804.

[0153] In some cases, the signal-to-noise ratio (SNR) of the detected electrical signal of the photomultiplier tube may be increased by installing a cable 2403, e.g., a rigid or flexible coaxial cable, configured to transmit a radio frequency (RF) electrical signal between the PMT 122 and the attenuation-amplification electronics 124. In some cases, the cable 2403 may provide an RF delay of RF signal reflections resulting from amplifying, attenuating, and detecting the electrical signal of the photomultiplier tube. The length of the cable 2403 may comprise a length of at least about 1 meter, at least about 2 meters, at least about 3 meters, or at least about 4 meters. The RF cable 2403 may transmit various sources of signal as well as noise (e.g., heat, shot, circuit, etc.) to the attenuation-amplification electronics 124. The RF cable may allow for movement of the PMT 122 relative to the position of the attenuation-amplification electronics 124. The length of the cable 2403 may be configured to prevent RF signal reflections from interfering with the detected electrical signal of the photomultiplier tube, thereby increasing the signal-to-noise detection of the electrical signal of the photomultiplier tube. The RF cable may improve the SNR of the detection of the photomultiplier electrical signal by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% when detecting the photomultiplier electrical signal, compared to the SNR of a system without the RF cable. In some cases, a rigid cable may be implemented in the fluorescence imaging system instead of a flexible (e.g., coiled) cable to maintain a compact system form factor. In some cases, the rigid cable may provide a better than expected improvement in SNR compared to commonly used flexible cables. The RF cable may provide a better expected result of signal-to-noise improvement in terms of the cable's length-dependent signal attenuation (e.g., about 1 dB to 3 dB loss per meter at 3 GHz).

[0154] In some cases, the intensity of the fluorescent light emitted from the tissue sample may be reduced by an acousto-optical modulator (AOM). The AOM may be controlled by a gain controller 221 to reduce the intensity of the fluorescent signal at the PMT when the fluorescent signal intensity exceeds and / or falls below the detectable range of the PMT. In some cases, the AOM may be placed between the light source 106 and the collection optics 118 of the optical scanning element 112. The AOM may reduce the intensity of the fluorescent light emitted from the tissue sample by redirecting the fluorescent light emitted from the tissue sample by an optical component that oscillates at an angle from the optical detection axis of the PMT 122. In some cases, the AOM may be electrically coupled and / or controlled by an FPGA connected to a DAC. In some cases, the FPGA connected to the DAC may provide an analog signal to the AOM driver, which then operates the AOM. In some cases, the AOM may be used to modulate the intensity of fluorescent light emitted from a tissue sample between a first area of ​​the tissue sample and a second area of ​​the tissue sample, which may or may not overlap. The AOM may have two functions: (1) if the fluorescence intensity exceeds the detectable range of the PMT, the AOM may reduce the fluorescence intensity by modulating the output light of the light source, and / or (2) dynamically adjust the fluorescence intensity incident on the PMT to alter gain of the PMT.

[0155] As an example of a gain system, in some cases, the tissue sample may be excited with multiple light pulses and the recorded data may be averaged and analyzed to determine if the signal from the tissue sample is too high or too low. The voltage provided to the PMT 122 from the voltage power supply 220 may then be adjusted by the gain controller 221 based on measurement feedback from the digitizer 234 and the system software. In some cases, a variable RF attenuator may be adjusted and / or controlled by the gain controller 221 when the signal exceeds and / or falls below the detectable range of the digitizer 234. The gain controller 221 may adjust the output voltage of the voltage power supply 220 of the PMT 122 through an analog electrical communication protocol. Such adjustments may be made manually or automatically, for example, by a processor and / or FPGA located on the gain controller 221. Such adjustments may be made iteratively until a desired signal level and / or signal-to-noise ratio is reached. The data may be recorded once the desired signal level and / or signal-to-noise ratio is reached.

[0156] In some cases, the system control electronics (128, 221, 222) may include a device controller 222. The device controller 222 (e.g., a microcontroller) may control or synchronize events such as movement of the stage 216, the position of the filters of the filter wheel 120 exposed to the collected emitted beam 117, the operating parameters of the light source 106, the gain controller 221, or any combination thereof. In some cases, the operating parameters of the light source may include controlling the output power, pulse width, pulse frequency, or any combination thereof of the light source 106. In some cases, the device controller 222 may receive inputs from and / or provide outputs to the digitizer (126, 234), the scan controller 2426, the gain controller 221, the extraction controller 2422, the light source 106, the computer system 804, and / or the computer system processor 810, or any combination thereof. In some cases, the device controller 222 may receive a universal serial bus (USB) input from the digitizer (126, 234).

[0157] In some cases, the fluorescence imaging system (300, 2300) may comprise one or more air flow features 2316 configured to draw and / or direct air flow from an exterior surface, into through the system's enclosure, and / or out of the imaging system enclosure. In some instances, the one or more air flow features may comprise one or more filters configured to filter environmental and / or atmospheric particles external to the imaging system enclosure prior to introduction into the imaging system enclosure.

[0158] In some cases, the one or more filters may filter particles from the external atmosphere prior to directing and / or assisting the transfer of atmosphere (e.g., air fluid atmosphere external to the system) into the enclosure of the imaging system. Particles filtered from the atmosphere, if not filtered, may adhere, precipitate, and / or land on one or more surfaces of the optical and / or electronic components of the imaging system, damaging the components and hindering their performance. In some cases, the filters may prevent particles from landing on one or more surfaces of optical components exposed to high pulsed energy from the light source, described elsewhere herein, which may ionize the particles and damage the optical components.

[0159] In some cases, the one or more airflow features 2316 may be configured to direct a flow of air from the atmosphere or environment external to the imaging system enclosure into the enclosure to maintain the temperature of the imaging system components, as seen in FIG. 24B. Maintaining an operating temperature for the one or more imaging system components may enable the one or more imaging system components to operate at peak efficiency, e.g., a laser cooled to an operating temperature of 28-35 degrees Celsius maintains nominal laser operation in terms of laser output power, repetition rate, and / or constant laser output spectral characteristics compared to a laser operating outside the operating temperature range. In some cases, the one or more airflow features may comprise airflow intake features, e.g., vents, slots, and / or openings otherwise disposed on a surface of the imaging system, in fluid communication with the atmosphere or environment external to the imaging system and the environment and / or atmosphere internal to the enclosure or imaging system. In some cases, the one or more features may comprise one or more baffles configured to direct and / or transport the flow of the external environment and / or atmosphere around the imaging system components within the imaging system enclosure. In some cases, the air flow intake feature and / or baffle may be positioned adjacent to the light source (e.g., laser), as described elsewhere herein, to direct air flow from the atmosphere and / or environment external to the imaging system toward the light source and maintain the operating temperature of the laser. In some cases, the light source may comprise a heat sink in contact with one or more surfaces of the light source, the heat sink configured to dissipate heat across a surface area greater than the surface area of ​​the light source to help maintain the operating temperature of the light source. In some cases, the air flow intake feature may be located on the top surface of the imaging system and / or a surface at its peak height, and the air flow outlet vent, i.e., exhaust, is located at the bottom or lowest height of the imaging system relative to a horizontal surface on which the imaging system is maintained.By placing air flow intake features on the top surface of the imaging system and / or on a surface at its peak height, and exhaust vents at the bottom, dispersion of potential contaminants present in the exhaust air into the sterile surgical field surrounding the imaging system can be limited in an operating room setting in which the imaging system is used.

[0160] In some embodiments, the fluorescence imaging system (300, 2300) may include a handle 2312 that allows one or more users of the fluorescence imaging system to transport the imaging system mounted on one or more wheels (e.g., casters). The one or more wheels of the system may comprise a material that allows the fluorescence imaging system (300, 2300) to be transported over uneven surfaces without damaging or misaligning one or more optical components of the fluorescence imaging system.

