Liquid crystal shutters engineered as dynamic long-pass optical filters.

Abstract

The present disclosure relates to a novel liquid crystal assembly for use in a multispectral imaging system. In particular, the liquid crystal assembly includes an engineered polarizer and liquid crystals. In various aspects, the liquid crystal assembly takes advantage of drawbacks found in conventional polarizers and liquid crystals that require complex temperature control. The present disclosure generally relates to a liquid crystal assembly for use with an imaging system having a light source. The assembly includes a polarizer positioned in the path of light from the light source.

Description

[Technical Field]

[0001] This disclosure relates generally to liquid crystal shutters engineered for use in multispectral imaging. Specifically, the shutters rely on the previously undesirable limitations of optical polarizers to increase the proportion of transmitted light available for imaging and analysis at video-rate switching speeds to one or more optical sensors. [Background technology]

[0002] Multicolor or "multispectral" imaging is a method of examining information from a wide range of different wavelengths of light and displaying them in a relatively narrow visible range. When processing spectral information, it is often necessary to apply different image analysis techniques to the different wavelengths of light in order to appropriately weight their values ​​before recombining them. To allow for independent processing of different color images, the spectrum is typically divided into multiple spectral bands, and images from those spectral bands are collected independently. The hardware used to image that wide spectrum may include multiple image sensors, each responsible for collecting a different spectral band.

[0003] Liquid crystal shutters are commonly used in liquid crystal displays and also to enable video-rate switching of digital light projection (DLP) systems, for example in theaters. Typically, when linear polarizers are used in liquid crystal shutters, the useful spectral range of the shutter is severely limited by the polarization extinction ratio of the polarizing filter and the limited transmission of the liquid crystal itself. Summary of the Invention

[0004] The present disclosure generally relates to a liquid crystal assembly for use with an imaging system having a light source. The assembly includes a polarizer disposed in a path of light from the light source. The polarizer is configured to have a light cutoff outside its polarization range. The assembly also includes a liquid crystal disposed in a path of light transmitted through the polarizer and a drive voltage source configured to apply first and second voltages to the liquid crystal. The first and second voltages establish high and low electric fields across the liquid crystal. The first and second voltages toggle the liquid crystal between an open state and a closed state. In one aspect, application of either the first or second voltage toggles the liquid crystal to the open state. Application of either the first or second voltage toggles the liquid crystal to the closed state.

[0005] In various embodiments, the polarizer is configured to be transparent above the light cutoff. In one embodiment, the light cutoff includes the longest wavelength of the spectral range, and the polarizer is configured to be ineffective and transparent above the longest wavelength. In some embodiments, the liquid crystal comprises a twisted nematic (TN) cell, a Pi cell, a thermotropic liquid crystal, a lyotropic liquid crystal, a liquid crystal display (LCD), a liquid crystal with photoconductor properties, or a liquid crystal that undergoes expansion and alignment under an electric field. In some embodiments, the second voltage induces backflow in the liquid crystal.

[0006] In various embodiments, one of the first voltage or the second voltage is 0 V. In another embodiment, the second voltage is a plurality of voltages including a bipolar square wave having an average value of 0 V. In one embodiment, the first voltage, or both the first and second voltages, include a plurality of voltages that produce a bipolar square wave having an average value of 0 V. In another embodiment, the bipolar square wave includes an amplitude of + / -0.1 to + / -5 Vp-p, + / -3 to + / -30 Vp-p, + / -16 Vp-p, + / -24 Vp-p, or + / -30 Vp-p, or an amplitude between + / -16 V (32 Vp-p) and + / -30 V (60 Vp-p), inclusive, or an amplitude between + / -30 V (60 Vp-p) and + / -100 V (200 Vp-p), inclusive. In a further aspect, the second voltage is a bipolar square wave having an amplitude of + / -0.1 to + / -5 Vp-p, + / -3 to + / -30 Vp-p, + / -16 Vp-p, + / -24 Vp-p, or + / -30 Vp-p, or an amplitude between + / -16 V (32 Vp-p) and + / -30 V (60 Vp-p) inclusive, or an amplitude between + / -30 V (60 Vp-p) and + / -100 V (200 Vp-p) inclusive.

[0007] In various embodiments, the bipolar square wave amplitude is modulated at the video capture rate of the imaging system. In another embodiment, the bipolar square wave amplitude is modulated at 500 to 2000 Hz. In some embodiments, the drive voltage source toggles the liquid crystal between the first and second voltages for a period of 1 ms to 40 ms (inclusive), or 2 ms to 60 ms (inclusive), or 60 ms to 100 ms (inclusive), and up to 300 ms. In some embodiments, the drive voltage source toggles the liquid crystal between the first and second voltages for a video rate sampling period of approximately 1 ms to 10 ms, 2 ms to 20 ms, or 6 ms.

[0008] In various embodiments, the liquid crystal is a twisted nematic (TN) liquid crystal. In one embodiment, the liquid crystal is heated to a temperature in the range of 30°C to 50°C. In another embodiment, the liquid crystal is enclosed in a housing of the imaging system, and the liquid crystal is heated by waste heat generated by other components of the imaging system. In one embodiment, the transition speed between the open and closed states is determined by the temperature of the liquid crystal and the voltage source drive level.

[0009] In various embodiments, the polarizer is an engineered dye polarizer. In one embodiment, the polarizer is configured to have a light cutoff in the range optimal for the fluorescence imaging process. In some embodiments, the polarizer is selected to lose effectiveness or cutoff at 650-800 nm, or between 600 nm and about 800 nm, or between about 700 nm and about 800 nm, about 800 nm and about 950 nm, about 800 nm and about 880 nm, between about 775 nm and about 795 nm, or about 785 nm. In another embodiment, the polarizer is selected to lose effectiveness or cut off by 700 nm, 725 nm, 750 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm, 805 nm, 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm, 895 nm, or 900 nm.

[0010] In various embodiments, the beam splitter further includes a beam splitter. In one embodiment, the beam splitter has a front surface and a rear surface. In some embodiments, the front surface has a front surface coating and the rear surface has a rear surface coating. In another embodiment, the front surface coating has a higher P-polarized reflectivity than the rear surface coating. In one embodiment, the front and rear coatings produce a P-polarized front-to-rear reflectivity ratio of at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 12:1, at least 14:1, at least 16:1, at least 18:1, or at least 20:1. In one embodiment, S-polarized reflections at the front and rear surfaces of the beam splitter are minimized by the front and rear coatings. In another embodiment, the polarizer is configured to block or attenuate ghost or secondary reflections reflected from the beam splitter. In one embodiment, the polarizer is configured to block S-polarized light from the beam splitter. In another embodiment, the polarizer is configured to allow P-polarized light to pass through the liquid crystal assembly to the imaging lens and / or camera when the liquid crystal is in the open state. In one embodiment, the first polarizer allows a front-to-back reflectance ratio from the beam splitter of at least 2:1, 4:1, 6:1, 8:1, 10:1, 12:1, 15:1, 17:1, or 20:1 to pass through to the imaging lens and / or camera. In some embodiments, the total reflectance from the beam splitter is about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, or about 3% or less.

[0011] In various embodiments, the liquid crystal assembly comprises two or more polarizers. In another embodiment, the liquid crystal assembly is compatible with a medical instrument, including a surgical microscope, a confocal microscope, a fluoroscope, an exoscope, an endoscope, or a surgical robot. In another embodiment, the liquid crystal assembly is compatible with the medical instrument, optionally using an optical gasket. In some embodiments, the medical instrument is a KINEVO system (e.g., KINEVO900), an OMPI PENTERO system (e.g., PENTERO900, PENTERO800), or a Leica FL800 system. In another embodiment, the liquid crystal assembly is used in a method for imaging emitted light emitted by a fluorophore.

[0012] The present disclosure also generally relates to a method for imaging emitted light emitted by a fluorophore in an imaging system including a liquid crystal assembly. The method includes enabling or directing visible light toward a sample, directing excitation light toward the sample, and directing emitted light and reflected visible light from the sample toward the liquid crystal assembly. In various aspects of the aforementioned method, the liquid crystal assembly includes an engineered polarizer disposed in the path of light from the sample, the polarizer configured to have an optical cutoff in the range of 600 nm to 900 nm. The focusing assembly also includes a liquid crystal disposed in the path of light transmitted through the polarizer and a drive voltage source configured to apply first and second voltages to the liquid crystal, the first and second voltages to toggle the liquid crystal between an open state and a closed state. The method further includes directing the emitted light and reflected visible light through the engineered polarizer, wherein reflected visible light having wavelengths below the optical cutoff passes through the polarizer with approximately 50% attenuation, and emitted light having wavelengths above the cutoff passes through the polarizer with minimal attenuation. The method further includes directing polarized visible light and unpolarized radiation at the liquid crystal, applying a first voltage to the liquid crystal to cause it to assume an open state during a first video rate sampling period, and detecting the polarized light with an imaging sensor. The method further includes applying a second voltage to the liquid crystal to cause it to assume a closed state to block polarized light from passing to the imaging sensor, and detecting the unpolarized radiation with the imaging sensor during the closed state during a second video rate sampling period.

[0013] In various embodiments of the disclosed method, the minimum attenuation of emitted light having wavelengths above the optical cutoff is between 0% and 15% attenuation. In one embodiment, reflected visible light having wavelengths below the optical cutoff passes through the polarizer with about 50% or less attenuation. In another embodiment, the first video-rate sampling period is between 1 ms and 10 ms, between 2 ms and 20 ms, or 6 ms. In some embodiments, the second video-rate sampling period is between 1 ms and 10 ms, between 2 ms and 20 ms, or 6 ms. In some embodiments, the first and second voltages achieve high and low electric fields across the liquid crystal, respectively.

[0014] In various aspects of the disclosed method, the first voltage and the second voltage toggle the liquid crystal between an open state and a closed state. In one aspect, the first voltage toggles the liquid crystal to the open state and the second voltage toggles the liquid crystal to the closed state, or vice versa. In another aspect, one of the first voltage or the second voltage is 0V. In one aspect, the second voltage is a plurality of voltages including a bipolar square wave having an average value of 0V. In another aspect, the first voltage, or both the first voltage and the second voltage, include a plurality of voltages that produce a bipolar square wave having an average value of 0V. In one embodiment, the bipolar square wave has an amplitude of + / -0.1 to + / -5 Vp-p, + / -3 to + / -30 Vp-p, + / -16 Vp-p, + / -24 Vp-p, or + / -30 Vp-p, or any value between + / -16 V (32 Vp-p) and + / -30 V (60 Vp-p), or any value between + / -30 V (60 Vp-p) and + / -100 V (200 Vp-p), inclusive. In another embodiment, the second voltage is a bipolar square wave having an amplitude of + / -0.1 to + / -5 Vp-p, + / -3 to + / -30 Vp-p, + / -16 Vp-p, + / -24 Vp-p, or + / -30 Vp-p, or an amplitude between + / -16 V (32 Vp-p) and + / -30 V (60 Vp-p), inclusive, or an amplitude between + / -30 V (60 Vp-p) and + / -100 V (200 Vp-p), inclusive. In some embodiments, the bipolar square wave amplitude is modulated at the video capture rate of the imaging system. In another aspect, the first voltage is a bipolar square wave having an amplitude of + / -0.1 to + / -5 Vp-p, + / -3 to + / -30 Vp-p, + / -16 Vp-p, + / -24 Vp-p, or + / -30 Vp-p, or an amplitude between + / -16 V (32 Vp-p) and + / -30 V (60 Vp-p) inclusive, or an amplitude between + / -30 V (60 Vp-p) and + / -100 V (200 Vp-p) inclusive.

[0015] In various embodiments of the disclosed method, the amplitude is modulated at 500 to 2000 Hz. According to various embodiments, the disclosed method further includes heating the liquid crystal using waste heat from the imaging system. In some embodiments, the polarizer is a dye-based polarizer. In other embodiments, the polarizer is configured to have an optical cutoff in a range optimal for the fluorescence imaging process. In various embodiments, the polarizer in the liquid crystal assembly is configured to have an optical cutoff in the range of about 600 nm to about 850 nm, about 700 nm to about 850 nm, about 800 nm to about 950 nm, about 800 nm to about 880 nm, about 775 nm to about 795 nm, or about 785 nm. In another embodiment, the polarizer in the liquid crystal assembly is selected to lose effectiveness or cut off by 700 nm, 725 nm, 750 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm, 805 nm, 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm, 895 nm, or 900 nm. In other embodiments, the polarizer of the liquid crystal assembly further comprises two or more polarizers.

[0016] According to various aspects of the disclosed method, the excitation light is engineered to enhance the signal-to-noise ratio (SNR) of the NIR image, producing controlled-energy excitation where the peak intensity and duration of the excitation light are below safety thresholds. According to various aspects, the disclosed method further includes directing the emitted light and reflected visible light to a beam splitter before directing the emitted light and reflected light to the liquid crystal assembly. In one aspect, the beam splitter has a front surface and a rear surface. In another aspect, the front surface has a front surface coating and the rear surface has a rear surface coating. In one aspect, the front surface coating has a higher P-polarized reflection than the rear surface coating. In another aspect, the front surface coating and the rear surface coating produce a P-polarized front-to-back reflectance ratio of at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 12:1, at least 14:1, at least 16:1, at least 18:1, or at least 20:1. In another embodiment, S-polarized light on the front and back surfaces of the beam splitter is minimized by front and back coatings. In a further embodiment, a polarizer is configured to block or attenuate ghost or secondary reflections reflected from the beam splitter. In one embodiment, a first polarizer is configured to block S-polarized light from the beam splitter. In another embodiment, the polarizer is configured to allow P-polarized light to pass through the liquid crystal assembly to the imaging lens or camera when the liquid crystal is in the open state. In one embodiment, the first polarizer allows a front to back reflectance ratio from the beam splitter of at least 2:1, 4:1, 6:1, 8:1, 10:1, 12:1, 15:1, 17:1, or 20:1 to pass through to the imaging lens and camera. In some embodiments, the total reflectance from the beam splitter is about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, or about 3% or less.

[0017] The present disclosure also relates to a liquid crystal assembly including any of the features described, in any configuration, either individually or in combination with any other feature, as disclosed herein.

[0018] The present disclosure also relates to a method for imaging emitted light emitted by a fluorophore in an imaging system including a liquid crystal assembly including any of the features described, in any configuration, either individually or in combination with any other feature, as disclosed herein.