[0161] In some cases, the internal LEDs and / or light sources of the electrical, opto-mechanical, and / or mechanical components of the imaging system may be covered and / or blocked from transmitting light to other components of the fluorescence imaging system. The LEDs and / or light sources of the electrical, opto-mechanical, and / or mechanical components may be covered and / or blocked from transmitting light to other components of the fluorescence imaging system to improve the signal-to-noise ratio of the detected fluorescence signal by the detector (e.g., photomultiplier tube) by reducing background light of the LEDs and / or light sources of the electrical, opto-mechanical, and / or mechanical internal system components from entering the optical detection path of the imaging system. In some cases, the LEDs and / or light sources of the electrical, opto-mechanical, and / or mechanical components of the imaging system may be covered with black optical tape or weather stripping. In some cases, orifices and / or openings between the internal surface of the imaging system and the external surface of the imaging system may be blocked and / or sealed to prevent stray light from the ambient environment surrounding the imaging system from entering the optical detection path of the imaging system. The blocked orifices and / or openings of the imaging system may increase the signal-to-noise ratio of the detection of the fluorescent signal by the detector by reducing background light provided by the surrounding environment of the imaging system. Computer Systems and Machine Learning Models

[0162] In some embodiments, the systems disclosed herein may comprise a computer system 804 suitable for implementing a machine learning model configured to analyze fluorescence data generated by an imaging system described elsewhere herein, as seen in FIG. 8. In some cases, the machine learning model may analyze, extract, summarize, reduce, predict, classify, or any combination thereof performed on the obtained data. In some cases, the fluorescence data may comprise autofluorescence data, fluorescence lifetime data, or any combination thereof. In some cases, the obtained data may comprise multiple autofluorescence or fluorescence lifetime images of a tissue sample.

[0163] In some embodiments, the systems disclosed herein may implement a machine learning algorithm configured to classify one or more autofluorescence or fluorescence lifetime characteristics and determine the presence or absence of cancer in a tissue sample. In some cases, the machine learning classification module may include performing cancer classification for each individual signal collection channel or for all channels together. The machine learning model may include a classification module that may take the collected / extracted features from the signal pre-processing step and classify the features. In some cases, the features may be extracted without a signal pre-processing step.

[0164] In some cases, machine learning algorithms may need to extract and draw relationships between features because traditional statistical techniques may not be sufficient. In some cases, machine learning algorithms may be used in conjunction with traditional statistical techniques. In some cases, traditional statistical techniques may provide pre-processed features to the machine learning algorithm.

[0165] In some embodiments, the plurality of features may be classified into any number of categories. One or more images generated by the systems described elsewhere herein may be classified as cancerous or non-cancerous images. In some cases, the plurality of features may be classified into 1-20 categories. Each category may also be divided into subcategories.

[0166] In some embodiments, a human may select and discard features prior to / during machine learning classification. In some cases, a computer may select and discard features. In some cases, features may be discarded based on a threshold.

[0167] In some embodiments, any number of features may be classified by a machine learning algorithm. The machine learning algorithm may classify at least 10 features. In some cases, the plurality of features may include between about 10 features and 200 features. In some cases, the plurality of features may include between about 10 features and 100 features. In some cases, the plurality of features may include between about 10 features and 50 features. In some embodiments, the machine learning algorithm may be, for example, an unsupervised learning algorithm, a supervised learning algorithm, or a combination thereof. The unsupervised learning algorithm may be, for example, clustering, hierarchical clustering, k-means, mixture models, DBSCAN, OPTICS algorithm, anomaly detection, local outlier factor methods, neural networks, autoencoders, deep belief networks, Hebbian learning, generative adversarial networks, self-organizing maps, expectation maximization algorithms (EM), method of moments, blind source separation techniques, principal component analysis, independent component analysis, non-negative matrix factorization, singular value decomposition, or a combination thereof. The supervised learning algorithm may be, for example, a support vector machine, a linear regression, a logistic regression, a linear discriminant analysis, a decision tree, a k-nearest neighbor algorithm, a neural network, similarity learning, or a combination thereof. In some embodiments, the machine learning algorithm may comprise a deep neural network (DNN). The deep neural network may comprise a convolutional neural network (CNN). The CNN may be, for example, U-Net, ImageNet, LeNet-5, AlexNet, ZFNet, GoogleNet, VGGNet, ResNet18, or ResNet, etc.Other neural networks may be, for example, deep feedforward neural networks, recurrent neural networks, LSTMs (long short-term memories), GRUs (gated recurrent units), autoencoders, variational autoencoders, adversarial autoencoders, denoising autoencoders, sparse autoencoders, Boltzmann machines, RBMs (restricted BMs), deep belief networks, generative adversarial networks (GANs), deep residual networks, capsule networks, or attention / transformer networks, etc.

[0168] In some cases, the machine learning model may comprise clustering, scalar vector machine, kernel SVM, linear discriminant analysis, quadratic discriminant analysis, neighborhood component analysis, manifold learning, convolutional neural network, reinforcement learning, random forest, naive Bayes, Gaussian mixture, hidden Markov model, Monte Carlo, restricted Boltzmann machine, linear regression, or any combination thereof.

[0169] In some cases, the machine learning algorithm may include ensemble learning algorithms such as bagging, boosting, and stacking. The machine learning algorithm may be applied to multiple features extracted per channel individually, such that each channel may have a separate iteration of the machine learning algorithm, or may be applied to multiple features extracted from all channels or a subset of channels at once.

[0170] In some embodiments, the system may apply one or more machine learning algorithms. In some embodiments, the method may apply one or more machine learning algorithms per channel.

[0171] The machine learning classification module may comprise any number of machine learning algorithms. In some embodiments, the random forest machine learning algorithm may be an ensemble of bagged decision trees. In some cases, the ensemble of bagged decision trees may classify each temporal data segment per channel as (1) cancer positive or (2) cancer negative. The ensemble may be at least about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 500, 1,000, or more bagged decision trees. The ensemble may be at least about 1,000, 500, 250, 200, 180, 160, 140, 120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or less bagged decision trees. The ensemble may be about 1-1,000, 1-500, 1-200, 1-100, or 1-10 bagged decision trees.

[0172] In some embodiments, the method may include applying the machine learning classifier to any number of channels. The method may include applying the machine learning classifier to at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 500, 1,000, or more channels. The method may include applying the machine learning classifier to at least about 1,000, 500, 100, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less channels. The method may include applying the machine learning classifier to about 1-1,000, 1-100, 1-25, or 1-5 channels.

[0173] In some cases, multiple autofluorescence or fluorescence lifetime signals may be collected across multiple channels. Machine learning algorithms may be applied to the features extracted per channel individually, such that each channel has a separate iteration of the machine learning algorithm, or may be applied to the features extracted from all channels or a subset of channels at once. Each channel may be applied with at least about 1, 2, 5, 10, 25, 50, or more machine learning algorithms. Each channel may be applied with at least about 50, 25, 10, 5, 2, or fewer machine learning algorithms.

[0174] In some embodiments, the method may include applying a machine learning classifier to a subset of channels. The subset of channels may be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, or more of the total set of channels. The subset of channels may be at least about 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less of the total set of channels. The subset of channels may be about 1%-50%, 1%-40%, 1%-30%, 1%-20%, 1%-10%, or 1%-5% of the total set of channels.

[0175] In some embodiments, the machine learning algorithm may have various parameters, such as a learning rate, a small batch size, a number of epochs to train on, momentum, a learning weight decay rate, or a neural network layer.

[0176] In some embodiments, the learning rate may be between about 0.00001 and 0.1.

[0177] In some embodiments, the small batch size may be about 16-128.

[0178] In some embodiments, the neural network may comprise neural network layers. The neural network may have at least about 2 to 1,000 or more neural network layers.

[0179] In some embodiments, the number of reference time points for training may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 500, 1,000, 10,000, or more.

[0180] In some embodiments, the momentum may be at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or more. In some embodiments, the momentum may be at least about 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or less.

[0181] In some embodiments, the learning weighted decay rate may be at least about 0.00001, 0.0001, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, or more. In some embodiments, the learning weighted decay rate may be at least about 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, 0.001, 0.0001, 0.00001, or less.

[0182] In some embodiments, the machine learning algorithm may use a loss function, which may be, for example, a regression loss, a mean absolute error, a mean bias error, a hinge loss, an Adam optimizer, and / or a cross-entropy.

[0183] In some embodiments, the parameters of the machine learning algorithm may be adjusted with the aid of a human and / or a computer system.

[0184] In some embodiments, the machine learning algorithm may prioritize certain features. The machine learning algorithm may prioritize features that may be more relevant for detecting seizures. A feature may be more relevant for detecting seizures if it is classified more frequently than another feature. In some cases, features may be prioritized using a weighting system. In some cases, features may be prioritized on a probability statistic based on the frequency and / or number of occurrences of the feature. The machine learning algorithm may prioritize features with the aid of a human and / or a computer system.

[0185] In some embodiments, one or more of the features may be used in conjunction with machine learning or traditional statistical techniques to determine whether the section is likely to contain artifacts. The identified artifacts may be the result of optical misalignment, sample movement during image acquisition, laser power instability, laser pulse frequency jitter, or any combination thereof, or movement, subject movement, subject eye movement or blinking, subject chewing, subject muscle tension, subject electrocardiogram artifacts, etc. In some cases, a movement sensor or other sensor may be used as an additional input to the artifact removal module. In some cases, the identified artifacts may be removed so that they are not used in cancer classification. In some cases, the identified artifacts may be reduced, erased, or eliminated, and the remaining areas of the tissue sample may still be processed for cancer classification.

[0186] In some cases, a machine learning algorithm may prioritize certain features to reduce computational cost, save processing power, save processing time, increase reliability, reduce random access memory usage, etc.

[0187] The computer system 804 may include a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 810, which may be a single-core or multi-core processor or multiple processors for parallel processing. The computer system 804 may further include a memory or memory location 808 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 806 (e.g., hard disk), a communication interface 814 (e.g., network adapter) for communicating with one or more other devices, and peripheral devices 812, such as cache, other memory, data storage devices, and / or electronic display adapters. The memory 808, the storage unit 806, the interface 814, and the peripheral devices (e.g., mouse, keyboard, etc.) 312 may communicate with the CPU 810 through a communication bus (solid lines) such as a motherboard. The storage unit 806 may be a data storage unit (or data repository) for storing data. The computer system 804 may be operatively coupled to a computer network ("network") 816 with the aid of the communication interface 814. The network 816 may be the Internet, an Internet and / or an extranet, or an intranet (e.g., an intranet of an imaging system) and / or an extranet in communication with the Internet. In some cases, the subsystem components, e.g., the processor, the controller, the optical scanning element driver, the light source, or any combination thereof, may communicate electrically with each other via Ethernet CAT-5, CAT-6, CAT-7 cables. The network 816 may be a telecommunications and / or data network in some cases. The network 816 may include one or more computer servers, which may enable distributed computing, such as cloud computing.The network 816 may, in some cases, implement a peer-to-peer network that may enable devices coupled to the computer system 804 to act as clients or servers, with the aid of the computer system 804.

[0188] The CPU 810 may execute sequences of machine-readable instructions, which may be embodied in a program or software. The instructions may be directed to the CPU 810, which may subsequently program or otherwise configure the CPU 810 to obtain and / or process data produced by the imaging system described elsewhere herein.

[0189] In some embodiments, the central processing unit of the computer system 804 may execute machine executable or machine readable code, which may be provided in the form of software, to transfer data generated by the imaging system to the network and / or cloud 816 for further processing, classification, data clustering, or any combination thereof. In some cases, the data may comprise individual image pixel data, where an image is comprised of one or more pixels. In some cases, the pixel data may comprise autofluorescence data, fluorescence lifetime data, or any combination thereof acquired by the imaging system. In some cases, the data may comprise a plurality of autofluorescence or fluorescence lifetime decay curves. In some cases, the data transfer of data generated by the imaging system to the network 816 may comprise a workflow 1901, as seen in FIG. 19A. In some cases, one or more imaging systems (1902, 1903, 1904) may obtain image data and transmit the data via the network and / or cloud 816 to a data server 1912 for raw image data, and / or transmit the data via the network and / or cloud 816 to an API gateway 1918. In some cases, the API Gateway 1918 may comprise one or more functions that may act on the data for processing. In some cases, the data received at the API Gateway 1918 may be acted upon by a user or operator of the imaging system (1902, 1903, 1994) post acquisition or in an asynchronous aspect to process the acquired data. In some cases, the asynchronous aspect may include acquiring data at a data acquisition rate of at least 30 pixels / second and simultaneously transmitting the data to the API Gateway 1918 or the raw data server 1912. In some cases, the API Gateway 1918 may direct the data towards one or more processing steps.In some cases, the one or more processing steps may comprise calibration 1914, pixel classification 1916, data image aggregation 1932, context classification 1928, image processing 1924, or any combination thereof.

[0190] In some cases, the calibration and pixel classification processing steps may be completed in an asynchronous or synchronous data transfer configuration. In some cases, the calibration processing step 1914 may correct for any system-specific calibrations referenced from machine-specific data headers included with each data point. In some cases, the calibration processing step 1914 may comprise one or more calibration processes 1920, which may comprise data processing actions for one or more imaging systems (1902, 1903, 1904). In some cases, the one or more calibration processes 1920 may include locating the calibrations in the calibration database 1908 and applying the calibrations to the one or more calibration processes 1920. After the calibration process 1920, the calibrated data from the one or more systems may then be classified asynchronously or synchronously with the pixel classification process 1916.

[0191] The pixel classification process 1916 may comprise one or more parallel pixel classification processes 1922 configured to identify a tissue or sub-tissue classification of a given stream of pixel data of one or more pixels. In some cases, the pixel classification process 1916 may determine a pixel classification of at least one pixel based on the pixel data. In some cases, the scan type database 1910 may comprise one or more tissue type classification sub-processes 1922 for one or more sets of classifiers 2006 configured to classify the pixel data into tissue types. In some cases, the tissue types may comprise cancerous tissue, healthy tissue, fat, muscle, cancerous tissue immersed in formalin, healthy tissue immersed in formalin, fat tissue immersed in formalin, muscle tissue immersed in formalin, or any combination thereof. In some cases, the pixel classification module may comprise one or more tissue type classification sub-processes 1922 as seen in FIG. 19B. In some cases, the one or more classification sub-processes 1922 may comprise at least one pre-processing pipeline 2004, at least one classifier 2006, or any combination thereof. In some cases, the at least one pre-processing pipeline 2004 may comprise z-score pixel data operations, pixel data outlier filtering, or any combination thereof. In some cases, the z-score pixel data operations may include normalizing at least one pixel data to a Gaussian distribution. In some cases, the various pre-processing pipelines may increase the signal-to-noise ratio for pixel data relative to background noise signals. In some cases, the at least one classifier 2006 may comprise a support vector machine (SVM), k-means clustering, neural network, linear regression, non-linear regression, random forest, or any combination thereof. In some cases, the classifier 2006 may classify tissues or tissue subtypes by one or more pixel data features, respectively.In some cases, the one or more pixel data features may comprise one or more subsamples of one or more fluorescence or autofluorescence decay emissions of the given pixel data, Laguerre coefficients of one or more fluorescence or autofluorescence decay emissions of the given pixel data, raw lifetime data of one or more fluorescence or autofluorescence decay emissions of the given pixel data, or any combination thereof. In some cases, the classifiers 2006 of each classification sub-process may provide a probability that the pixel data may be classified as one of the tissue types dictated by the scan type database 1910. In some cases, the probability may comprise a value from about 0 to about 1. In some cases, the probabilities generated for each classifier 2006 may be weighted by a value stored in the scan type database 1910 and correlated to each classifier 2006. In some cases, the pixel classification process may arrive at a pixel classification tissue type 2022 by calculating an index of the maximum argument 2020 of all the weighted probabilities of each classifier 2006. In some instances, the index may comprise an indicator of which tissue type classification sub-process 1922 yielded the highest probability among all tissue type classification sub-processes 1922.