[0019] The present disclosure also relates to a liquid crystal assembly for use with an imaging system having a light source for providing excitation light. The liquid crystal assembly includes a first polarizer disposed in a path of light from the light source, the first polarizer configured to have a light cutoff outside its polarization range, a liquid crystal disposed in a path of light transmitted through the first polarizer, and a second polarizer disposed in a path of light transmitted through the liquid crystal. The second polarizer is configured to have a light cutoff outside its polarization range. The liquid crystal assembly also includes a drive voltage source configured to apply a first voltage and a second voltage to the liquid crystal. The first voltage and the second voltage toggle the liquid crystal between an open state and a closed state.

[0020] In various embodiments, the first polarizer and the second polarizer are cross-polarized. In one embodiment, when one of the first voltage or the second voltage is applied to the liquid crystal, the liquid crystal is in an open state. In another embodiment, one of the first voltage or the second voltage is 0 V. In some embodiments, when one of the first voltage or the second voltage is applied to the liquid crystal, the liquid crystal is in a closed state. In another embodiment, the second voltage is a bipolar square wave. In one embodiment, the bipolar square wave has an amplitude of + / -0.1 to + / -5 Vp-p, + / -3 to + / -30 Vp-p, + / -16 Vp-p, + / -24 Vp-p, or + / -30 Vp-p.

[0021] In various embodiments, the liquid crystal assembly further includes an imaging lens in the path of light transmitted through the second polarizer and a camera in the path of light transmitted through the imaging lens. In another embodiment, when the liquid crystal is in the open state, the liquid crystal rotates the polarization of polarized visible light transmitted through the first polarizer, allowing the polarized visible light to pass through the second polarizer to the imaging lens with an attenuation of about 50% or less. In some embodiments, when the liquid crystal is in the closed state, the liquid crystal does not rotate the polarization of the polarized visible light, and the polarized visible light transmitted through the first polarizer is blocked by the second polarizer.

[0022] In various embodiments, the light cutoff range of the first and second polarizers is where the polarizer extinction ratio is insufficient or minimal. In other embodiments, light outside the light cutoff range of the first and second polarizers passes through the first and second polarizers with no or minimal attenuation to the imaging lens. In one embodiment, the minimum attenuation is 15% or less. In some embodiments, the light cutoff range of the first polarizer is from about 700 nm to about 800 nm, or about 700 nm, 725 nm, 750 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm, 805 nm, 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm, 895 nm, or 900 nm. In another embodiment, the light cutoff range of the second polarizer is about 700 nm to about 800 nm, or about 700 nm, 725 nm, 750 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm, 805 nm, 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm, 895 nm, or 900 nm. In another embodiment, the light cutoff ranges of the first polarizer and the second polarizer are greater than the longest wavelength of the visible light spectrum.

[0023] According to various embodiments, when the liquid crystal is in an open state, the liquid crystal assembly is configured to see visible light. In another embodiment, when the liquid crystal is in a closed state, the liquid crystal assembly is configured to block visible light. In one embodiment, the blocked visible light allows for fluorescence imaging from the fluorophore. In some embodiments, the fluorophore provides emitted light emitted by the fluorophore. In one embodiment, the fluorophore is provided with excitation light. In some embodiments, the excitation light is white light, NIR light, IR light, or any other type of excitation light. In one embodiment, the excitation light is near-infrared light provided by a laser diode.

[0024] In various embodiments, the liquid crystal assembly further includes a beamsplitter. In some embodiments, the beamsplitter has a front surface and a rear surface. In one embodiment, the front surface has a front surface coating and the rear surface has a rear surface coating. In some embodiments, the front surface coating has a higher P-polarized light reflectivity than the rear surface coating. In other embodiments, the front and rear coatings produce a P-polarized front-to-rear reflectivity ratio of at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 12:1, at least 14:1, at least 16:1, at least 18:1, or at least 20:1. In one embodiment, S-polarized light on the front and rear surfaces of the beamsplitter is minimized by the front and rear coatings. In another embodiment, the first polarizer is configured to block or attenuate ghost or secondary reflections reflected from the beamsplitter. In some embodiments, the first polarizer is configured to block S-polarized light from the beamsplitter. In another aspect, the first polarizer and the second polarizer are configured to allow P-polarized light to pass through the liquid crystal assembly to the imaging lens or camera when the liquid crystal is in the open state. In one aspect, the first polarizer allows a front-to-back reflectance ratio from the beam splitter of at least 2:1, 4:1, 6:1, 8:1, 10:1, 12:1, 15:1, 17:1, or 20:1 to pass through to the imaging lens and camera. In another aspect, the total reflectance from the beam splitter is about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, or about 3% or less.

[0025] In various embodiments, the imaging lens and camera are configured to view the fluorescence image using a video rate sampling period of 1 ms to 10 ms, 2 ms to 20 ms, or 6 ms. In one embodiment, the fluorescence image has reduced motion blur.

[0026] The present disclosure also relates to a method for imaging emitted light emitted by a fluorophore with an imaging system including a liquid crystal assembly. The method includes enabling or directing visible light toward a sample, directing excitation light toward the sample, and directing emitted light and reflected visible light toward a liquid crystal assembly. The liquid crystal assembly includes a first engineered polarizer in the path of light from the sample. The first engineered polarizer is configured to have a light cutoff in the range of 600 nm to 900 nm or 800 nm to 880 nm. The liquid crystal assembly also includes a liquid crystal disposed in the path of light transmitted through the first engineered polarizer. The liquid crystal assembly also includes a second engineered polarizer in the path of light transmitted through the liquid crystal. The second engineered polarizer is configured to have a light cutoff in the range of 600 nm to 900 nm or 800 nm to 880 nm. In one embodiment, the first engineered polarizer and the second engineered polarizer are cross-polarized. The liquid crystal assembly also includes a drive voltage source configured to apply a first voltage and a second voltage to the liquid crystal, the first voltage and the second voltage to toggle the liquid crystal between an open state and a closed state. In one aspect, the disclosed method also includes directing the emitted light and the reflected visible light through a first engineered polarizer, the first engineered polarizer polarizing the reflected visible light below a light cutoff range to generate polarized reflected visible light. The disclosed method further includes directing the emitted light and the polarized reflected visible light through the liquid crystal, applying a first voltage to the liquid crystal to cause it to assume an open state during a first video-rate sampling period, and rotating the polarization of the polarized reflected visible light using the liquid crystal in the open state. The disclosed method further includes directing the emitted light and the polarized reflected visible light through a second engineered polarizer, the second engineered polarizer allowing the polarized reflected visible light to pass through the second engineered polarizer. The disclosed method further includes detecting, with the imaging sensor, a first portion of the polarized reflected visible light that passes through the second engineered polarizer while in the open state.The disclosed method further includes applying a second voltage to the liquid crystal to cause it to assume a closed state during a second video rate sampling period, passing the emitted light and the polarized reflected visible light through the liquid crystal without rotating the emitted light and the polarized reflected visible light, blocking the polarized reflected visible light with a second engineered polarizer, and detecting a portion of the emitted light with an imaging sensor during the closed state.

[0027] In various aspects of the disclosed methods, the method further includes using a first polarizer to block secondary reflections of the reflected visible light. In one aspect, the excitation light is provided to the sample via a laser diode.

[0028] In various aspects of the disclosed method, the method further includes turning off the laser diode to stop providing the excitation light, providing a second voltage to the liquid crystal to cause it to assume a closed state during a third video rate sampling period, detecting a dark background image, and subtracting the dark background image of the sample from the detected portion of the emitted light to generate a fluorescence image.

[0029] In various aspects of the disclosed method, the first video rate sampling period is between about 2 ms and about 10 ms. In one aspect, the second video rate sampling period is between about 8 ms and about 16 ms. In another aspect, the third video rate sampling period is between about 8 ms and about 16 ms.

[0030] In various embodiments of the disclosed methods, one of the first voltage or the second voltage is about 0 V. In other embodiments, the second voltage is a bipolar square wave. In one embodiment, the bipolar square wave has an amplitude of + / -0.1 to + / -5 Vp-p, + / -3 to + / -30 Vp-p, + / -16 Vp-p, + / -24 Vp-p, or + / -30 Vp-p, or an amplitude between + / -16 V (32 Vp-p) and + / -30 V (60 Vp-p), inclusive, or an amplitude between + / -30 V (60 Vp-p) and + / -100 V (200 Vp-p), inclusive.

[0031] In various aspects of the disclosed method, the fluorescence image has reduced motion blur. In one aspect, the second video-rate sampling period and the third video-rate sampling period allow for post-processing with digital gain. In another aspect of the disclosed method, the disclosed method further includes orienting the first engineered polarizer to block undesired polarized light. In one aspect, the second video-rate sampling period and the third video-rate sampling period enhance the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of the fluorescence image. In one aspect, the enhanced CNR allows for fast frame rates. In another aspect, the disclosed method provides real-time display of reflected visible light and fluorescence images.

[0032] In various aspects of the disclosed method, the disclosed method further includes directing the emitted and reflected light to a beam splitter before directing the emitted and reflected light to the liquid crystal assembly. In one aspect, the beam splitter has a front surface and a rear surface. In another aspect, the front surface has a front surface coating and the rear surface has a rear surface coating. In some aspects, the front surface coating has a higher P-polarized reflectivity than the rear surface coating. In one aspect, the front and rear coatings produce a P-polarized front-to-rear reflectivity ratio of at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 12:1, at least 14:1, at least 16:1, at least 18:1, or at least 20:1. In another aspect, S-polarized light on the front and rear surfaces of the beam splitter is minimized by the front and rear coatings. In another aspect, the first polarizer is configured to block or attenuate ghost or secondary reflections reflected from the beam splitter. In one embodiment, the first polarizer is configured to block S-polarized light from the beam splitter. In another embodiment, the first polarizer and the second polarizer are configured to allow P-polarized light to pass through the liquid crystal assembly to the imaging lens and / or camera when the liquid crystal is in the open state. In some embodiments, the first polarizer allows a front-to-back reflectance ratio from the beam splitter of at least 2:1, 4:1, 6:1, 8:1, 10:1, 12:1, 15:1, 17:1, or 20:1 to pass through to the imaging lens and camera. In various embodiments, the total reflectance from the beam splitter is about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, or about 3% or less.

[0033] The present disclosure also relates to a method of imaging abnormal tissue, cancer, tumor, vasculature, or structure in a sample from a subject, the method comprising generating an image of the vasculature or structure by imaging fluorescence using a liquid crystal assembly disclosed herein.

[0034] The present disclosure also relates to a method of imaging abnormal tissue, cancer, tumor, vasculature, or structure in a sample from a subject according to any of the methods disclosed herein, the method comprising generating an image of the abnormal tissue, cancer, tumor, vasculature, or structure by imaging fluorescence using an imaging system including a liquid crystal assembly.

[0035] In various aspects, the imaged fluorescence is autofluorescence, a contrast or imaging agent, a chemical agent, a radiolabeled agent, a radiosensitizer, a photosensitizer, a fluorophore, a therapeutic agent, an imaging agent, a diagnostic agent, a protein, a peptide, a nanoparticle, or a small molecule, or any combination thereof, or any combination thereof. In one aspect, the imaged fluorescence is autofluorescence, a contrast or imaging agent, a chemical agent, a radiolabeled agent, a radiosensitizer, a photosensitizer, a fluorophore, a therapeutic agent, an imaging agent, a diagnostic agent, a protein, a peptide, a nanoparticle, or a small molecule, or any combination thereof. In one aspect of the disclosed method, the method further comprises administering an imaging or imaging agent to the subject.

[0036] The present disclosure also relates to a method of imaging abnormal tissue, cancer, tumor, vasculature, or structure within a fluorophore from a subject using any of the disclosed liquid crystal assemblies, which method includes administering a contrast or imaging agent to the subject and imaging the contrast or imaging agent using an imaging system to generate an image of the abnormal tissue, cancer, tumor, vasculature, or structure.

[0037] The present disclosure also relates to a method of imaging abnormal tissue, cancer, tumor, vasculature, or structure within a fluorophore from a subject according to any of the disclosed methods. The disclosed method further includes administering a contrast or imaging agent to the subject and imaging the contrast or imaging agent using an imaging system to generate an image of the abnormal tissue, cancer, tumor, vasculature, or structure. In one aspect, the contrast or imaging agent includes a dye, a fluorophore, a fluorescent biotin compound, a luminescent compound, a chemiluminescent compound, or any combination thereof.

[0038] In various embodiments of the disclosed methods, the imaging or contrast agent further comprises a protein, a peptide, an amino acid, a nucleotide, a polynucleotide, or any combination thereof. In one embodiment, the imaging or contrast agent further comprises tozurelitide. In another embodiment, the imaging or contrast agent absorbs at wavelengths between about 200 nm and about 900 nm. In other aspects, the contrast or imaging agent is selected from the group consisting of DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, or indocyanine green (ICG), and derivatives of any of the foregoing; fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanate or FITC, naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanines, merocyanines, styryl dyes, oxonol dyes, phylloerythrin, litrosin, eosin, rhodamine dyes (e.g., calcitonin, These dyes include carboxytetramethylrhodamine (TAMRA), carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine (TMR), etc.; coumarins, coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA)), etc.; Oregon Green dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514), Texas Red, Texas Red-X, Spectrum Red, Spectrum Green, and cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY-5).5, etc.), ALEXA FLUOR dyes (e.g., ALEXA FLUOR350, ALEXA FLUOR488, ALEXA FLUOR532, ALEXA FLUOR546, ALEXA FLUOR568, ALEXA FLUOR594, ALEXA FLUOR633, ALEXA FLUOR660, ALEXA FLUOR680, etc.), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY530 / 550, BODIPY558 / 568, BODIPY564 / 570, BODIPY576 / 589, BODIPY581 / 591, BODIPY630 / 650, BODIPY650 / 665, etc.), IRDye (e.g., IRD40, IRD700, IRD800, etc.), 7-aminocoumarin, dialkylaminocoumarin reactive dyes, 6,8-difluoro-7-hydroxycoumarin fluorophores, hydroxycoumarin derivatives, Alkoxycoumarin derivatives, succinimidyl esters, pyrene succinimidyl esters, pyridyloxazole derivatives, aminonaphthalene dyes, dansyl chloride, dapoxyl dyes, dapoxyl sulfonyl chloride, amine-reactive dapoxyl succinimidyl esters, carboxylic acid-reactive dapoxyl (2-aminoethyl) sulfonamides, bimane dyes, bimane mercaptoacetic acid, NBD dyes, QsY35, or any combination thereof.