[0192] In some cases, the pixel classification texture type 2022 may then be stored in the processed data server 1936 for further processing and analysis. In some cases, the pixel classification texture type 2022 may then reach the data image aggregation process 1932. In some cases, the data image aggregation process 1932 may include one or more sub-image data aggregation processes 1934, each of which may aggregate pixel data of one or more imaging systems (1902, 1903, 1904) in parallel. In some cases, each sub-image data aggregation process 1934 may combine one or more pixel data locations and corresponding pixel classification texture type 2022 into a matrix. The matrix for each sub-image data aggregation process 1934 may be stored in the processed data server 1936 for further processing and analysis. In some cases, the aggregated pixel classification texture type matrix may then be sent to the context classification process 1928 for further processing.

[0193] In some instances, the context classification process may comprise one or more sub-context classification processes 1930, each of which may contextually classify one or more pixel classification tissue types 2022 of one or more imaging systems (1902, 1903, 1904) in parallel. In some cases, the sub-context classification process 1930 may determine a classification of one or more nearby local pixel classification tissue types 2022 (e.g., adjacent pixels or pixels within a defined neighborhood) based, at least in part, on the distribution of the classification tissue types 2022 of pixels within a local neighborhood. The context classifications for each sub-context classification process 1930 may be stored in the processed data server 1936 for further processing and analysis. In some cases, the contextually classified pixel data may then be sent to the image processing process 1924 to generate a representative pseudo-colored image indicative of all pixel classification tissue types 2022 of pixels in the image dataset.

[0194] In some cases, the contextually classified pixel data may be converted by the image processing process 1924 into a pseudocolored image 1938 showing the pixel classification tissue type 2022 of each pixel. The image processing process 1924 may comprise one or more sub-image processing processes 1926 configured to process images of pixel data of one or more imaging systems (1902, 1903, 1904) in parallel. In some cases, the sub-image processing process 1926 may interpolate pixel classification tissue types 2022 between tissues to generate high resolution images from low resolution images. In some cases, the sub-image processing process 1926 may also overlay a pseudocolor map to spatially distinguish various pixel classification tissue types 2022 per pixel in the image dataset. In some cases, the processed image datasets of the one or more sub-image processing processes 1926 may be stored in the processed data server 1936 for further processing and analysis. In some cases, the processed image data set 1938 of one or more sub-image processing processes 1926 may then be displayed on one or more imaging systems (1902, 1903, 1904) from which the image pixel data originated.

[0195] In some embodiments, the CPU 810 may be part of a circuit, such as an integrated circuit. One or more other components of the system 804 may be included within the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0196] The storage unit 806 may store files such as drivers, libraries, and saved programs. The storage unit 806 may store the acquired autofluorescence data, fluorescence lifetime data, or any combination thereof. The computer system 804 may include one or more additional data storage units that are external to the computer system 804, such as located on a remote server that communicates with the computer system 804 through an intranet or the Internet in some cases. In some cases, the computer system may include a communication channel 2448 configured to acquire and / or transfer the acquired autofluorescence data, fluorescence lifetime data, or any combination thereof. In some cases, the communication channel may provide an input and / or output interface of the computer system configured to allow the remote server and / or cloud-based server to push updates (e.g., operating system parameters) to the imaging system. In some cases, the communication channel 2448 may provide a user remote access to the system. In some cases, the communication channel may provide a data link between the imaging system hardware and the memory of the computer system 804 for further processing. In some cases, the communication channel 2448 may be used to stream the autofluorescence and / or fluorescence lifetime data acquired using the imaging system as well as a data container (e.g., virtualization of memory and computing power) for sorting the autofluorescence and / or fluorescence lifetime data as described elsewhere herein. In some instances, the data container may be located locally on the computer system 806 and / or in the cloud 816. In some cases, the data container may be a data container that allows for management and hosting of one or more data containers.

[0197] Methods as described herein may be implemented using machine (e.g., computer processor) executable code stored on an electronic storage location of the computing device 804, such as on the memory 808 or the electronic storage unit 806. The machine executable or machine readable code may be provided in the form of software. During use, the code may be executed by the processor 810. In some cases, the code may be read from the storage unit 806 and stored on the memory 808 for easy access by the processor 806. In some cases, the electronic storage unit 806 may be excluded and the machine executable instructions are stored on the memory 808.

[0198] The code may be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or may be compiled during run-time. The code may be provided in a programming language that may be selected to allow the code to be executed in a pre-compiled or as-compiled manner.

[0199] Aspects of the systems and methods provided herein, such as the computer system 804, may be embodied in programming. Various aspects of the technology may be considered as "products" or "articles of manufacture," typically in the form of machine (or processor) executable code and / or associated data carried on or embodied in some type of machine-readable medium. The machine executable code may be stored on an electronic storage unit, such memory (e.g., read-only memory, random access memory, flash memory), or hard disk. A "storage" type medium may include any and all tangible memory of a computer, processor, equivalent, or their associated modules, such as various semiconductor memories, tape drives, disk drives, and the like, which may provide non-transitory storage for software programming from time to time. All or portions of the software may be communicated over the Internet or various other telecommunications networks from time to time. Such communication may, for example, enable loading of the software from one computer or processor to another, for example, from a management server or host computer to a computer platform of an application server. Thus, other types of media that may carry software elements include optical, electrical, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical terrestrial networks, and via various wireless links. Physical elements that carry such waves, such as wired or wireless links, optical links, or the like, may also be considered software-bearing media. As used herein, unless limited to non-transitory tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0200] Thus, a machine-readable medium such as a computer-executable code may take many forms, including, but not limited to, a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media may include, for example, optical or magnetic disks, such as any of the storage devices in any computer or equivalent, such as those that may be used to implement databases, etc. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables, copper wire, and optical fibers, including the wires that comprise a bus in a computing device. Carrier wave transmission media may take the form of electric or electromagnetic signals or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer readable media thus include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with a pattern of holes, a RAM, a ROM, a PROM and EPROM, a flash EPROM, any other memory chip or cartridge, a carrier wave that transports data or instructions, a cable or link that transports such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0201] The computer system may include or communicate with an electronic display 301 with a user interface (UI) 130 for viewing the raw autofluorescence data, raw fluorescence lifetime data, autofluorescence image 1802, fluorescence lifetime image 1802, visible light image 1800, or any combination thereof, as seen in FIGS. 18A-18C. In some cases, the computer system may transmit and / or relay system data via an electronic display operator 2402, which displays the data on the UI 130 of the display 301. In some cases, the computer system 804 may transmit system control information (e.g., system operating parameters) via a control operator 2404, which displays the system control information on the UI 130 controls of the imaging system. In some cases, user interaction with the system, for example, through use of a touch screen, keyboard, and / or mouse, may be transmitted to the computer system through a user interaction control operator 2406. In some cases, the autofluorescence or fluorescence lifetime image 1802 may provide visualization of a suspected cancer 1803 that may not be visible in the visible light image 1800. After processing and classifying pixel image data obtained by the system of the present disclosure described elsewhere herein, the image data may be pseudocolored (1802) to indicate one or more classifications of tissue type. In some cases, the pseudocolored image may be pseudocolored with one or more colors in the visible spectrum (e.g., red, green, blue, yellow, purple, orange, etc.). As can be seen in Figures 18A-C, a corresponding histopathology image 1804 showing a depiction of cancerous tissue 1805 of a tissue sample corresponds to the cancer morphology 1803 shown in the autofluorescence or fluorescence lifetime image 1802. In some cases, the UI 130 may also include a number of control buttons, slides, radio buttons, dialogs, or any combination thereof to control the operation of the imaging system. User Interface

[0202] Aspects of the system of the disclosure provided herein may include a user interface 301, as seen in FIGS. 4A-4B. The UI 130 may include an image display, such as a flat screen panel or touch screen display. The UI 130 may enable visualization of data acquired from a tissue sample. The user interface may provide actionable information for health care personnel to guide surgical dissection or resection of a patient's cancer. In some cases, the user interface 130 may display a visible light video (402, 404) or a visible light still image of the tissue sample being imaged. In some cases, the user interface may include a view 412 showing an average fluorescence lifetime per acquisition channel of a plurality of acquisition channels. In some cases, the user interface may include a view 414 showing raw fluorescence lifetime data (e.g., FIG. 5) for each acquisition channel of a plurality of acquisition channels. In some instances, the user interface 130 may include multiple views 416 of the spatial distribution of acquired fluorescence lifetimes for each individual channel. In some cases, the user interface may display a fluorescence map 406, which represents a combined or averaged image of the spatial distribution of fluorescence lifetimes for multiple points across the tissue sample. The user interface may also include a view 408 that displays an image of the tissue sample being imaged overlaid with the PMT intensity signal.

[0203] In some embodiments, the user interface 130 may comprise function buttons, switches, editable dialog boxes, slides, radio buttons, or any combination thereof. In some cases, the user interface may comprise one or more displays that allow the user to configure device parameters, such as scan speed, manual scan position of the stage, resolution, or any combination thereof. The user interface may comprise function buttons that may toggle between various overlay signal processing pseudocolor maps that may show the user areas of the tissue sample that may have cancer. In some cases, the user interface may comprise function buttons that allow scanning, stopping scanning, emergency stopping scanning, pausing scanning, resuming scanning, or any combination thereof.

[0204] In some embodiments, the user interface 130 may include a touch screen interface that allows the user to tap on the screen to select an action, and / or may be operated or interacted with a keyboard and / or mouse. In some cases, the touch screen interface may be displayed on one or more monitors and / or displays (301, 2304, 2302), as seen in FIGS. 3A-3B and 24A-24C. In some cases, the touch screen monitor 2304 and / or display (301, 2302) may be positioned at arm level of an individual, such as a user, physician, scrub medical personnel, nurse, or any combination thereof, for ergonomic use of such touch screen monitor and / or display. In some cases, the imaging system may include one or more monitors and / or displays configured to display raw, processed, analyzed, or any combination thereof categories of fluorescence imaging data to an individual, such as a user, physician (e.g., scrub physician), scrub medical personnel, nurse, or any combination thereof. In some cases, one or more monitors (301, 2302) may be equipped with mechanisms for adjusting the tilt, three-dimensional position, and / or rotation of the monitor and / or display.

[0205] In some cases, the imaging system may include one or more test and / or calibration phantoms and / or targets that may be analyzed upon initialization, calibration, and / or start-up of the imaging system. In some cases, the one or more test and / or calibration phantoms and / or targets may include a fluorescence intensity imaging resolution target, a fluorescence lifetime imaging resolution target, one or more vials of dye with known fluorescence lifetime measurements, or any combination thereof. In some cases, the one or more test and / or calibration phantoms and / or targets may be embedded within the imaging system. In some cases, one or more vials of dye with known fluorescence lifetimes may be used to test the impulse response function, accuracy, and / or precision of the lifetime measurements of the imaging system. In some cases, the fluorescence intensity imaging resolution target may include a material as described elsewhere herein, for example, a polymer (e.g., plastic) with known fluorescence lifetimes overlaid with a metal coating configured to reflect the excitation light source provided to spatially isolate the area of ​​fluorescence lifetime measurements. By spatially isolating one or more regions of varying fluorescence intensity of the calibration phantom and / or target, the emitted fluorescence signal intensity may be measured and taken into account for future system optical alignment adjustments and / or software compensation (e.g., compensation of fluorescence decay curve measurements, spatial alignment of scans and / or visible images, adjustment of performance parameters associated with the galvanic scanning mirror and / or motorized stage, or adjustment of automatic gain performance parameters). Parameters associated with the galvanic scanning mirror and / or motorized stage may comprise resolution, speed, step size, acceleration profile, etc., or any combination thereof. Automatic gain performance parameters may comprise weighting and amount of PMT gain, AOM attenuation, RF attenuation, or time characteristics associated with the automatic gain performance parameters.In some cases, the fluorescence lifetime imaging resolution target may include a first material with a first lifetime overlaid and / or inlaid with a geometric shape of a second polymeric material with a second lifetime (e.g., a triangular or polygonal shape with straight edges). By imaging the fluorescence lifetime imaging resolution target, the boundary between the first material and the second material may be measured and used for system calibration and / or adjustment (e.g., compensation of measurements of the fluorescence lifetime signal, spatial alignment of the scanned and / or visible images, adjustment of performance parameters associated with the galvanic scanning mirror and / or motorized stage, or adjustment of automatic gain performance parameters).

[0206] In some cases, the fluorescence intensity imaging resolution target and / or the fluorescence lifetime imaging resolution target may include a material overlaid with a transparent space and / or resolution target (e.g., USAF-1951) that is metal coated except in the area of ​​the resolution target feature. The metal coated areas may have various levels of optical attenuation. In some cases, the fluorescence intensity imaging resolution target may include a material with a spatially varying fluorescence lifetime and / or intensity. Such a resolution target may allow a light source of the imaging system to be transmitted through the resolution target and excite the material below the line target, thereby providing a spatial fluorescence emission in a well-defined pattern. The well-defined pattern of fluorescence emission may be analyzed and taken into account when calibrating and / or adjusting system parameters to improve system performance, as described elsewhere herein. In some cases, the phantom and / or target may be integrated into the imaging system to simplify user operation of the system. In some cases, the phantom and / or target may be used during system power-on self-test (POST) and built-in self-test (BIST). method

[0207] Aspects of the disclosure provided herein may comprise a scanning method for imaging a tissue sample to identify or characterize the presence or absence of cancer in the tissue sample, as described elsewhere herein. The scanning method may provide better than expected results in terms of reduced imaging time, imaging resolution (i.e., high speed high numerical aperture imaging), reduced imaging noise, and / or facilitated reconstruction of imaging data. The scanning method may reduce imaging time by completing a scan of a first area of ​​the sample, then translating the optical scanning element 112 to a second area of ​​the sample, where the first and second areas of the sample do not overlap, but instead continuously scan an area 2100 (i.e., swath) and / or strip 2116 of data consisting of one or more sections 2118 (i.e., columns) with a width 2112 of data across the sample, as shown in FIG. 20A. In some cases, an emission channel of one or more emission channels, described elsewhere herein, may be utilized to collect fluorescent emission from a light source scanned across an area 2100, i.e., a swath and / or strip 2116, scanned across the sample. In some cases, the scanning of one or more additional areas 2100, swaths, and / or strips 2116 may be repeated to collect fluorescent emission for one or more of the other emission channels. By collecting data for a single emission channel, the gain voltage of the detector (e.g., PMT) may remain at a constant value, increasing the signal-to-noise ratio and / or imaging resolution of the fluorescent emission detected in the emission channel of the one or more emission channels. In some cases, the signal-to-noise ratio and imaging resolution may be increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% compared to scanning methods that do not utilize the scanning methods described herein. In some cases, the scanning methods may reduce the scan time that would otherwise be required to start and stop the motion of the optical scanning element 112.In some embodiments, the scanning time may be reduced by the scanning method by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% compared to a scanning method that does not utilize the scanning method described herein. In addition, the scanning method may reduce noise introduced into the imaging data by mechanical jitter or motion artifacts caused by starting and stopping the motion of the optical scanning element 112. In some cases, the scanning method may reduce noise introduced into the imaging data by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% compared to a scanning method that does not utilize the scanning method described herein. In some cases, the scanning method may facilitate co-registration of areas, swaths, and / or strips of data scanned and collected across a sample by scanning an area section (i.e., a swath and / or strip) across the length of the sample, as compared to multiple localized discrete areas scanned in traditional mosaic scanning methods. By scanning and collecting data for strips, swaths, and / or areas of data, the scanning method may reduce the number of data points for co-registration as well as the complexity of the data interface.