[0039] In various aspects of the disclosed methods, administering includes intravenous administration, intramuscular administration, subcutaneous administration, intraocular administration, intraarterial administration, intraperitoneal administration, intratumoral administration, intradermal administration, or any combination thereof. In one aspect, imaging includes tissue imaging, ex vivo imaging, intraoperative imaging, or any combination thereof. In one aspect, the sample is an in vivo sample, an in situ sample, an ex vivo sample, or an intraoperative sample. In another aspect, the sample is an organ, organ substructure, tissue, or cell. In various aspects, the sample is autofluorescent. In one aspect, the autofluorescence of the sample includes an ocular fluorophore, tryptophan, or protein present in a tumor or malignancy. In one aspect, the method is used to visualize vascular flow or vascular patency.

[0040] In various embodiments of the disclosed methods, the abnormal tissue, cancer, tumor, vasculature, or structure comprises a blood vessel, lymphatic vasculature, neurovasculature, or CNS structure. In one embodiment, the imaging is angiography, arteriography, lymphography, or cholangiography. In another embodiment, the imaging comprises detecting a vascular abnormality, vascular malformation, vascular lesion, organ or organ substructure, cancer, or diseased region, tissue, structure, or cell. In another embodiment, the vascular abnormality, vascular malformation, or vascular lesion is an aneurysm, arteriovenous malformation, cavernous malformation, venous malformation, lymphatic malformation, telangiectasia, mixed vascular malformation, spinal dural arteriovenous fistula, or a combination thereof. In one embodiment, the organ or organ substructure is the brain, heart, lung, kidney, liver, or pancreas.

[0041] In various aspects of the disclosed methods, the disclosed methods further include performing surgery on the subject. In one aspect, the surgery includes angioplasty, cardiovascular surgery, aneurysm repair surgery, valve replacement surgery, aneurysm surgery, arteriovenous or cavernous malformation surgery, venous malformation surgery, lymphatic malformation surgery, telangiectasia surgery, mixed vascular malformation surgery, or spinal dural arteriovenous fistula surgery, fistula surgery, repair or bypass, arterial bypass, organ transplant, plastic surgery, eye surgery, reproductive system surgery, stent insertion or replacement, plaque ablation, removal of cancerous or diseased areas, tissues, structures, or cells in a subject, or any combination thereof.

[0042] In various embodiments of the disclosed methods, imaging includes imaging vascular abnormalities, cancerous or diseased areas, tissues, structures, or cells in a subject after surgery. In various embodiments, the disclosed methods further include treating cancer in a subject. In one embodiment, the disclosed methods further include repair of intracranial CNS vascular defects, spinal CNS vascular defects, peripheral vascular defects, removal of abnormally vascularized tissue, ocular imaging and repair, anastomosis, reconstructive or plastic surgery, plaque ablation or treatment or restenosis in atherosclerosis, repair or resection (including selective resection), preservation (including selective preservation), intraoperative identification and management (optionally preserving, optionally selective resection) of a vital organ or structure such as a nerve, kidney, thyroid, parathyroid, liver segment, or ureter, diagnosis and treatment of limb ischemia, or treatment of a chronic wound. [Brief explanation of the drawings]

[0043] [Figure 1] FIG. 1 is a diagram of a liquid crystal shutter assembly shown with a camera, according to one embodiment. [Figure 2] 1 illustrates the spectral response of an exemplary camera, according to one aspect. [Figure 3] 1 illustrates the spectral response of an exemplary camera to light attenuated by a long-pass filter, according to one embodiment. [Figure 4] 1 illustrates the change in extinction ratio of light for a liquid crystal cell in an open and closed state, according to one embodiment. [Figure 5] 1 shows the closed-state transmission of a liquid crystal cell in combination with various polarizers, according to one embodiment. [Figure 6] 1 illustrates the transition speed of a liquid crystal cell in response to different applied voltages, according to one embodiment. [Figure 7] 1 illustrates the transition speed of a liquid crystal cell at different temperatures, according to one embodiment. [Figure 8] 1 illustrates light blocking in a liquid crystal cell in response to an applied voltage, according to one embodiment. [Figure 9]1 illustrates the average power delivered by pulsed or controlled excitation energy delivered to a tissue sample compared to the average power of exemplary clinical levels of energy delivered to a tissue sample, according to one embodiment. [Figure 10] 1 illustrates exemplary signal-to-noise ratios obtained using a pulsed excitation light source or a controlled excitation light source for an exemplary clinical laser excitation light source, according to one embodiment. [Figure 11] 1 illustrates a liquid crystal shutter assembly, according to one embodiment. [Figure 12] 1 shows the transmittance of a liquid crystal medium in the open and closed states according to one embodiment. [Figure 13] 1 shows a diagram of a liquid crystal shutter assembly shown with a camera, according to one embodiment. [Figure 14A] 1 shows a visible light image captured with the liquid crystal shutter assembly in an open state, according to one embodiment. [Figure 14B] 1 shows a visible light image captured with a liquid crystal shutter assembly in a closed state, according to one embodiment. [Figure 15] 1 illustrates contrast to noise ratio using drive voltages supplied to a liquid crystal assembly, according to one embodiment. [Figure 16] 1 illustrates the exposure rate required for post-processing via digital gain using a drive voltage supplied to a liquid crystal assembly with a contrast-to-noise ratio of 5, according to one embodiment. [Figure 17] FIG. 1 shows a diagram for reducing integration time of a fluorescent image using a liquid crystal shutter assembly, according to one embodiment. [Figure 18A] 1 shows an image of a fluorescent cube moving on a linear stage acquired using bright background subtraction, according to one embodiment. [Figure 18B] 1 shows an image of a phosphor cube moving on a linear stage acquired using a liquid crystal shutter assembly, according to one embodiment. [Figure 19] 1 shows the use of a liquid crystal shutter assembly configured to visualize a peptide-fluorophore conjugate (tozrelitide). [Figure 20A]An example of a front-to-back reflectance ratio (FBR) of approximately 10:1 for the visible spectrum is shown. The FBR of a beamsplitter designed to be filtered by an LC shutter over a range spanning the visible spectrum (i.e., wavelengths from 400 nm to 700 nm) is shown for either S-polarized light (FBR S) or P-polarized light (FBR P), respectively. Note the minimum FBR of 10:1 in this example. [Figure 20B] This figure shows the effect of a polarizer on the total reflectance of a beam splitter designed to be filtered by an LC shutter. When the FBR of a beam splitter designed to be filtered by an LC shutter is targeted at a different FBR (e.g., an FBR of approximately 20:1), the FBR curve still varies with wavelength but has an average value of 20 across the visible spectrum. This figure shows the effect of a polarizer on the total reflectance ("total reflectance") of a beam splitter designed to be filtered by an LC shutter across the visible spectrum (i.e., wavelengths from 400 nm to 700 nm), compared to the minimum reflectance (11%) without a polarizing filter. DETAILED DESCRIPTION OF THE INVENTION

[0044] Multispectral imaging is an important technology for capturing the spectral characteristics of light in a single image. The most common example of multispectral imaging is color vision. The human eye contains three different chemicals in its rods and cones, which allow it to capture different colors of light. These three colors, red, green, and blue, are then displayed as a monochromatic image in the human brain. Digital color cameras emulate the biological properties of the eye by using different color filters on each pixel of the sensor. Assigning different pixels to different colors effectively trades spatial resolution for spectral information. The benefits of using color bands to add information to an image can be applied to imaging technology. Furthermore, the capabilities of multispectral imaging can be extended to wavelengths of light that are not directly visible to the eye. As the range of spectral information expands, an increasing amount of information can be condensed into a visible image. An interesting application of multispectral imaging is blending visible (VIS) and near-infrared (NIR) images. This combination is enhanced by CCD (charge-coupled device) and CMOS (complementary metal-oxide semiconductor) silicon cameras, which have good sensitivity in both the visible and NIR ranges. Mixing of these two spectral bands can be achieved with multiple cameras or, for example, using a single camera with dedicated NIR pixel filters on the sensor. Furthermore, in some embodiments, more than one sensor is present in the system to detect these various spectral bands. Alternatively, time multiplexing can be used to acquire full-resolution images of each spectral band with a single image sensor, but this is time-consuming because each image must be acquired sequentially. Filter wheels or motorized mirrors are traditionally used to switch filters, but are typically too slow for the optimal video rate switching speeds required for real-time imaging. Time multiplexing can also be applied to multiple imaging sensors in a system (e.g., a multi-camera or multi-sensor system), but the same limitations on time cost and video rate apply.

[0045] Multiplexed spectral bands are also used in fluorescence imaging. Fluorescence is an energy transfer mechanism in which light of one color (wavelength) is absorbed and then emitted as a different color (wavelength). The emitted photons are typically lower in energy than the absorbed photons and therefore have a longer wavelength (Stokes shift) than the absorbed photons. Fluorescence is not perceptible to the human eye because the excitation wavelength is in the UV, but it is often observed by the human eye when emitting visible light. Fluorescence can also be observed by instruments and sensors whose excitation wavelength is in any spectrum not perceptible by the instrument or sensor, but which emits wavelengths in the range of the instrument or sensor designed to receive a detectable fluorescence emission signal.

[0046] Fluorophores are molecules that absorb and then emit light; therefore, a particular fluorophore possesses both excitation and emission spectral bands. For example, one family of fluorophores, including the cyanine dyes and indocyanine green dye family (ICG), absorbs and emits light in the near-infrared spectrum. These NIR fluorophores are particularly important for imaging biological samples because their absorption and emission spectra are both near 830 nm, which is considered the primary "biological window" for penetration within tissue. ICG is also useful in medical and diagnostic applications. Combining fluorescence imaging with visible spectrum imaging can be an insightful tool for such biological applications. While infrared and near-infrared are particularly useful wavelength ranges for medical imaging, other fluorescent dyes and moieties can be used in the detection systems using the liquid crystal shutter assemblies described herein.

[0047] Additionally, fluorescence imaging can be captured in real or near real time using video image processing to detect changes over time. While video-rate switching between visible and NIR fluorescence often requires multiple sensors or cameras, in some instances, a single camera system may be employed. Furthermore, when used during NIR fluorescence, fluorophore excitation can be turned on and off to control known temporal states, enabling background subtraction. Liquid crystal ("LC") shutters are assemblies consisting of two linear polarizing filters with a liquid crystal medium interposed between them. The polarizing filter elements have the ability to block light with a polarization different from the orientation of the filters. While the liquid crystal material does not block light, it has the ability to rotate the polarization of light passing through the material. The orientation of the crystals in the LC material changes the degree to which the polarization of light is rotated, thus changing whether the polarization of the passing light is rotated or not. The combination of two filters and an LC medium, along with an applied electric field, can be assembled to create a shutter that blocks or transmits light depending on the applied electric field. When the polarization direction of the light passing through the LC material is aligned with the exit polarizer, the light passes with minimal attenuation. This is considered the open state of the shutter. When the electric field is adjusted so that the polarization direction of the light changes, so that the light is rotated 90° from the exit polarizer, the light is blocked. This is considered the closed state of the LC shutter assembly.

[0048] Due to the spectral bandwidth limitations of the polarizing elements incorporated into the liquid crystal shutter assembly, light outside the spectral band of the high extinction ratio range of the polarizing elements incorporated into the liquid crystal shutter assembly (e.g., outside the spectral bandwidth limit, also called out-of-band light) is conventionally blocked or diverted by other spectral elements (filters) before entering the first polarizing element. The use of these additional spectral filters ensures the extinction ratio of the liquid crystal shutter.

[0049] In the disclosed methods and systems, rather than diverting out-of-band incident light away from the liquid crystal assembly, as would normally occur, the out-of-band light can be intentionally directed into the polarizing element and thus into the liquid crystal shutter assembly. The out-of-band light passes through the shutter because it is unaffected by the orientation of the polarizer or the liquid crystal medium. Designing optical systems in this manner traditionally results in insufficient or minimal extinction ratios, thereby impairing the functionality of the liquid crystal shutter assembly. In the disclosed methods and systems, the spectral characteristics of the polarizing element are engineered so that light leaking through the shutter assembly is aligned with the NIR emission of the fluorophore, and therefore selected so that the leaked light is intentional and beneficial. This configuration contradicts conventional good practice for liquid crystal shutter applications. Liquid crystal shutters are used to attenuate light in a controlled manner by adjusting the level of an applied electric field. Liquid crystal shutters used in this manner have a limited effective spectral range, most commonly limited by the polarization technology of the polarizing filter. According to various aspects, the disclosed liquid crystal shutter assemblies utilize various wavelength ranges that are not polarized by the polarizing filter. Alternatively, a polarizing beam splitter may be used in combination with or in place of the polarizing filter.

[0050] Wavelengths of light not polarized by a linear polarizing filter are typically considered to be outside the operating range of a liquid crystal shutter assembly. Rather than operating within the typical operating range of a liquid crystal shutter assembly, the liquid crystal shutter assemblies of the present disclosure utilize various wavelength ranges that are outside the effective range of linear polarizers, and therefore utilize spectral ranges previously considered harmful in typical liquid crystal applications.

[0051] The terms "liquid crystal," "LC," "LC cell," "LC medium," "LC material," "liquid crystal cell," "liquid crystal material," or "liquid crystal medium," and the like, may be used interchangeably herein to refer to non-limiting liquid crystal materials, media, or cells, including, for example, thermotropic and lyotropic liquid crystals, Pi cells, twisted nematic ("TN") cells, liquid crystal displays (LCDs), liquid crystals with photoconductive properties, or materials, media, or cells containing liquid crystals that undergo development and adjustment under an electric field. It is understood that liquid crystals can occur in states between a crystalline (solid) state and an isotropic (liquid) state. There are many types of liquid crystal states, depending on the amount of order in the liquid crystal material.

[0052] The terms "liquid crystal shutter," "liquid crystal assembly," "liquid crystal shutter assembly," "LC shutter," "LC assembly," or "LC shutter assembly" may be used interchangeably herein and refer to liquid crystals used in conjunction with, with, including, or carrying one or more polarizers. Exemplary LC shutters are described herein.

[0053] The terms "polarizing," "polarizer," or "polarizing," particularly when used to describe a component or element, are understood to describe various components or elements that can be functionally interchangeable and used in a manner to transmit light of a particular polarization orientation. For example, the LC assemblies described herein can use various polarizing components, including, but not limited to, one or more polarizing beam splitters, polarizing filters, alone or in combination with other polarizing components or elements.

[0054] The terms "excitation energy" and "excitation light" are used interchangeably in this disclosure to mean providing excitation light to a fluorophore or causing emission light from a fluorophore. Excitation energy or excitation light can also be used to irradiate a fluorophore or other substance or molecule, causing emission light.

[0055] The terms "video rate," "video-rate imaging," "video frame rate," "frame rate," or "real-time" and "in real time" in the context of images, video, frame rate, and video rate, particularly when used to describe the speed, visualization, and clarity of an image or imaging system, refer to the number of unique or distinct frames or images used in time. Video rate is often measured in frames per second ("fps"), which defines the number of frames or images contained in a video in any given second. Video rate may also be measured in bitrate standards. Real-time frame rates can be approximately 25 fps, or 30-50 fps. Real-time video frame rates can also range from 16-120 fps (inclusive). Video rate is a measure that affects a system's responsiveness to motion. For example, the higher the video rate, the clearer the images in a video appear. A higher frame rate means that more images are displayed per second, resulting in smoother and sharper video quality and potentially sharper edges in the images.

[0056] The terms “video rate sampling period” and “exposure time” may be used interchangeably in this disclosure. A video rate sampling period or exposure time may be the period during which an imaging sensor or camera images a sample. A video rate sampling period may also be described with respect to the LC shutters and LC assemblies described herein, for example, when describing the rate at which the LC shutters toggle between open and closed states (i.e., the period between the open and closed states), or when two voltage sources or drive voltage sources in a system are toggled. The voltage supplied to the system may toggle the LC shutters between open and closed states at a video rate sampling period ranging from 1 ms to 10 ms, or from 2 ms to 20 ms, or between open and closed states at approximately 1 ms, 2 ms, 3 ms, 4 ms, 5 ms, 6 ms, 7 ms, 8 ms, 9 ms, 10 ms, 11 ms, 12 ms, 13 ms, 14 ms, 15 ms, 16 ms, 17 ms, 18 ms, 19 ms, or 20 ms. By shortening the time between toggling between open and closed states in the LC shutters and LC assemblies described herein, the video rate sampling period can be shortened, effectively increasing the video rate, resulting in more images displayed per second and therefore smoother, sharper video quality. A higher video rate results in a shorter video rate sampling period, which improves video quality, reduces motion blur, sharper images, better defined image boundaries, and fewer artifacts.

[0057] The liquid crystal shutter assemblies and methods disclosed herein may be used in a variety of imaging systems and methods. By way of example and not limitation, the liquid crystal shutter assemblies of the present disclosure may be used in conjunction with any of the patent applications described in PCT / US2014035203, entitled "Systems and methods for recording simultaneously visible light image and infrared light image from fluorophores," published on October 30, 2014 as WO2014 / 176375; PCT / US2019 / 024689, entitled "Systems and methods for simultaneous near-infrared light and visible light imaging," published on October 3, 2019 as WO2019 / 191497; PCT / US2020 / 053746, entitled "Systems and methods for vascular and structural imaging," published on April 8, 2021 as WO2021 / 067563; and PCT / US2021 / 263159, entitled "Systems and methods for simultaneous near-infrared light and visible light imaging." The liquid crystal shutter assemblies of the present disclosure may be incorporated into any one or more systems and methods for fluorescence imaging known in the art, including, but not limited to, PCT / US2021 / 039177 entitled "Fluorescence Imaging" (collectively referred to as the "Blaze Bioscience System"), which are incorporated herein by reference in their entireties. It should also be understood that the liquid crystal shutter assemblies of the present disclosure may be incorporated into any one or more systems and methods for fluorescence imaging known in the art.

[0058] In one aspect of the present disclosure, it is desirable to use a liquid crystal shutter assembly 100 in combination with at least one sensor or camera 102 that has sufficient sensitivity in the visible and NIR spectral ranges. As shown in Figure 1, the LC shutter assembly 100 further includes a polarizer 104 and a liquid crystal medium 106 within the LC shutter assembly 100.

[0059] According to one embodiment, two polarizers 104 are integrated into the LC shutter 100, as shown in FIG. 1 . In other embodiments, one or more polarizers 104 may be separate components positioned in the same optical path and aligned on either side of the LC medium cell 106. In yet other embodiments, multiple LC medium cells 106, each associated with one or more polarizers 104, may be used to form the LC assembly 100. This stacking of polarizers and liquid crystal media may be used to increase the extinction ratio compared to a single-cell LC shutter assembly. Alternatively, one or more LC assemblies 100 including one or more LC cells 106 and one or more polarizers 104 may be used in conjunction with any combination of additional LC cells 106 and / or polarizers 104.

[0060] According to one embodiment, the two polarizers 104 may each include a substrate layer 1102(a) and 1102(b), as shown in FIG. 11 . The first substrate layer 1102(a) of the first polarizer and the second substrate layer 1102(b) of the second polarizer may be glass substrate layers. The glass substrate layers may be configured to allow light to pass through. As shown in FIG. 11 , the first polarizing film 1101(a) may be attached to the first substrate layer 1102(a), and the second polarizing film 1101(b) may be attached to the second substrate layer 1102(b). The first polarizing film 1101(a) and the second polarizing film 1101(b) may be cross-polarized (e.g., oppositely oriented).

[0061] In one embodiment, the first substrate layer 1101(a) may have a conductive layer 1103(a) located on the side of the substrate layer 1102(a) opposite the first polarizing film 1101(a) and adjacent to the LC medium 106. The second substrate layer 1102(b) may have a conductive layer 1103(b) located on the side of the substrate layer 1102(b) opposite the second polarizing film 1101(b) and adjacent to the LC medium 106.

[0062] In one embodiment, the first conductive layer 1103(a) and the second conductive layer 1103(b) can both comprise indium tin oxide (“ITO”). As used herein, a transparent conductive film or layer is understood to include any thin film of an optically transparent, conductive material. In other examples, the first conductive layer 1103(a) can include any transparent conductive film or layer, including those comprising indium tin oxide (“ITO”), low-resistivity or high-resistivity transparent conductive films, transparent conductive oxides (“TCO”), indium zinc oxide (“IZO”), other tin oxide (SnO)-based films including indium-free undoped tin dioxide, Cu- and Ga-doped zinc oxide (ZnO) films (“GZO”), aluminum zinc oxide (“AZO”), and cadmium stannate (“CTO”). The second conductive layer 1103(b) may comprise any transparent conductive film or layer, including indium tin oxide ("ITO"), low-resistivity or high-resistivity transparent conductive films, transparent conductive oxides ("TCO"), indium zinc oxide ("IZO"), other tin oxide (SnO)-based films including indium-free undoped tin dioxide, Cu- and Ga-doped zinc oxide (ZnO) films ("GZO"), aluminum zinc oxide ("AZO"), and cadmium stannate ("CTO"). In some examples, the first conductive layer 1103(a) and the second conductive layer 1103(b) may comprise the same material. In other examples, the first conductive layer 1103(a) and the second conductive layer 1103(b) may comprise different materials. The first conductive layer 1103(a) may be soldered to the first substrate layer 1102(a). The second conductive layer 1103(b) may be soldered to the second substrate layer 1102(b).

[0063] In another aspect, the first conductive layer 1103(a) and the second conductive layer 1103(b) can be kept thin to maintain high transmission of NIR light through the LC shutter 100. In some embodiments, the conductive layer (whether the first or second layer) can be 10-50 nm thick (inclusive), less than 25 nm thick, less than 50 nm thick, 50 nm-250 nm thick, 75 nm-165 nm thick, or greater than 250 nm thick, depending on the material used for the transparent conductive film or layer. In some embodiments, the transparent conductive film or layer is 10 to 30 nm thick, 30 to 50 nm thick, 50 to 70 nm thick, 70 to 90 nm thick, 90 to 110 nm thick, 110 to 130 nm thick, 130 to 150 nm thick, 150 to 170 nm thick, 170 to 190 nm thick, 190 to 210 nm thick, 210 to 230 nm thick, or 230 to 250 nm thick. In some aspects, the transparent conductive film or layer is 20 nm (+ / - 5 nm) thick, 30 nm (+ / - 5 nm) thick, 40 nm (+ / - 5 nm) thick, 50 nm (+ / - 5 nm) thick, 60 nm (+ / - 5 nm) thick, 70 nm (+ / - 5 nm) thick, 80 nm (+ / - 5 nm) thick, 90 nm (+ / - 5 nm) thick, 100 nm (+ / - 5 nm) thick, 110 nm (+ / - 5 nm) thick, 120 nm (+ / - 5 nm) thick, 130 nm (+ / - 5 nm) thick, 140 nm (+ / - 5 nm) thick, 150 nm (+ / - 5 nm) thick, 160 nm (+ / - 5 nm) thick, or 170 nm (+ / - 5 nm) thick. In some embodiments, the conductive film or layer may or may not be heated.

[0064] In another embodiment, the LC medium 106 may be disposed between a first conductive layer 1103(a) and a second conductive layer 1103(b), as shown in FIG. 11 . The LC medium 106 may be sealed within the LC shutter 100 by the first conductive layer 1103(a), the second conductive layer 1103(b), the first spacer 1104(a), and the second spacer 1104(b). The LC shutter 100 may include a first electrical connector 1106(a) and a second electrical connector 1106(b). The first electrical connector 1106(a) may be connected to the first conductive layer 1103(a). The second electrical connector 1106(b) may be connected to the second conductive layer 1103(b). The first electrical connector 1106(a) may be configured to supply a voltage from a driving voltage source to the first conductive layer 1103(a). The second electrical connector 1106(b) may be configured to supply a voltage from a driving voltage source to the second conductive layer 1103(b). When a voltage is supplied to the first conductive layer 1103(a) and the second conductive layer 1103(b), an electric field may be applied to the LC medium 106. The voltage supplied by the driving voltage source to the first conductive layer 1103(a) and the second conductive layer 1103(b) may be a bipolar square wave.

[0065] When no electric field (e.g., no voltage) is applied to the LC medium 106, the LC medium 106 may rotate the polarization of light passing through it. When the first polarizing film 1101(a) and the second polarizing film 1101(b) are cross-polarized (e.g., of opposite orientations), this state of the LC shutter 100 is referred to as the open state. By rotating the polarization of the light, the light can pass through the second polarizing film 1101(b) because it has the same polarization as the second polarizing film 1101(b) (e.g., light is polarized by the first polarizing film 1101(a) and then rotated by the LC medium 106 to reach the same polarization at the second polarizing film 1101(b)). In this example, the two polarizing films may appear aligned with each other. The LC shutter 100 may appear transparent (e.g., no attenuation of light) when no electric field is applied to the LC medium 106. When an electric field is applied to the LC medium 106, the LC medium 106 does not rotate light passing through it. If the LC medium 106 did not rotate light, the light would have the polarization of the first polarizing film 1101(a) when it reached the second polarizing film 1101(b) and would therefore be blocked by the cross-oriented second polarizing film 1101(b). This state of the LC shutter 100 is the closed state. An applied voltage can toggle the LC shutter between the open and closed states.

[0066] In another embodiment, the first polarizing film 1101(a) and the second polarizing film 1101(b) may have the same polarization (e.g., the same orientation). In this example, when no electric field (e.g., no voltage) is applied, the LC medium 106 may rotate the polarization of light passing through it. In this example, by rotating the polarization of the light, the rotated polarization of the light has the opposite polarization to that of the second polarizing film 1101(b), so the light may be blocked by the second polarizing film 1101(b). In this example, when an electric field is applied to the LC medium 106, the LC medium 106 does not rotate the light. In this example, when the LC medium 106 does not rotate the light, the light has the polarization of the first polarizing film 1101(a) and therefore the polarization of the second polarizing film 1101(b), allowing the light to pass through the polarizing film 1101(b). In this example, by changing the orientation of the polarizing films 1101(a), 1101(b), the effect of applying a voltage can be reversed (e.g., no voltage provides a closed state, and applying a voltage provides an open state).

[0067] In various embodiments, the camera 102 incorporates one or more color filters that are highly transmissive in the NIR spectrum. As shown in FIG. 2 , which illustrates an exemplary camera spectral response 200, the camera 102 may include blue, green, and red filters, generally designated 202, 204, and 206, at the sensor pixels to separate incident light of the aforementioned wavelengths. According to various embodiments, the camera 102 is a multispectral camera. In other embodiments, the camera is configured for hyperspectral imaging. In various embodiments, the polarizer 104 and LC cell 106 can block or pass light of shorter wavelengths in the visible spectrum based on an applied electric field, thereby effectively functioning as a long-pass filter to control the light received by the camera 102. In various embodiments, unattenuated visible and emitted light may be directed to the LC shutter 100.

[0068] As shown in Figure 3, multispectral imaging systems typically include a NIR long-pass filter used to separate visible and NIR light. The long-pass filter allows only NIR light from the light source to pass through to an imaging sensor, such as camera 102. In various aspects of multispectral imaging, the use of a NIR long-pass filter is desirable in that all three depicted color pixels 202-206 operate like a monochrome camera, transmitting the full resolution of the sensor. To time-multiplex the visible and NIR images, the long-pass filter is typically moved in and out of the image path. In practice, moving a physical filter in and out of the image path can be achieved using a rapidly rotating filter wheel or a rotary solenoid, but these methods are difficult to perform accurately at video speeds and are prone to synchronization errors, audible and sub-audible noise, thermal noise, and vibration (i.e., any of the aforementioned commonly referred to as noise, or the specific noise required by the context), as well as mechanical failure. As a result, the liquid crystal shutter assembly of the present disclosure can function as a long-pass filter used to address these limitations and reduce or eliminate the aforementioned challenges, including synchronization errors, noise, and mechanical failure, for image acquisition at video rates. According to various aspects of the present disclosure, the reduced extinction ratio at long wavelengths effectively transforms the liquid crystal assembly into a long-pass filter with variable attenuation of short-wavelength light within the effective range of the polarizer. Linear polarizing filters often suffer from undesirable drawbacks. The first drawback is that at least 50% of unpolarized light is lost to absorption or reflection, leaving only polarized light. For liquid crystal assemblies using such polarizers, this light is lost from the first polarizer. Furthermore, in low-light applications, such as those found during NIR fluorescence imaging, signal attenuation of approximately 50% or more can degrade the signal-to-noise ratio (SNR) to such an extent that increased exposure times are required to capture useful data. Consequently, the need for longer exposure times is often detrimental to capturing data at video rates.

[0069] A second drawback to using these types of polarizers is that they are not effective for all wavelengths of incident light. As can be easily understood, a polarizer is effectively transparent outside of its polarization range. In other words, a polarizer may not be effective at polarizing light outside of its polarization wavelength range. Therefore, at certain wavelengths, the polarizer cannot polarize the incident light.