[0208] In some cases, the total scanned area of ​​the sample is approximately 1 mm 2 ~ approx. 6,400mm 2 In some cases, the total scanned area of ​​the sample is about 1 mm 2 ~ approx. 50mm 2 , about 1mm 2 ~ approx. 100mm 2 , about 1mm 2 ~about 200mm 2 , about 1mm 2 ~ approx. 400mm 2 , about 1mm 2~ approx. 600mm 2 , about 1mm 2 ~ approx. 800mm 2 , about 1mm 2 ~ approx. 1,000mm 2 , about 1mm 2 ~Approx. 1,200mm 2 , about 1mm 2 ~ approx. 1,600mm 2 , about 1mm 2 ~Approx. 3,200mm 2 , about 1mm 2 ~ approx. 6,400mm 2 , about 50 mm 2 ~ approx. 100mm 2 , about 50 mm 2 ~about 200mm 2 , about 50 mm 2 ~ approx. 400mm 2 , about 50 mm 2 ~ approx. 600mm 2 , about 50 mm 2 ~ approx. 800mm 2 , about 50 mm 2 ~ approx. 1,000mm 2 , about 50 mm 2 ~Approx. 1,200mm 2 , about 50 mm 2 ~ approx. 1,600mm 2 , about 50 mm 2 ~Approx. 3,200mm 2 , about 50 mm 2 ~ approx. 6,400mm 2 , about 100 mm 2 ~about 200mm 2 , about 100 mm 2 ~ approx. 400mm 2 , about 100 mm 2 ~ approx. 600mm 2 , about 100 mm 2 ~ approx. 800mm 2 , about 100 mm 2 ~ approx. 1,000mm 2 , about 100 mm 2 ~Approx. 1,200mm 2 , about 100 mm 2 ~ approx. 1,600mm 2 , about 100 mm 2 ~Approx. 3,200mm 2 , about 100 mm2 ~ approx. 6,400mm 2 , about 200mm 2 ~ approx. 400mm 2 , about 200mm 2 ~ approx. 600mm 2 , about 200mm 2 ~ approx. 800mm 2 , about 200mm 2 ~ approx. 1,000mm 2 , about 200mm 2 ~Approx. 1,200mm 2 , about 200mm 2 ~ approx. 1,600mm 2 , about 200mm 2 ~Approx. 3,200mm 2 , about 200mm 2 ~ approx. 6,400mm 2 , about 400 mm 2 ~ approx. 600mm 2 , about 400 mm 2 ~ approx. 800mm 2 , about 400 mm 2 ~ approx. 1,000mm 2 , about 400 mm 2 ~Approx. 1,200mm 2 , about 400 mm 2 ~ approx. 1,600mm 2 , about 400 mm 2 ~Approx. 3,200mm 2 , about 400 mm 2 ~ approx. 6,400mm 2 , about 600 mm 2 ~ approx. 800mm 2 , about 600 mm 2 ~ approx. 1,000mm 2 , about 600 mm 2 ~Approx. 1,200mm 2 , about 600 mm 2 ~ approx. 1,600mm 2 , about 600 mm 2 ~Approx. 3,200mm 2 , about 600 mm 2 ~ approx. 6,400mm 2 , about 800 mm 2 ~ approx. 1,000mm 2 , about 800 mm 2 ~Approx. 1,200mm 2 , about 800 mm2 ~ approx. 1,600mm 2 , about 800 mm 2 ~Approx. 3,200mm 2 , about 800 mm 2 ~ approx. 6,400mm 2 , about 1,000 mm 2 ~Approx. 1,200mm 2 , about 1,000 mm 2 ~ approx. 1,600mm 2 , about 1,000 mm 2 ~Approx. 3,200mm 2 , about 1,000 mm 2 ~ approx. 6,400mm 2 , approx. 1,200mm 2 ~ approx. 1,600mm 2 , approx. 1,200mm 2 ~Approx. 3,200mm 2 , approx. 1,200mm 2 ~ approx. 6,400mm 2 , approx. 1,600 mm 2 ~Approx. 3,200mm 2 , approx. 1,600 mm 2 ~ approx. 6,400mm 2 , or approximately 3,200 mm 2 ~ approx. 6,400mm 2 In some cases, the total scanned area of ​​the sample is about 1 mm 2 , about 50 mm 2 , about 100 mm 2 , about 200mm 2 , about 400 mm 2 , about 600 mm 2 , about 800 mm 2 , about 1,000 mm 2 , approx. 1,200mm 2 , approx. 1,600 mm 2 , approx. 3,200mm 2 , or approximately 6,400 mm 2 In some cases, the total scanned area of ​​the sample is at least about 1 mm 2 , about 50 mm 2 , about 100 mm 2 , about 200mm 2 , about 400 mm 2 , about 600 mm 2 , about 800 mm2 , about 1,000 mm 2 , approx. 1,200mm 2 , approx. 1,600 mm 2 , or approximately 3,200 mm 2 In some cases, the total scanned area of ​​the sample is at most about 50 mm 2 , about 100 mm 2 , about 200mm 2 , about 400 mm 2 , about 600 mm 2 , about 800 mm 2 , about 1,000 mm 2 , approx. 1,200mm 2 , approx. 1,600 mm 2 , approx. 3,200mm 2 , or approximately 6,400 mm 2 The present invention may also include:

[0209] A scanning method for imaging a sample to identify or characterize the presence or absence of cancer in the sample may include (a) translating a light source (e.g., as described elsewhere herein) emitted from an optical scanning element with a first mirror along a first axis 2110 across the sample 2102, (b) translating the optical scanning element along a second axis 2114 perpendicular to the first axis 2110, and (c) actuating the second mirror to compensate for the motion of the optical scanning element along the second axis. In some embodiments, the compensation may maintain the position of the light source along the axis. In some cases, the compensation may allow for smearing of the light source along the second axis.

[0210] In some cases, the scan length along the first axis may comprise a length of about 2 pixels to about 2,200 pixels. In some cases, the scan length along the first axis may comprise a length of about 2 pixels to about 10 pixels, about 2 pixels to about 25 pixels, about 2 pixels to about 50 pixels, about 2 pixels to about 100 pixels, about 2 pixels to about 200 pixels, about 2 pixels to about 300 pixels, about 2 pixels to about 400 pixels, about 2 pixels to about 500 pixels, about 2 pixels to about 1,000 pixels, about 2 pixels to about 2,000 pixels, about 2 pixels to about 2,200 pixels, about 10 pixels to about 25 pixels, about 10 pixels to about 50 pixels. 10 pixels to 100 pixels, 10 pixels to 200 pixels, 10 pixels to 300 pixels, 10 pixels to 400 pixels, 10 pixels to 500 pixels, 10 pixels to 1,000 pixels, 10 pixels to 2,000 pixels, 10 pixels to 2,200 pixels, 25 pixels to 50 pixels, 25 pixels to 100 pixels, 25 pixels to 200 pixels, 25 pixels to 300 pixels, 25 pixels to 400 pixels 25 pixels to 500 pixels, 25 pixels to 1,000 pixels, 25 pixels to 2,000 pixels, 25 pixels to 2,200 pixels, 50 pixels to 100 pixels, 50 pixels to 200 pixels, 50 pixels to 300 pixels, 50 pixels to 400 pixels, 50 pixels to 500 pixels, 50 pixels to 1,000 pixels, 50 pixels to 2,000 pixels, 50 pixels to 2,200 pixels, 100 pixels to approx. 200 pixels, about 100 pixels to about 300 pixels, about 100 pixels to about 400 pixels, about 100 pixels to about 500 pixels, about 100 pixels to about 1,000 pixels, about 100 pixels to about 2,000 pixels, about 100 pixels to about 2,200 pixels, about 200 pixels to about 300 pixels, about 200 pixels to about 400 pixels, about 200 pixels to about 500 pixels, about 200 pixels to about 1,000 pixels, about 200 pixels to about 2,000 pixels, about 200 pixels to about 2,The length may be about 200 pixels, about 300 pixels to about 400 pixels, about 300 pixels to about 500 pixels, about 300 pixels to about 1,000 pixels, about 300 pixels to about 2,000 pixels, about 300 pixels to about 2,200 pixels, about 400 pixels to about 500 pixels, about 400 pixels to about 1,000 pixels, about 400 pixels to about 2,000 pixels, about 400 pixels to about 2,200 pixels, about 500 pixels to about 1,000 pixels, about 500 pixels to about 2,000 pixels, about 500 pixels to about 2,200 pixels, about 1,000 pixels to about 2,000 pixels, about 1,000 pixels to about 2,200 pixels, or about 2,000 pixels to about 2,200 pixels. In some cases, the scan length along the first axis may comprise a length of about 2 pixels, about 10 pixels, about 25 pixels, about 50 pixels, about 100 pixels, about 200 pixels, about 300 pixels, about 400 pixels, about 500 pixels, about 1,000 pixels, about 2,000 pixels, or about 2,200 pixels. In some cases, the scan length along the first axis may comprise a length of at least about 2 pixels, about 10 pixels, about 25 pixels, about 50 pixels, about 100 pixels, about 200 pixels, about 300 pixels, about 400 pixels, about 500 pixels, about 1,000 pixels, or about 2,000 pixels. In some cases, the scan length along the first axis may comprise a length of up to about 10 pixels, about 25 pixels, about 50 pixels, about 100 pixels, about 200 pixels, about 300 pixels, about 400 pixels, about 500 pixels, about 1,000 pixels, about 2,000 pixels, or about 2,200 pixels.