[0070] Contrary to prevailing beliefs, according to various aspects, the systems and methods of the present disclosure take advantage of these previously undesirable characteristics or shortcomings. The LC shutter assembly 100 can operate in three states. These include "State 1," also referred to as the "open state," in which unpolarized light within the functional range of the linear polarizers is partially attenuated by the first linear polarizer but can pass through the second polarizer, generally based on the polarization rotation of the liquid crystal. The open state can be used to image visible light (e.g., reflected visible light) from a sample. "State 2," also referred to as the "closed state," is a state in which unpolarized light is partially attenuated by the first linear polarizer and then nearly completely blocked or attenuated by the second linear polarizer due to the alternating rotation of the liquid crystal phase states. The ability to attenuate light in this closed state depends on the effectiveness of the linear polarizer to fully polarize light. In various aspects, the properties of the polarizing filters enable the LC shutter to achieve an extinction ratio of two to four orders of magnitude within the spectral range of the polarizing filters when comparing the open and closed states. In an exemplary embodiment of visible and NIR spectral imaging, the polarizing filters in the systems and methods of the present disclosure are selected to be effective in the visible spectrum, so that the open and closed states only affect visible light. Alternatively, a polarizing beam splitter can be used in place of the polarizing filter.

[0071] Finally, "State 3" refers to a state in which light passing through an LC shutter assembly, such as, but not limited to, LC assembly 100, has spectral characteristics outside the effective operating range of the polarizer and therefore passes through the shutter assembly with little attenuation. In the spectral range of State 3, unpolarized light is not polarized or attenuated, and therefore the LC phase state is not important. In this spectral range, transmission greater than 90% can be achieved with appropriate anti-reflection coatings on the surface of the polarizing filter. State 3, traditionally considered a failure state due to its low extinction ratio, becomes a highly transmissive state regardless of the liquid crystal phase state.

[0072] According to one embodiment, the LC shutter assembly 100 utilizes both incident light polarized by the polarizer 104 (State 1) and incident light that is not polarized by the filter and reaches the LC cell 106 largely unattenuated due to filtering through the LC shutter 100 in either the open or closed state (States 1 and 2). As shown in FIG. 4, the extinction ratio of light entering the LC shutter 100 between the open and closed states of the shutter, designated as 400, decreases due to the inability of the polarizer to attenuate light in the NIR spectrum. As shown in FIG. 12, when the LC shutter 100 is in the open state 404 (State 1), light in the visible spectrum (e.g., having a wavelength between 400 nm and 700 nm) can be transmitted through the LC shutter 100. When the LC shutter 100 is in the closed state 406 (State 2), light in the visible spectrum (e.g., having a wavelength between 400 nm and 700 nm) cannot be transmitted through the LC shutter 100. The same general method works for other optical systems with light sources with similar spectral characteristics. In systems with different spectral contributions to the light source, other polarizers may be selected that provide the appropriate light blocking / attenuation.

[0073] As shown, the attenuation caused by the polarizer 104 fails at longer wavelengths. The LC cell 106 no longer receives approximately 50% of the incident (unpolarized) light due to the 50% attenuation failure at those longer wavelengths. In some instances, the attenuation failure is the result of a light blocking range specifically engineered to see the desired light (e.g., fluorescent light). Therefore, the contrast from the open state 404 to the closed state 406, generally shown as 402, also decreases.

[0074] According to various aspects of the present disclosure, this reduction in contrast becomes a beneficial feature during State 3 operation when a visible light image is overlaid on a NIR fluorescence image. The resulting overlaid composite image(s) and / or video(s) provide medical personnel with greater clarity, detail, and information regarding the presence or absence of pathologies (e.g., including the presence of tumors, cancers, vascular malformations, inflammation, or other), variations in tissue and sub-tissue structure, the anatomical structure of interest, and the surrounding tissues or samples described above, whether examined in vivo or ex vivo.

[0075] In yet another aspect, the amount of visible light can be adjusted by operating the LC phase intermediate between State 1 (open state) and State 2 (closed state). These intermediate states are actually controlled by adjusting the applied voltage and therefore the electric field across the LC cell, allowing for variable attenuation of the light without affecting the light in the failure state (State 3).

[0076] According to various embodiments, an LC shutter assembly, such as the LC shutter assembly 100 of the present disclosure, can be used in a variety of states and can even operate intermediately between various open and closed states to allow for the selection of desired wavelengths in the failure zone depending on the application of the disclosed system or method. In another embodiment, one "intermediate" operating state has at least 50% attenuation and up to 100% maximum attenuation based on the filter's extinction ratio. Operating intermediately between the open and closed states allows for the adjustment or customization of the ratio of visible to NIR light. NIR signals are typically much lower in intensity than the visible light present in a system, and therefore typically require different gain settings or exposure times on the image sensor. Adjusting the amount of visible light attenuation between the open and closed states allows the visible light level to be scaled to match the level of the weak NIR fluorescence signal, thus allowing the same camera settings to be used in both spectral ranges.

[0077] In another embodiment, the video-rate sampling period (i.e., exposure time) may be different for capturing visible light images and fluorophore-emission light (e.g., fluorescence or fluorescent images). The liquid crystal shutter assembly may be placed in an open state for capturing visible light images. The liquid crystal shutter assembly may remain in the open state for about 2 ms to about 6 ms relative to the video-rate sampling period for capturing visible light images. The liquid crystal shutter assembly may be placed in a closed state for capturing fluorophore-emission light. The liquid crystal shutter assembly may remain in the closed state for about 8 ms to about 16 ms relative to the video-rate sampling period for capturing fluorophore-emission light. In other examples, the video-rate sampling period for visible light images may be about 1 ms, about 2 ms, about 3 ms, about 4 ms, about 5 ms, about 6 ms, about 7 ms, about 8 ms, about 9 ms, about 10 ms, about 11 ms, about 12 ms, about 13 ms, about 14 ms, about 15 ms, about 16 ms, or longer. In another example, the video rate sampling period for imaging emitted light from a fluorophore may be about 5 ms, about 6 ms, about 7 ms, about 8 ms, about 9 ms, about 10 ms, about 11 ms, about 12 ms, about 13 ms, about 14 ms, about 15 ms, about 16 ms, about 17 ms, about 18 ms, about 19 ms, about 20 ms, about 25 ms, about 30 ms, or more.

[0078] Engineered polarizers To fully utilize the failed polarizer state (i.e., when the incident wavelength is outside the polarizer's polarization range), the polarizer 104 can be specially manufactured or engineered to cut off (e.g., light cutoff) specific desired wavelengths. For example, when performing fluorescence imaging using indocyanine green (ICG) dye, the spectral emission is in the 750 nm to 950 nm range, or the 800 nm to 880 nm range, peaking at approximately 830 nm. For example, in some embodiments, the polarizer 104 of the liquid crystal shutter assembly 100 is selected to lose effectiveness between 650 and 800 nm, or between 600 nm and about 850 nm, or between about 700 nm and about 850 nm, about 800 nm and about 950 nm, about 800 nm and about 880 nm, about 775 nm to about 795 nm, or about 785 nm. In additional embodiments, polarizer 104 (or two polarizers) is configured to lose effectiveness, or have a light cutoff range, at about 600 nm to about 620 nm, about 620 nm to about 640 nm, about 640 nm to about 660 nm, about 660 nm to about 680 nm, about 680 nm to about 700 nm, about 700 nm to about 720 nm, about 720 nm to about 740 nm, about 740 nm to about 760 nm, about 760 nm to about 780 nm, about 780 nm to about 800 nm, about 800 nm to about 820 nm, about 820 nm to about 840 nm, about 840 nm to about 860 nm, about 860 nm to about 880 nm, or about 880 nm to about 900 nm. In other embodiments, polarizer 104 (or two polarizers) may be configured to lose effectiveness or have a light cutoff range between about 100 nm and about 150 nm, about 150 nm and about 200 nm, about 200 nm and about 250 nm, about 250 nm and about 300 nm, about 300 nm and about 350 nm, about 350 nm and about 400 nm, about 400 nm and about 450 nm, or about 450 nm and about 500 nm. In other embodiments, the liquid crystal shutter assembly is selected to lose effectiveness at 800 nm.

[0079] According to one aspect of the LC shutter assembly 100, approximately 50% of the fluorescent light may be lost due to the polarizer 500, as shown in FIG. 5. In contrast, a specially configured or engineered polarizer 104A may be used in accordance with various embodiments. The engineered polarizer 104A is specifically selected and / or manufactured so that the cutoff wavelength of the polarizer can be tuned or engineered to a specific wavelength. For example, if ICG is the detectable fluorophore of interest, the polarizer can be tuned to cutoff at a desired wavelength within the range of 790-900 nm. In another example, a different ICG fluorophore, or any other fluorophore suitable for fluorescence imaging in the near-infrared range (approximately 780 nm-2500 nm), may be used in conjunction with the engineered polarizer 104 according to a specific desired cutoff wavelength. FIG. 5 illustrates the different NIR transmission levels between the engineered polarizer 104A and the polymer polarizer 500 in combination with an LC shutter in the closed state.

[0080] By way of example and not limitation, polarizer 104 may be a dye polarizer, which is highly effective in the visible range but falls off rapidly in the NIR wavelength range. According to various embodiments, dye polarizers are preferred because they have a high damage threshold suitable for absorbing large amounts of light in the visible spectrum.

[0081] In one embodiment, the liquid crystal shutter assembly minimizes attenuation of light outside the light cutoff range of the polarizer in State 3. In some examples, the light outside the light cutoff range can be near-infrared (NIR) or infrared (IR) light. In other examples, the light outside the light cutoff range can be light having wavelengths above the visible light spectrum, within the visible light spectrum, or below the visible light spectrum. Minimal attenuation provides the maximum intensity of emitted light to the imaging sensor and / or camera, resulting in optimal imaging of fluorescence from the fluorophores. In some examples, the emitted light from the fluorophores can have an attenuation of 0% to 5% when passing through the liquid crystal shutter assembly 100. In other examples, the emitted light from the fluorophores can have an attenuation of about 0% to about 5%, about 5% to about 10%, or about 10% to about 15% when passing through the liquid crystal shutter assembly 100.

[0082] Liquid crystal cell According to various embodiments, the LC shutter 100 can use any of several liquid crystal materials. By way of example, the LC shutter 100 can include a Pi cell or a twisted nematic ("TN") cell. Typically, a Pi LC cell has faster opening and closing times than a twisted nematic (TN) cell. Furthermore, a Pi cell can cause color shifts in the image hue. Pi cells are typically used in DLP projectors to provide video-rate switching. However, DLP projectors require temperature-compensated voltage control to avoid color drift. While effective, the use of a Pi cell may be less desirable for use in uncooled, simplified imaging systems. In one embodiment of a twisted nematic liquid crystal, applying a non-zero voltage induces backflow in the liquid crystal, causing the cell to momentarily "open." In yet another embodiment, the backflow defines a partially open, transmissive state.

[0083] According to another aspect of the LC shutter assembly 100, TN cells are preferred. While TN cells typically shutter slower than comparable Pi cells, TN cells have more desirable spectral characteristics in that their absorption is relatively neutral within the operating spectral range. Therefore, for compact, uncooled imaging systems, the color-natural characteristics of TN cells are more desirable. The robust spectral characteristics of TN cells offer significant advantages. TN cells result in slower shutter opening times, which can be improved with temperature.

[0084] According to various embodiments, the closing time of a TN cell may be increased by increasing the applied voltage of the TN cell above its normal operating voltage. In one example, a TN cell (e.g., LC medium 106) that may be used in the LC shutter 100 may be configured to be open when no electric field is applied. In this example, the TN cell shutter may achieve very fast shutter speeds and high-contrast extinction ratios. An additional characteristic of TN cells that may typically be considered a drawback is that when driven with high voltages, the TN cell exhibits "backflow" when opening. Specifically, high voltages induce backflow in the TN cell, causing the LC shutter to briefly open, fully close, and eventually fully open.

[0085] For example, FIG. 6 shows a TN cell driven with two different voltages: low-voltage drive 600 and high-voltage drive 602. The low-voltage drive 600 is the one recommended by the manufacturer to avoid backflow. As shown, a 6 ms box 604 indicates an exemplary video-rate sampling period (i.e., exposure time) for video-rate imaging. The low-voltage drive 600 induces a more open state within the 6 ms duration, but the closing time is slower, necessitating early termination of the voltage drive to close the cell. In contrast, the high-voltage drive 602 induces a faster initial opening before backflow closure 606 occurs. While the total light transmission using the high-voltage drive 602 is less than that of the low-voltage drive 600 within the 6 ms window, the performance of the TN cell can be further modified by heating the LC cell 106.

[0086] Referring to FIG. 7, the open time of a TNLC shutter (i.e., an LC shutter constructed using a TN cell as the liquid crystal cell) can be a function of temperature. Specifically, the open times of the TNLC shutter at temperatures of 20°C (700), 25°C (702), 35°C (704), 45°C (706), 55°C (708), 65°C (710), and 75°C (712) are shown. The open time of a Pi cell, designated 714, is also shown for comparison. As shown, heating the LC cell 106 reduces the viscosity of the liquid crystal material within the cell. As a result, the response speed of the LC shutter increases, thereby shortening the duration of the backflow phenomenon and ultimately increasing the speed at which the LC shutter 100 fully opens. In some examples, the transition speed between the open and closed states is determined by the temperature of the liquid crystal and the drive voltage source. In some embodiments, the LC cell 106 is heated, for example, between 30°C and 50°C.

[0087] According to one embodiment, the LC shutter can be heated by a dedicated heater that can be controlled to directly regulate the performance of the TN cell. According to another embodiment, the LC shutter 100 can be placed inside a sealed housing that provides heat generation through waste heat generated by other components of the imaging system. As shown in Figure 6, in response to a heated environment, the reduced backflow transmission and partial open state are sufficiently high for real-time visible imaging. By placing the LC cell 106 or the entire LC shutter assembly 100 in a sealed environment, such as, but not limited to, the imaging system housing disclosed in the Blaze Bioscience system mentioned above, the increased internal temperature generated by other components of the imaging system helps mitigate the backflow associated with the TN cell. By way of example and not limitation, the LC cell 106 can be maintained at approximately 35-40°C. In many embodiments, an additional benefit of driving the TN cell at a higher voltage is increased light blocking, as shown in Figure 8. For example, TN cells are typically driven at 0 V in the open state and at some nominal low voltage in the closed state. However, the higher voltage used for fast closing times also helps increase the blocking capability of the cell. As shown, higher peak-to-peak ("pp") drive voltages work better for the application. The LC cell 106 can be driven by a DC drive voltage or an AC drive voltage.

[0088] According to one embodiment, the LC cell 106 is driven with a high voltage ranging from + / -3 Vp-p to + / -60 Vp-p [peak-to-peak]. In one exemplary embodiment, the LC shutter cell is driven with + / -24 V (48 Vp-p). In one exemplary embodiment, the LC shutter cell is driven with any value in the range between + / -16 V (32 Vp-p) and + / -30 V (60 Vp-p), inclusive. In one exemplary embodiment, the LC shutter cell is driven with any value in the range between + / -30 V (60 Vp-p) and + / -100 V (200 Vp-p), inclusive. According to various embodiments, the LC shutter is driven with a square wave voltage having a frequency of 1-2 kHz and a 50% duty cycle. In one example, the square wave voltage ranges from a minimum of 0 V in the open state to a maximum of + / - 24 V (48 Vp-p) in the closed state. In another embodiment, the square wave drive voltage is provided as a DC drive voltage ranging from -24 V to 24 V, resulting in a drive voltage of approximately 48 V peak-to-peak. In another embodiment, the amplitude of the bipolar square wave ranges from + / - 3 to + / - 30 Vp-p. In one exemplary embodiment, the amplitude of the bipolar square wave ranges anywhere between + / - 16 V (32 Vp-p) and + / - 30 V (60 Vp-p), inclusive. In one exemplary embodiment, the amplitude of the bipolar square wave ranges anywhere between + / - 30 V (60 Vp-p) and + / - 100 V (200 Vp-p), inclusive. In another aspect, the second voltage includes a plurality of voltages that produce a bipolar square wave voltage with an average value of 0V. In this aspect, the voltage is held at a full 24V for a video-rate image in the closed state. In this embodiment, there is no switching of the voltage bias polarity during frame exposure, and therefore the LC shutter maintains a higher level of light attenuation for its closed state. The voltage polarity is reversed for the next frame, which requires a closed state to maintain the health and reliability of the LC shutter.

[0089] According to various aspects, the LC shutter may have a first voltage for toggling the LC shutter to an open state and a second voltage for toggling the LC shutter to a closed state. In some examples, the first voltage may be 0 V. Applying the first voltage may cause the liquid crystal shutter assembly to assume an open state. In other examples, the first voltage may be a bipolar square wave having an amplitude ranging from + / -0.1 Vp-p to + / -5 Vp-p. If the liquid crystal is a twisted nematic liquid crystal cell, the first voltage may be 0 V. If the liquid crystal is a Pi cell, the first voltage may be a bipolar square wave having an amplitude ranging from + / -0.1 Vp-p to + / -5 Vp-p. In other examples, the first voltage may be a bipolar square wave having an amplitude ranging from + / -3 Vp-p to + / -60 Vp-p. In other examples, the first voltage is about + / -12 Vp-p to + / -15 Vp-p, about + / -15 Vp-p to + / -18 Vp-p, about + / -18 Vp-p to + / -21 Vp-p, about + / -21 Vp-p to + / -24 Vp-p, about + / -24 Vp-p to + / -27 Vp-p, about + / -30 Vp-p to + / -33 Vp-p, about + / -36 Vp-p The second voltage may be a bipolar square wave having an amplitude in the range of + / -3 Vp-p to + / -39 Vp-p, about + / -42 Vp-p to + / -45 Vp-p, about + / -45 Vp-p to + / -48 Vp-p, about + / -51 Vp-p to + / -54 Vp-p, about + / -54 Vp-p to + / -57 Vp-p, about + / -57 Vp-p to + / -60 Vp-p, or more. The second voltage may be a bipolar square wave having an amplitude in the range of + / -3 Vp-p to + / -60 Vp-p. Application of the second voltage may enable the liquid crystal shutter assembly to assume a closed state.In other examples, the second voltage is about + / -12 Vp-p to + / -15 Vp-p, about + / -15 Vp-p to + / -18 Vp-p, about + / -18 Vp-p to + / -21 Vp-p, about + / -21 Vp-p to + / -24 Vp-p, about + / -24 Vp-p to + / -27 Vp-p, about + / -30 Vp-p to + / -33 Vp-p, about + / -36 Vp-p The second voltage may be a bipolar square wave having an amplitude of about + / -39 Vp-p, about + / -42 Vp-p to + / -45 Vp-p, about + / -45 Vp-p to + / -48 Vp-p, about + / -51 Vp-p to + / -54 Vp-p, about + / -54 Vp-p to + / -57 Vp-p, about + / -57 Vp-p to + / -60 Vp-p, or more. In a further example, the second voltage may be 0 V. In another example, the second voltage may be a bipolar square wave having an amplitude in the range of + / -0.1 Vp-p to + / -5 Vp-p.

[0090] In one embodiment, the bipolar square wave can be modulated at 500 to 2000 Hz. In some examples, the drive voltage source toggles the liquid crystal between a first voltage (e.g., an open state) and a second voltage (e.g., a closed state) for periods of 2 ms to 40 ms (inclusive), 1 ms to 40 ms (inclusive), or 2 ms to 60 ms (inclusive), 60 ms to 100 ms (inclusive), and up to 300 ms. In other examples, the drive voltage source toggles the liquid crystal between a first voltage (e.g., an open state) and a second voltage (e.g., a closed state) for a period of about 2 ms to about 4 ms, about 4 ms to about 6 ms, about 6 ms to about 8 ms, about 8 ms to about 10 ms, about 10 ms to about 12 ms, about 12 ms to about 14 ms, about 14 ms to about 16 ms, about 16 ms to about 18 ms, about 18 ms to about 20 ms, about 20 ms to about 22 ms, about 22 ms to about 24 ms, about 24 ms to about 26 ms, about 26 ms to about 28 ms, about 28 ms to about 30 ms, about 30 ms to about 32 ms, about 32 ms to about 34 ms, about 34 ms to about 36 ms, about 36 ms to about 38 ms, or about 38 ms to about 40 ms.

[0091] With respect to the first and second voltages described herein, it is understood that the first and second voltages may be applied or generated in any order or configuration in the liquid crystal assemblies described herein (e.g., regardless of whether the first or second is in an open or closed state, or has 0 V or + / - voltages applied, as described herein). For example, the polarizers in the LC assembly may be similarly configured (e.g., oriented) to affect whether the first or second is in an open or closed state, or has 0 V or + / - voltages applied, as described herein. For example, when the polarizers are cross-polarized, a voltage of 0 V may be applied to cause the LC cell to assume an open state (e.g., the LC cell rotates the polarization of light transmitted from the first polarizer so that the light passes through the second polarizer). When the polarizers are cross-polarized, a + / - voltage may be applied to cause the LC cell to assume a closed state (e.g., the LC cell does not rotate the polarization of light transmitted from the first polarizer so that the light is blocked or attenuated by the second polarizer). For example, if the polarizers have the same polarization (e.g., the same orientation), a + / - voltage may be applied to cause the cell to assume an open state (e.g., the LC cell does not rotate the polarization of light transmitted from the first and second polarizers, and the light passes through the second polarizer). If the polarizers have the same polarization (e.g., the same orientation), a voltage of 0V may be applied to cause the cell to assume a closed state (e.g., the LC cell rotates the polarization of light transmitted from the first polarizer, and the light is blocked or attenuated by the second polarizer).

[0092] According to one embodiment of the LC shutter assembly 100, a dye polarizer 104 is paired with a twisted nematic (TN) liquid crystal cell 106, which can be tuned to a desired separation between the visible and NIR spectral bands. When used with a TN cell as the LC cell 106, the dye polarizer 104 allows for high attenuation in the visible band with only about 15% attenuation in the ICG emission band when used in ICG fluorescence imaging. Thus, the engineered dye polarizer provides approximately 85% transmission of the ICG emission band whether the LC shutter 100 is in the closed or open state. This increased emission light transmission is particularly useful when generating composite image(s) by overlaying visible light with available NIR fluorescence.

[0093] Adjustment to fluorescent dyes In many aspects, the cutoff wavelength of the polarizer may be tuned or engineered to a specific wavelength optimized for a particular fluorescent molecule targeted for detection by a system utilizing the liquid crystal shutter assembly of the present disclosure. In such embodiments, the polarizer can be configured to have a light cutoff in a range tuned to the emission range of the applicable fluorescent moiety. In some examples, the light cutoff range of the polarizer can include the longest wavelengths in the visible spectral range. In some examples, the light cutoff range of the polarizer can be within the visible spectral range. The polarizer can be ineffective and transparent above the longest wavelengths in the visible spectral range. In another example, the polarizer can be ineffective and transparent below the shortest wavelengths in the visible spectral range. For example, the systems and methods of the present disclosure can be configured or optimized for fluorophores in the NIR range and / or for fluorophores outside the NIR range. For example, using an ICG dye, the polarizer can be configured to have a cutoff in the long wavelength spectral range applicable to the excitation light, e.g., about 600 nm to about 850 nm, or about 700 nm to about 800 nm, about 800 nm to about 950 nm, about 800 nm to about 880 nm, about 775 nm to about 795 nm, or about 785 nm. Also, using ICG dyes, polarizers can be configured to have a wavelength cutoff by 800 nm, or alternatively, can be configured to have wavelength cutoff by 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm, 805 nm, 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm, 895 nm, or 900 nm. A polarizer with an effective cutoff minimum, for example in the range of 800 nm to 880 nm, allows the emission band (e.g., from an excited fluorophore) to pass unpolarized wavelengths that are collected if the polarizer cutoff occurs at 800 nm.In other embodiments, fluorophores and / or fluorescent dyes that emit light at wavelengths in the infrared range or higher, or in the visible range or below the visible range, may be used with engineered polarizers and LC shutters driven with appropriate voltages to capture light at video switching rates based on their corresponding emission spectra.

[0094] For example, the fluorescent molecule can include an ultraviolet (UV) dye, a blue dye, or both. Exemplary UV and blue dyes for fluorophores include ALEXA FLUOR 350 and AMCA dyes (e.g., AMCA-X dyes), derivatives of 7-aminocoumarin dyes, reactive versions of dialkylaminocoumarin ALEXA FLUOR 350 dyes, ALEXA FLUOR 430 (a reactive UV dye that absorbs between 400 nm and 450 nm and has significant fluorescence above 500 nm in aqueous solution), Marina Blue and Pacific Blue dyes (based on the 6,8-difluoro-7-hydroxycoumarin fluorophore), hydroxycoumarin and alkoxycoumarin derivatives that exhibit bright blue fluorescence emission near 460 nm, Zenon ALEXA FLUOR 350, Zenon ALEXA FLUOR 430, and Zenon Pacific Blue, succinimidyl esters of Pacific Orange dye, Cascade Blue acetyl azide, and other pyrene derivatives, ALEXA Fluorescent molecules include FLUOR405 and its derivatives, pyrene succinimidyl esters, cascade yellow dyes, PyMPO and pyridyloxazole derivatives, aminonaphthalene dyes and dansyl chloride, dapoxyl dyes (e.g., dapoxyl sulfonyl chloride, amine-reactive dapoxyl succinimidyl esters, carboxylic acid-reactive dapoxyl(2-aminoethyl)sulfonamide), bimane dyes (e.g., bimane mercaptoacetic acid) and its derivatives, NBD dyes and its derivatives, QsY35 dyes and its derivatives, and fluorescein and its derivatives. Fluorescent molecules can include infrared dyes, near-infrared dyes, or both.Exemplary infrared and near-infrared dyes for fluorophores include DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, or indocyanine green (ICG) and any derivatives of the foregoing, cyanine dyes, acridine orange or yellow, ALEXA FLUOR and any derivatives thereof, 7-actinomycin D, 8-anilinonaphthalene-1-sulfonic acid, ATTO dye and any derivatives thereof, auramine rhodamine stain and any derivatives thereof, benthanone, bimane, 9,10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)naphthacene, bisbenzimide, Brainbow, calcein, carboxyfluorescein and any derivatives thereof, 1-chloro-9,10-bis(phenylethynyl)anthracene and any derivatives thereof, DAPI, DiOC6, DyLight Fluor and any derivatives thereof, epicocconone, ethidium bromide, FlAsH-EDT2, Fluo dyes and any derivatives thereof, FluoProbe and any derivatives thereof, fluorescein and any derivatives thereof, Fura and any derivatives thereof, Gel Green and any derivatives thereof, Gel Red and any derivatives thereof, fluorescent proteins and any derivatives thereof, m isoform proteins and any derivatives thereof, such as mCherry, hetamethine dyes and any derivatives thereof, Hoechst stain, iminocoumarin, Indian Yellow, Indo-1 and any derivatives thereof, Laurdan, Lucifer Yellow and any derivatives thereof, Luciferin and any derivatives thereof, luciferase and any derivatives thereof, merocyanine and any derivatives thereof, Nile dyes and any derivatives thereof, perylene, phloxine, phycodyes and any derivatives thereof, propium iodide, pyranine, rhodamine and any derivatives thereof, RiboGreen, RoGFP, rubrene, stilbene and any derivatives thereof, sulforhodamine and any derivatives thereof, SYBR and any derivatives thereof, synapto-pHluorin, tetraphenylbutadiene, tetrasodium tris, Texas Red, titanium yellow, TSQ, umbelliferone, violanthrone, yellow fluorescent protein, and YOYO-1.Other suitable fluorescent dyes include fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanines, merocyanines, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine, etc.), and the like. B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon green dyes (e.g., Oregon green 488, Oregon green 500, Oregon green 514, etc.), Texas red, Texas red-X, spectrum red, spectrum green, cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY-5.5, etc.), ALEXA Fluor dyes (e.g., ALEXA FLUOR 350, ALEXA FLUOR 488, ALEXA FLUOR 532, ALEXA FLUOR 546, ALEXA FLUOR 568, ALEXA FLUOR 594, ALEXA FLUOR 633, ALEXA FLUOR 660, ALEXA FLUOR 680, etc.), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, etc.), IRDyes (e.g., IRD40, IRD 700, IRD 800, etc.), etc. Additional suitable detectable agents are known and are described in International Patent Application PCT / US2014 / 056177, which is incorporated by reference in its entirety.

[0095] Fluorescent molecules used to detect samples with the systems and methods herein can include one or more dyes, two or more, three, four, five, and up to ten or more such dyes in a given sample, using any type of dye (e.g., ultraviolet (UV) dyes, blue dyes, infrared dyes, or near-infrared dyes) in any combination. In addition to fluorescence, the disclosed systems and methods are also useful for photoluminescence applications. Band gaps associated with aluminum nitride have emissions similar to fluorophores in the visible spectrum, while band gaps associated with aluminum gallium arsenide have emissions similar to NIR fluorophores. Photoluminescence imaging has applications in, but not limited to, optoelectronic semiconductor processing and defect detection in organic and inorganic LED displays. The systems and methods herein can be used to detect autofluorescence within organs, organ substructures, tissues, targets, cells, or samples.