[0211] In some cases, the scan length along the first axis may comprise a length of about 0.01 mm to about 300 mm. In some cases, the scan length along the first axis may comprise a length of about 0.01 mm to about 0.1 mm, about 0.01 mm to about 0.5 mm, about 0.01 mm to about 1 mm, about 0.01 mm to about 5 mm, about 0.01 mm to about 10 mm, about 0.01 mm to about 50 mm, about 0.01 mm to about 100 mm, about 0.01 mm to about 150 mm, about 0.01 mm to about 200 mm, about 0.01 mm to about 250 mm, about 0.01 mm to about 300 mm, about 0.1 mm to about 0.5 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 10mm, approximately 0.1mm to approximately 50mm, approximately 0.1mm to approximately 100mm, approximately 0.1mm to approximately 150mm, approximately 0.1mm to approximately 200mm, approximately 0.1mm to approximately 250mm, approximately 0.1mm to approximately 300mm, approximately 0.5mm to approximately 1mm, approximately 0.5mm to approximately 5mm, approximately 0.5mm~10mm, 0.5mm~50mm, 0.5mm~100mm, 0.5mm~150mm, 0.5mm~200mm, 0.5mm~250mm, 0.5mm~300mm, 1mm~5mm, 1mm~10 mm, approx. 1 mm to approx. 50 mm, approx. 1 mm to approx. 100 mm, approx. 1 mm to approx. 150 mm, approx. 1 mm to approx. 200 mm, approx. 1 mm to approx. 250 mm, approx. ~150mm, 5mm~200mm, 5mm~250mm, 5mm~300mm, 10mm~50mm, 10mm~100mm, 10mm~150mm, 10mm~200mm, 10mm~250mm, 10mm The length may be about 300 mm, about 50 mm to about 100 mm, about 50 mm to about 150 mm, about 50 mm to about 200 mm, about 50 mm to about 250 mm, about 50 mm to about 300 mm, about 100 mm to about 150 mm, about 100 mm to about 200 mm, about 100 mm to about 250 mm, about 100 mm to about 300 mm, about 150 mm to about 200 mm, about 150 mm to about 250 mm, about 150 mm to about 300 mm, about 200 mm to about 250 mm, about 200 mm to about 300 mm, or about 250 mm to about 300 mm.In some cases, the scan length along the first axis may comprise a length of about 0.01 mm, about 0.1 mm, about 0.5 mm, about 1 mm, about 5 mm, about 10 mm, about 50 mm, about 100 mm, about 150 mm, about 200 mm, about 250 mm, or about 300 mm. In some cases, the scan length along the first axis may comprise a length of at least about 0.01 mm, about 0.1 mm, about 0.5 mm, about 1 mm, about 5 mm, about 10 mm, about 50 mm, about 100 mm, about 150 mm, about 200 mm, or about 250 mm. In some cases, the scan length along the first axis may comprise a length of at most about 0.1 mm, about 0.5 mm, about 1 mm, about 5 mm, about 10 mm, about 50 mm, about 100 mm, about 150 mm, about 200 mm, about 250 mm, or about 300 mm.

[0212] In some cases, the scan length along the second axis may comprise a length of about 0.01 mm to about 300 mm. In some cases, the scan length along the first axis may comprise a length of about 0.01 mm to about 0.1 mm, about 0.01 mm to about 0.5 mm, about 0.01 mm to about 1 mm, about 0.01 mm to about 5 mm, about 0.01 mm to about 10 mm, about 0.01 mm to about 50 mm, about 0.01 mm to about 100 mm, about 0.01 mm to about 150 mm, about 0.01 mm to about 200 mm, about 0.01 mm to about 250 mm, about 0.01 mm to about 300 mm, about 0.1 mm to about 0.5 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 10mm, approximately 0.1mm to approximately 50mm, approximately 0.1mm to approximately 100mm, approximately 0.1mm to approximately 150mm, approximately 0.1mm to approximately 200mm, approximately 0.1mm to approximately 250mm, approximately 0.1mm to approximately 300mm, approximately 0.5mm to approximately 1mm, approximately 0.5mm to approximately 5mm, approximately 0.5mm~10mm, 0.5mm~50mm, 0.5mm~100mm, 0.5mm~150mm, 0.5mm~200mm, 0.5mm~250mm, 0.5mm~300mm, 1mm~5mm, 1mm~10 mm, approx. 1 mm to approx. 50 mm, approx. 1 mm to approx. 100 mm, approx. 1 mm to approx. 150 mm, approx. 1 mm to approx. 200 mm, approx. 1 mm to approx. 250 mm, approx. ~150mm, 5mm~200mm, 5mm~250mm, 5mm~300mm, 10mm~50mm, 10mm~100mm, 10mm~150mm, 10mm~200mm, 10mm~250mm, 10mm The length may be about 300 mm, about 50 mm to about 100 mm, about 50 mm to about 150 mm, about 50 mm to about 200 mm, about 50 mm to about 250 mm, about 50 mm to about 300 mm, about 100 mm to about 150 mm, about 100 mm to about 200 mm, about 100 mm to about 250 mm, about 100 mm to about 300 mm, about 150 mm to about 200 mm, about 150 mm to about 250 mm, about 150 mm to about 300 mm, about 200 mm to about 250 mm, about 200 mm to about 300 mm, or about 250 mm to about 300 mm.In some cases, the scan length along the second axis may comprise a length of about 0.01 mm, about 0.1 mm, about 0.5 mm, about 1 mm, about 5 mm, about 10 mm, about 50 mm, about 100 mm, about 150 mm, about 200 mm, about 250 mm, or about 300 mm. In some cases, the scan length along the second axis may comprise a length of at least about 0.01 mm, about 0.1 mm, about 0.5 mm, about 1 mm, about 5 mm, about 10 mm, about 50 mm, about 100 mm, about 150 mm, about 200 mm, or about 250 mm. In some cases, the scan length along the second axis may comprise a length of at most about 0.1 mm, about 0.5 mm, about 1 mm, about 5 mm, about 10 mm, about 50 mm, about 100 mm, about 150 mm, about 200 mm, about 250 mm, or about 300 mm.