[0096] Use of controlled excitation energy by liquid crystal shutters. According to various embodiments, integrating LC shutters, such as but not limited to LC shutter 100, into a wide variety of mixed visible and NIR systems, along with appropriate tuning of spectral filters, such as but not limited to polarizer 104, enables the capture of NIR images without an overwhelming contribution of visible light. These improvements can be further enhanced when paired with techniques that reduce the effects of light pollution within the emission band being collected.

[0097] Optical pumping of fluorescence and photoluminescence relies on a powerful source to excite the material of interest. In particular, increasing the excitation power can increase the signal. This type of signal enhancement is effective up to the point of saturation of the fluorophore or non-radiative recombination loss of the photoluminescent conjugate.

[0098] Increasing excitation energy typically has the traditional undesirable effect of adding heat to the sample being measured. This undesirable effect can be mitigated by reducing the duration of excitation so that the average wattage delivered to the measured sample is maintained below a predetermined level or at a safe level required for the application. For example, in in vivo applications using the systems and methods disclosed herein to image organs, organ substructures, tissues, targets, cells, or samples, the excitation energy delivered to the tissue must be below known safety standards and thresholds to minimize heating and / or damage. In various embodiments, the excitation energy is controlled by controlling the peak intensity and duration of the excitation energy. The controlled excitation energy may be further adjusted to enhance the signal-to-noise ratio (SNR) of the NIR image while maintaining boundaries imposed by damage thresholds or safety concerns.

[0099] Referring to Figure 9, patient safety criteria can be quantified as the area under the curve plotting energy delivered to tissue over time or duration. In physiological applications, there is a safe level of energy that can enter the tissue that will not cause damage to the tissue. As shown, A CLINCAL The area shown under 900 illustrates an example of a "safe" exposure to energy (e.g., biological tissue), as indicated by the area under the curve formed by the threshold level or intensity of energy indicated by 902, which may be applied for a short or long period of time (e.g., an extended amount of time). The system can use higher intensity energy for a short period of time to achieve energies below safe levels, which has the advantage of enhancing the photons available for imaging while still meeting or exceeding patient safety in clinical applications where tissue is not damaged. As shown in Figure 9, A FLASH The burst-controlled intense excitation energy identified as 906 represents an intense excitation energy pulse that can be applied for a short period of time using the systems and methods disclosed herein. As shown, the area under the curve (A) defined by the controlled excitation energy at higher intensity is FLASH906) is shown by 908 with a flash duration indicated at 910. As shown, because the flash duration 910 is much shorter than the clinical duration 904, the intensity of the flash energy 908 is much higher than the intensity of the clinical energy intensity 902, but the area under the curve (A FLASH 906) is curve A CLINCAL The area below 900 is smaller than the area below 900, which meets or exceeds the safety criteria (for tissue not being damaged). FLASH 906The area under the curve is A CLINCAL 900 curve or less, so that the total energy imparted to the tissue by the controlled excitation energy is below the applicable safety threshold. In certain embodiments used herein, the systems and methods of the present invention provide (e.g., by adjusting the intensity of the energy and the duration of the excitation energy pulse) FLASH The area under the curve (AUC) of 906 is CLINCAL It is configured to have an AUC of 900 or less.

[0100] It will be appreciated that the methods described herein using controlled excitation energy can be applied to methods and systems using the LC shutter assemblies of the present disclosure and / or with the Blaze Bioscience system.

[0101] In various aspects of the above-described method, the liquid crystal assembly includes an engineered polarizer disposed in the path of light from the sample, the polarizer configured to have an effective spectral range such that the long-wavelength cutoff range is between 600 nm and 900 nm or between about 800 and 880 nm. The method may further include directing the emitted light and reflected visible light through the liquid crystal assembly, wherein reflected visible light having wavelengths within the effective spectral range of the engineered polarizer may be blocked or allowed to pass depending on the state of the liquid crystal. Regarding the state of the liquid crystal, emitted light incident on the liquid crystal has wavelengths outside the effective spectral range of the engineered polarizer and passes through substantially unattenuated, regardless of the state of the liquid crystal.

[0102] Furthermore, for certain applications of video-rate imaging, the unique sequencing of individual frames time-multiplexed with LC shutters pairs well with controlled and / or pulsed energy excitation. In some examples, the controlled and / or pulsed energy excitation can be white light, NIR light, IR light, or any other type of excitation light. In addition to generating stronger excitation relative to ambient light pollution in the emission spectral band, controlled excitation energy has the additional advantage of increasing the signal-to-background or ambient light ratio, allowing for shorter exposure times (i.e., video-rate sampling periods). According to various aspects, the shorter exposure times enabled by the use of controlled excitation energy have the secondary benefit of reducing the amount of thermal noise integrated into the image from the sensor, thus improving the SNR of the image. In particular, the SNR increases significantly as the laser pulse width and corresponding integration time decrease. Referring to FIG. 10, the SNR of pulsed or controlled excitation energy 1000 is greater than the SNR of a lower-intensity energy pulse 1002. It is further shown that the signal to noise continues to increase as the laser duration is reduced.

[0103] A type of SNR for image analysis is the contrast-to-noise ratio (CNR). The CNR of an image can be improved when the object intended to be detected is bright and the surrounding area is dark. When the object intended to be detected is fluorescent, the amount of available light is much less than the ambient light. Furthermore, visible lighting added to assist in delicate tasks such as surgical procedures is often much brighter than the general ambient light. Therefore, the fluorescence to be detected may be several orders of magnitude lower than the ambient background light, and the CNR of the fluorescence image falls below the usable range. To improve the CNR, the fluorescence can be increased. Two known methods for increasing fluorescence are to increase the amount of fluorescent material or to increase the excitation energy. However, increasing the amount of fluorescent material may be impractical in biological materials due to physiological barriers. Furthermore, increasing the amount of excitation energy may be impractical due to the possibility of thermal damage to the biological material.

[0104] Traditionally, CNR has been improved by reducing background light (e.g., visible light) to make the fluorescence more visible. Ideally, the background light source is removed or intermittently turned off. If the background light source cannot be removed or turned off, the excitation source is modulated and dark frames are collected to subtract the background light from the signal. This method results in the detection of fluorescence at the expense of filling much of the camera's dynamic range with bright background light that must be removed by subtraction. This results in high image noise levels and long sequence times, resulting in slower video frame rates.

[0105] While the frame subtraction system is an improvement over conventional systems, the use of the LC shutter 100 can significantly improve the video frame rate by reducing the sequence time (e.g., the required video-rate sampling period), blocking interfering light (e.g., background light), improving and increasing the CNR, and enhancing the SNR in bright ambient light settings. The enhanced CNR and / or SNR may allow for faster or increased frame rates. The LC shutter 100 can be used to intermittently block background light, as shown in FIG. 17. When the LC shutter is in an open state (e.g., a first voltage of 0 V is applied), bright background light (e.g., reflected visible light from the sample) is allowed to pass through the LC shutter for visible light imaging (e.g., a first video-rate sampling period). Imaging the visible light may occur during the first video-rate sampling period (e.g., when the LC shutter is in the open state). In some examples, the first video rate sampling period (i.e., first exposure time) can be about 1 ms to about 2 ms, about 2 ms to about 3 ms, about 3 ms to about 4 ms, about 4 ms to about 5 ms, about 5 ms to about 6 ms, about 6 ms to about 7 ms, about 7 ms to about 8 ms, about 8 ms to about 9 ms, or about 9 ms to about 10 ms. In one example, the first video rate sampling period can be about 2 ms to about 6 ms.

[0106] When the LC shutter is in a closed state (e.g., a second voltage is applied), bright background light (e.g., visible light) is blocked from the camera, allowing only NIR light to pass, enabling imaging of fluorescence from the sample for a second video-rate sampling period (e.g., a second exposure time). While the LC shutter 100 is in the closed state, a laser diode may be turned on to provide excitation energy to the fluorophores, allowing imaging of fluorescence light (i.e., emitted light from the fluorophores). In some examples, the second video rate sampling period (i.e., second exposure time) can be about 5 ms to about 6 ms, about 6 ms to about 7 ms, about 7 ms to about 8 ms, about 8 ms to about 9 ms, about 9 ms to about 10 ms, about 10 ms to about 11 ms, about 11 ms to about 12 ms, about 12 ms to about 13 ms, about 13 ms to about 14 ms, about 14 ms to about 15 ms, 15 ms to about 16 ms, or about 16 ms to about 17 ms. In some examples, the second video rate sampling period can be about 12 ms to about 16 ms, or about 13 ms to about 17 ms.

[0107] After imaging the fluorescence, the laser diode can be turned off for a short period of time (e.g., a third video rate sampling period) while the LC shutter remains closed, allowing imaging of a dark background image. When the laser diode is turned off, it stops providing excitation energy. In some examples, the third video rate sampling period (i.e., the third exposure time) can be about 5 ms to about 6 ms, about 6 ms to about 7 ms, about 7 ms to about 8 ms, about 8 ms to about 9 ms, about 9 ms to about 10 ms, about 10 ms to about 11 ms, about 11 ms to about 12 ms, about 12 ms to about 13 ms, about 13 ms to about 14 ms, about 14 ms to about 15 ms, 15 ms to about 16 ms, or about 16 ms to about 17 ms. In some examples, the third video rate sampling period can be about 12 ms to about 16 ms, or about 13 ms to about 17 ms.

[0108] In one embodiment, a dark background image can be subtracted from the fluorescence image to provide a clear fluorescence image. When closed, the LC shutter 100 blocks any light outside the emission band, spectrally blocking interfering light (bright background light) from being captured. Removing bright background light (e.g., visible light) allows for the use of higher analog gain during image capture to fill the camera's dynamic range. Increasing the camera's gain allows for much shorter integration times. In this embodiment, the LC shutter 100 acts as a temporal spectral filter, allowing fluorescence to be displayed when in the closed state and visible light to be displayed when in the open state. The reduced integration time required for minimum CNR enables real-time video of fluorescence images, even in the presence of bright background light.

[0109] As shown in Figure 8, as the driving voltage to the LC medium 106 increases, the blocking of visible light increases. When visible light is blocked, the fluorescent image buried beneath the visible image becomes visible. As shown in Figure 15, the CNR value of the fluorescent image increases with increasing driving voltage to the LC medium, allowing for better capture of the fluorescent image.

[0110] The increased CNR achieved by using the LC shutter 100 allows for post-processing of the fluorescence images with digital gain, as opposed to requiring longer exposure times. Post-processing with digital gain and shorter exposure times results in an increased frame rate for the fluorescence images. As shown in Figure 16, the exposure time required to maintain a CNR of 5 decreases as the voltage increases. When supplying the LC shutter 100 with a voltage of approximately 13 V or higher, exposure times of less than 5 ms can be achieved.

[0111] As shown in Figures 18A-18B, the reduced brightness of the dark image with the LC shutter assembly closed reduces artifacts in the subtracted image, as described herein. Additionally, the reduced exposure time (i.e., video-rate sampling period) of the dark frame (laser off) minimizes motion blur in the image. Furthermore, the use of the LC shutter assembly reduces the brightness of the dark image, thereby reducing the intensity of the dark frame and the ratio of dark frames to NIR frames. As shown in Figure 18A, fluorescence images acquired with a linear stage (e.g., rotating or moving) using bright background subtraction resulted in images with unclear boundaries due to the subtracted image showing errors in how the dark image was translated relative to the NIR image. Additionally, in Figure 18A, the longer exposure time of the dark frame results in an image that appears to capture motion and does not accurately depict the location of the fluorescence. As shown in Figure 18B, fluorescence images acquired with the LC shutter 100 blocking visible light and a linear stage (e.g., rotating or moving) resulted in images with clear boundaries. By utilizing the LC shutter 100 to block visual light and reduce exposure time, clear fluorescent images can be generated that provide precise location of the fluorescence, which is crucial in surgical procedures where surgeons need precise location of various body components to perform safe, efficient, and effective surgery.

[0112] Use of liquid crystal shutters to reduce ghosting or unwanted light As shown in FIG. 13, the imaging system may include a beam splitter 1302. The function of a beam splitter is to split incident light into a reflected beam and a transmitted beam. One undesirable feature of certain types of beam splitters is the occurrence of secondary reflections. Secondary reflections can create "ghost" images. As shown in FIG. 13, light from an object plane 1301 is partially reflected off the front surface of the beam splitter 1302, producing a front surface reflection 1304. The back surface of the beam splitter produces a back surface reflection 1305 (e.g., a "source" image). The remainder of the light is transmitted light 1303 without any "ghost" images. Both the front surface reflection 1304 and the back surface reflection 1305 of the beam splitter 1302 have a dominant S polarization relative to the orientation of the beam splitter surface. Traditionally, rear surface coatings attempt to minimize rear reflection 1305 with anti-reflection (AR) coatings, but front surface coatings create a front surface reflection 1304 (i.e., primary reflection) of the beamsplitter 1302. Traditionally, the front surface reflection 1304 needs to have at least 10 times the reflectivity of the rear surface reflection 1305 to make the rear reflection 1305 imperceptible or "invisible" relative to the front surface reflection 1304. In other words, having a front reflectivity (e.g., the reflectivity of the front surface reflection 1304) that is 10 times higher than the rear reflectivity (e.g., the reflectivity of the rear reflection 1305), or a 10:1 ratio (front reflection / rear reflection), is considered a desirable result for reducing image lag in systems that include the beamsplitter 1302. It is technically difficult to suppress S-polarized light and achieve a back surface reflectivity (e.g., reflectivity of the back surface reflection 1305) of less than 1% across the entire visible spectrum, therefore intentionally increasing the reflectivity of the front surface reflection 1304 is the only mechanism to achieve a 10:1 reflectivity ratio. This limits the minimum total reflectivity of the beamsplitter to 11% (by adding both the front and back reflectivities). In systems seeking to keep total reflectivity very low, this 11% lower limit is undesirable.