[0213] In some cases, the scanning method may include repeating steps (a)-(c) one or more times as the optical scanning element translates in the first direction along the second axis 2114. In some cases, when steps (a)-(c) are repeated in the first direction 2128 along the second axis, the light source may be translated along the first axis in the first direction 2104 or the second direction 2106, and the first and second directions are opposite. In some cases, the scanning method may include repeating steps (a)-(c) as the optical scanning element translates along the second axis in a second direction 2126 opposite to the first direction 2128 along the second axis. In some cases, when steps (a)-(c) are repeated in a second direction along the second axis, the light source may be translated along the first axis in a first direction 2104 or a second direction 2106, the first direction and the second direction being opposite to each other. In some cases, the first mirror, the second mirror, and / or the optical scanning element may be provided with a motion control waveform that drives the motion of the individual components. In some cases, the first mirror may be provided with a first waveform 2124, the first waveform may comprise a sawtooth, triangular, or parabolic waveform. In some cases, the second mirror may be provided with a second waveform 2122, the second waveform may comprise a linear waveform. In some cases, the second waveform may comprise a waveform that compensates for the period of the motion of the first mirror when the first mirror is transitioning between translations in the first and second directions along the first axis. In some cases, the optical scanning element may be provided with a third waveform, which may comprise a linear waveform. In some cases, the first waveform, the second waveform, and / or the third waveform may be generated and / or provided to the scanning optical element by a field programmable gate array (FPGA).

[0214] In some cases, the scanning methods provided herein may comprise super-resolution (e.g., imaging beyond the diffraction limit of light) scanning. In some cases, one or more pulses of a pulsed light source, as described elsewhere herein, may be provided to the sample across a pixel. In some cases, the pixel may comprise a length and / or width of at least about 125 μm or a pixel value as described elsewhere herein. In some cases, at least about 32 pulses of the light source may be provided when imaging a single pixel of data of the sample. In some cases, one or more pulses of the pulses may cover a length and / or width of at least about 3.9 μm of the pixel. In some cases, super-resolution scanning may be achieved by aggregating and / or averaging (e.g., running average) the emitted fluorescent imaging data of the sample over one or more pulses across the pixel. In some cases, at least about 1 pulse, at least about 2 pulses, at least about 3 pulses, at least about 4 pulses, at least about 5 pulses, at least about 6 pulses, at least about 7 pulses, at least about 8 pulses, at least about 9 pulses, at least about 10 pulses, at least about 11 pulses, at least about 12 pulses, at least about 13 pulses, at least about 14 pulses, at least about 15 pulses, at least about 16 pulses, at least about 18 pulses, at least about 19 pulses, at least about 20 pulses, at least about 21 pulses, at least about 22 pulses, at least about 23 pulses, at least about 24 pulses, at least about 25 pulses, at least about 26 pulses, at least about 27 pulses, at least about 28 pulses, at least about 29 pulses, at least about 30 pulses, at least about 31 pulses, or at least about 32 pulses may be averaged across a pixel. In some cases, the fluorescence imaging data of one or more pulses may be processed by moving averaging, filtering, convolution, ND convolution to image features at distances below the diffraction limit of the imaging system and / or light source.In some cases, a super-resolution scan may be completed along a first axis and / or a second axis of a scanning method described elsewhere herein.

[0215] Aspects of the disclosure provided herein may include methods (600, 608, 700, 708) for imaging a tissue sample to identify or characterize the presence or absence of cancer in the tissue sample, as seen in Figures 6A-6B and 7A-7B. In some cases, the tissue sample may include a resected tissue sample or a biopsy obtained during surgical resection of a tumor. In some cases, the methods provided herein may analyze tissue sample margins to identify margins that may comprise cancer to further inform or guide surgical dissection of the tumor. In some cases, the methods provided herein may be completed on a system described elsewhere herein.

[0216] In some embodiments, the method may comprise a method 600 for determining the presence of disease in a tissue sample by autofluorescence characteristics of the excised tissue sample, as seen in Figure 6 A. In some cases, the method may include (a) receiving 602 a tissue sample excised from a subject in a fluorescence imaging system, (b) imaging 604 the excised tissue sample and determining one or more autofluorescence characteristics of the excised tissue sample, and (c) determining 606 the presence of a tissue or cell type of interest in the excised tissue sample in the imaged excised tissue. In some cases, the method may further include (i) confirming 652 that the tissue sample contains the tissue or cell type of interest, (ii) confirming 654 that the tissue sample margin does not contain any tissue or cell type of interest, (iii) performing 656 an additional resection in the subject's body in an area corresponding to where the tissue or cell type of interest is present in the sample margin, and (iv) repeating 658 the above steps with respect to the additional resection based on the presence or absence of the tissue or cell type of interest in the sample tissue margin. In some cases, the tissue or cell type of interest may include diseased tissue or diseased cells. In some cases, the diseased tissue or diseased cells may include cancerous tissue or cancerous cells. In some cases, the subject may suffer from or be suspected of suffering from a disease. In some cases, the disease may be cancer.

[0217] In ...

Claims

1. 1. A device for determining the presence of a tissue or cell type in an excised tissue sample, said device comprising: (a) a stage coupled to a surface for receiving a tissue sample excised from a subject; (b) a light source configured to emit an excitation signal; (c) an optical assembly in optical communication with the light source for directing the excitation signal to the tissue sample received on the surface and collecting autofluorescent light emitted from the tissue sample in response; (d) a detector in optical communication with the optical assembly configured to capture the autofluorescent light emitted from the tissue sample; (e) a processor in communication with the detector for generating at least one image of the autofluorescent light emitted from the tissue sample; A device comprising:

2. The device described in claim 1, wherein the surface includes a surface of a tissue sample carrier, and the tissue sample carrier has an alignment feature.

3. The device described in claim 2, wherein the tissue sample carrier has one or more protruding or recessed features to prevent movement of the tissue sample.

4. The device described in claim 2, further comprising a barrier coupled to the tissue sample carrier, the barrier having one or more kinematic features for coupling or interlocking with the tissue sample carrier.

5. The device described in claim 4, wherein the surface includes a surface of a drawer.

6. The device described in claim 5, wherein the barrier has flanged or edge features to seal the interface between the barrier and the surface of the drawer.

7. The device described in claim 5, wherein the stage includes a linear actuator that lifts the surface with the tissue sample toward the optical assembly.

8. The device described in claim 7, wherein the drawer is provided with a lock, and when the linear actuator lifts the surface with the tissue sample, the drawer is locked in position by the lock.

9. The device described in claim 1, further comprising a stage coupled to the light source.

10. The device of claim 1, further comprising a programmable attenuator electrically coupled to the detector.

11. 1. A method for determining the presence of a tissue or cell type of interest in a tissue sample, the method comprising: (a) receiving a tissue sample excised from a subject on a surface coupled to the stage; (b) imaging the excised tissue sample to determine one or more autofluorescence characteristics of the excised tissue sample; (c) determining the presence of the tissue or cell type of interest in the excised tissue sample based on at least the one or more autofluorescence properties; and A method comprising:

12. The method of claim 11, wherein the surface comprises a surface of a tissue sample carrier, the tissue sample carrier having an alignment feature.

13. The method of claim 12, wherein the tissue sample carrier has one or more protruding or recessed features to prevent movement of the tissue sample.

14. The method of claim 12, further comprising a barrier coupled to the tissue sample carrier, the barrier having one or more kinematic features for coupling or engaging with the tissue sample carrier.

15. The method of claim 14, wherein the surface includes a surface of a drawer.

16. The method of claim 15, wherein the barrier has flanged or edge features to seal the interface between the barrier and the surface of the drawer.

17. The method described in claim 16, wherein the stage includes a linear actuator that lifts the surface with the tissue sample toward an optical assembly.

18. The method of claim 17, wherein the drawer is provided with a lock, and when the linear actuator lifts the surface with the tissue sample, the drawer is locked in position by the lock.

19. The method described in claim 11, wherein imaging includes providing an excitation signal from a light source to the excised tissue sample, the light source being coupled to a second stage different from the stage.

20. The method of claim 19, wherein imaging includes detecting autofluorescence light emitted from the excised tissue sample in response to the excitation signal of the light source using a detector, and a programmable attenuator is electrically coupled to the detector.

Images