[0113] In contrast to S-polarized light, it is much easier to control the P-polarized light on either the front surface reflection 1304 or the back surface reflection 1305 down to values ​​near zero. The disclosed LC shutter offers the advantage that only linearly polarized light within the polarizer's effective spectral range passes through the shutter. Therefore, the LC shutter can be oriented to block S-polarized light reflected by the beam splitter. By orienting the LC shutter's polarizer to block S-polarized light, only P-polarized light is imaged. The coatings on the front and back surfaces of the beam splitter can be configured to minimize S-polarized light reflections on both the front and back surfaces of the beam splitter. By adding a small amount of P-polarized light only to the front surface coating, the described LC shutter can easily achieve a reflectivity ratio of 10:1 or more while imaging only P-polarized light. The S-polarized light is then simply engineered down to the lowest reasonable level. By properly designing the coatings, the ratio of the front surface reflection 1304 to the back surface reflection 1305 for P-polarized light can exceed 10:1 while keeping the total reflectivity of the beam splitter 1302 below a minimum of 11%. Although the front-to-back ratio for S-polarized light does not achieve a 10:1 ratio, this light is subsequently filtered out by a properly oriented polarizing filter in the LC shutter of the present disclosure. The first polarizing film 1101(a) of the LC shutter 100 may be oriented to block S-polarized light from both the back reflection 1305 and the front reflection 1304, and pass only P-polarized light. The intensity ratio between the front reflection 1304 and the back reflection 1305 may be increased as the P-polarized light is passed through the imaging lens 1307 and then through the camera 1308. By filtering out the S-polarized light and imaging only with P-polarized light (relative to the angle of the beamsplitter), various front-to-back reflection ratios can be achieved depending on the application by engineering the reflection of the P-polarized light where a lower reflectance value is desired.

[0114] In some embodiments, a front-to-back reflection ratio of at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 11:1, at least 12:1, at least 14:1, at least 15:1, at least 16:1, at least 17:1, or at least 20:1 (inclusive) (i.e., any ratio between 2:1 and 20:1) can be achieved accordingly, any of which is effective in reducing ghosting. This technique of designing the beamsplitter coating to avoid linear polarization takes advantage of the LC shutter's ability to cut or reduce the total reflectance of the beamsplitter 1302 well below the nominal minimum of 11% achievable without a polarizer. The system can be configured to achieve a total reflectance of the beamsplitter of 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, or 3% or less. FIG. 20A illustrates an example of a reflectance ratio (FBR) of around 10:1 across the visible spectrum. As shown in FIG. 20A, the P-polarized FBR 2000 can maintain an FBR of 10:1 or greater across the entire visible spectrum. As shown in FIG. 20A, the S-polarized FBR 2002 is below the desired FBR of 10:1 across the entire visible spectrum, but the S-polarized light is attenuated or blocked by the first polarizing film 1101(a). As shown in FIG. 20B, using an LC shutter to filter out the S-polarized light from the beamsplitter can result in a total reflectance of less than 11% across the entire visible spectrum.

[0115] As shown in FIG. 14B, removing S-polarized light from the rear reflection 1305 can eliminate the “G host” image 1400. As shown in FIG. 14A, if the LC medium 106 is oriented such that the first polarizer attenuates P-polarized light, a ghost image 1400 may be present at the bottom edge of the image. As shown in FIG. 14B, if the LC medium 106 is oriented such that the first polarizer attenuates S-polarized light, the ghost image is eliminated. In some examples, the first polarizing film 1101(a) can be engineered to attenuate or block 100% of S-polarized light. By engineering the beamsplitter to block S-polarized light and achieve a reflectance ratio (front reflection to rear reflection) of 10:1 or greater for P-polarized light, the ghost image is eliminated or significantly reduced, resulting in a clearer image. In other examples, the first polarizing film 1101(a) can be oriented to block other types of polarized light, thereby filtering out undesired polarized light before it passes through the liquid crystal shutter assembly. As shown in Figures 14A-14B, removal of ghost images results in less signal strength (e.g., signal strength drops from μ = 212 to μ = 131) due to attenuation of the S reflection. The resulting image may lose some intensity. However, the image also defines a clear boundary without "ghost" images 1400, which is crucial when detailed imaging is required.

[0116] In some embodiments, the polarizer 104 of the LC shutter 100 can be configured to degrade in any situation where desired light has a different polarization than undesired light. In one example, a laser illuminator may be used in the imaging system. The laser illuminator can generate highly polarized light. In some examples, it may be beneficial to block the laser light so that other light, such as fluorescent light, can be viewed. Typically, light from a laser illuminator is blocked using a spectrally specific filter. Using polarized light can be useful to assist in blocking the laser light, provided that the polarizer is at least partially effective at the laser wavelength. In some examples, a typical spectral filter can be configured to block polarized laser light and allow only fluorescent light to be viewed. A typical spectral filter can be configured to block polarized laser light outside the desired fluorescence range, allowing fluorescence to be viewed preferentially at specific wavelengths tuned to the fluorescent molecules. For example, using such a configuration, it is possible to obtain wavelengths of 650 to 850 nm, or 600 nm to about 850 nm, or about 700 nm to about 850 nm, about 800 nm to about 950 nm, about 800 nm to about 880 nm, about 775 nm to about 795 nm, or about 785 nm, 700 nm, 725 nm, 750 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm, 805 nm, The fluorescent molecules described herein can be visualized using a polarizing film 1101(a), including fluorescent molecules or moieties in the NIR spectrum at wavelengths between about 780 nm and about 790 nm, including 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 845 nm, 850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm, 895 nm, or 900 nm. For example, the first polarizing film 1101(a) can be configured to block polarized laser light having a wavelength between about 780 nm and about 790 nm, while allowing emitted light from the fluorescent molecules to pass with minimal attenuation. It will be understood that a variety of laser illuminators can be used within the system, including gas lasers, solid-state lasers, semiconductor lasers, chemical lasers, dye lasers, metal vapor lasers, or other types of lasers.It will be appreciated that the first polarizing film 1101(a) may be oriented such that different undesired polarizations from various laser illuminators may be blocked. Furthermore, by adjusting the orientation of the first polarizing film 1101(a), undesired polarizations may be blocked.

[0117] The first polarizing film 1101(a) and the second polarizing film 1101(b) may be configured to block highly polarized laser light while allowing fluorescent light to pass through the LC shutter 100, improving the signal-to-noise ratio of the resulting fluorescent image. In one aspect, the first polarizing film 1101(a) and the second polarizing film 1101(b) may be configured to be effective at the wavelength of light from the laser illuminator. In one example, the first polarizing film 1101(a) may have an effective range of 420-790 nm and therefore be configured to be effective in helping to block laser light from 780-790 nm.

[0118] FIG. 19 illustrates the use of a liquid crystal shutter assembly described herein configured to visualize a peptide-fluorophore conjugate (tozrelitide). In this example, the liquid crystal shutter assembly is configured to block highly polarized light from a laser illuminator using a first polarizing film 1101(a). The first polarizing film 1101(a) may be effective in the visible light spectrum 1900 and a portion of the NIR spectrum 1902. To block highly polarized laser light 1906 from the laser illuminator, the first polarizing film 1101(a) is oriented to attenuate the highly polarized laser light 1906 from the laser illuminator. The highly polarized laser light 1906 may have a wavelength between about 780 nm and about 790 nm. The first polarizing film 1101(a) may be configured to be effective up to about 800 nm and then lose effectiveness at wavelengths above 800 nm. Blocking the highly polarized laser light 1906 optimizes visualization of fluorescent molecules (e.g., peptide-fluorophore conjugates (tozrelitide)) at wavelengths of approximately 800 nm to 840 nm. Because the fluorescence 1908 is outside the light cutoff (e.g., effective range) of the polarizer, the fluorescence can be seen regardless of the state of the liquid crystal shutter assembly.

[0119] As shown in FIG. 19, visible light (e.g., wavelengths between approximately 420 nm and 700 nm) can be completely attenuated (e.g., blocked) by the liquid crystal shutter assembly in the closed state 406. In the open state 404, the liquid crystal shutter assembly can be configured to allow visible light to pass through the liquid crystal shutter assembly with approximately 50% attenuation, enabling visible light imaging. In the visible light spectrum 1900, undesired polarized light (e.g., ghost images from S-polarized light from a beamsplitter, as described herein) can be attenuated or blocked by orienting the first polarizing film 1101(a) of the first polarizer to block the undesired polarized light. Blocking or attenuating undesired polarized light during visible light imaging can result in clearer visible images (e.g., reducing shadows or ghost images).

[0120] Detection Method In another aspect, disclosed herein is a method for imaging a sample, the method including: emitting infrared or near-infrared light by a light source, which induces fluorescence from the sample; directing the infrared or near-infrared light toward the sample by a plurality of optical elements; receiving the fluorescence from the sample at a detector by a plurality of optical elements, where the infrared or near-infrared light is directed toward the sample substantially coaxially with the fluorescence received from the sample to reduce shadows; and forming a fluorescence image of the sample and a visible light image of the sample on the detector. In some embodiments, the method herein includes using an imaging system disclosed herein. In some embodiments, the sample is an organ, organ substructure, tissue, or cell. In some embodiments, the method of imaging an organ, organ substructure, tissue, or cell includes imaging the organ, organ substructure, tissue, or cell with an imaging system described herein. In some aspects, the method further includes detecting a cancerous or diseased region, tissue, structure, or cell. In some embodiments, the method further includes performing surgery on the subject. In some embodiments, the method further includes treating the cancer. In some embodiments, the method further includes removing the cancerous or diseased area, tissue, structure, or cell of the subject. In some embodiments, the method further includes imaging the cancerous or diseased area, tissue, structure, or cell of the subject after surgical removal. In some embodiments, the detection is performed using fluorescent imaging. In some embodiments, the fluorescent imaging detects the detectable agent, and the detectable agent comprises a dye, a fluorophore, a fluorescent biotin compound, a luminescent compound, or a chemiluminescent compound. In some embodiments, the detectable agent comprises any one or more fluorophores described herein. In some embodiments, the detectable agent comprises ICG or tozurelitide.

[0121] In another aspect, disclosed herein are methods of treating or detecting in a subject in need thereof, the method comprising administering a companion diagnostic, therapeutic, or imaging agent, wherein the companion diagnostic or imaging agent is detected by the systems and methods described herein. In another embodiment, the method of administering the companion diagnostic comprises any one of the various methods using the systems described herein. In another embodiment, the diagnostic or imaging agent comprises a chemical agent, a radiolabeled agent, a radiosensitizer, a fluorophore, an imaging agent, a photosensitizing agent, a sonosensitizing agent, a diagnostic agent, a protein, a peptide, a nanoparticle, or a small molecule. In another embodiment, the system incorporates radiology or fluorescence, including x-ray, magnetic resonance imaging (MRI), ultrasound, endoscopy, elastography, tactile imaging, thermography, flow cytometry, medical photography, nuclear medicine functional imaging techniques, positron emission tomography (PET), single-photon emission computed tomography (SPECT), surgical instruments, surgical microscopes, confocal microscopes, fluoroscopes, extracorporeal endoscopes, or surgical robots. In another embodiment, the systems and methods are used to detect therapeutic agents or evaluate the safety and physiological effects of drugs. In yet another embodiment, the safety and physiological effects detected by the systems and methods are to evaluate drug bioavailability, uptake, concentration, presence, distribution and clearance, metabolism, pharmacokinetics, localization, blood levels, tissue levels, ratios, blood and / or tissue concentration measurements, therapeutic window, range and optimization.

[0122] In various aspects, the imaging systems disclosed herein can be used in methods for imaging abnormal tissue, cancer, tumor, vasculature, or structures within a subject using a fluorophore. In one aspect, the method includes generating an image of the vasculature or structure by imaging fluorescence using an imaging system provided herein. In another aspect, the method includes generating an image of the abnormal tissue, cancer, tumor, vasculature, or structure by imaging fluorescence using an imaging system including the liquid crystal assembly systems and methods disclosed herein. In some examples, the imaged fluorescence can be autofluorescence, a contrast or imaging agent, a chemical agent, a radiolabeled agent, a radiosensitizer, a photosensitizing agent, a fluorophore, a therapeutic agent, an imaging agent, a diagnostic agent, a protein, a peptide, a nanoparticle, or a small molecule, or any combination thereof, or any combination thereof. In some examples, the method can further include administering the contrast or imaging agent to the subject. In some examples, the contrast or imaging agent can be a dye, a fluorophore, a fluorescent biotin compound, a luminescent compound, a chemiluminescent compound, or any combination thereof.

[0123] In various embodiments, the imaging agent further comprises a protein, a peptide, an amino acid, a nucleotide, a polynucleotide, or any combination thereof. In some examples, the imaging agent can be tozurelitide. In some examples, the imaging agent can absorb light at wavelengths between about 200 nm and about 900 nm. In some examples, the contrast or imaging agent is DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, or indocyanine green (ICG), and derivatives of any of the foregoing; fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanate or FITC, naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanines, merocyanines, styryl dyes, oxonol dyes, phylloerythrin, litrosin, eosin, rhodamine dyes (e.g., camcorders, These dyes include carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine (TMR), etc.; coumarins and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA)), etc.; Oregon Green dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514, etc.); Texas Red, Texas Red-X, Spectrum Red, Spectrum Green, and cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY-5.5, etc.), ALEXA FLUOR dyes (e.g., ALEXA FLUOR350, ALEXA FLUOR488, ALEXA FLUOR532, ALEXA FLUOR546, ALEXA FLUOR568, ALEXA FLUOR594, ALEXA FLUOR633, ALEXA FLUOR660, ALEXA FLUOR680, etc.), BODIPY dyes (e.g., BODIPYFL, BODIPYR6G, BODIPYTMR, BODIPYTR, BODIPY530 / 550, BODIPY558 / 568, BODIPY564 / 570, BODIPY576 / 589, BODIPY581 / 591, BODIPY630 / 650, BODIPY650 / 665, etc.), IRDye (e.g., IRD40, IRD700, IRD800, etc.), 7-aminocoumarin, dialkylaminocoumarin reactive dyes, 6,8-difluoro-7-hydro The dyes may include hydroxycoumarin fluorophores, hydroxycoumarin derivatives, alkoxycoumarin derivatives, succinimidyl esters, pyrene succinimidyl esters, pyridyloxazole derivatives, aminonaphthalene dyes, dansyl chloride, dapoxyl dyes, dapoxyl sulfonyl chloride, amine-reactive dapoxyl succinimidyl esters, carboxylic acid-reactive dapoxyl (2-aminoethyl) sulfonamides, bimane dyes, bimane mercaptoacetic acid, NBD dyes, QsY35, or any combination thereof.

[0124] In various aspects, administering the contrast or imaging agent to the subject can include intravenous administration, intramuscular administration, subcutaneous administration, intraocular administration, intraarterial administration, intraperitoneal administration, intratumoral administration, intradermal administration, or any combination thereof.

[0125] In another aspect, the imaging may include tissue imaging, ex vivo imaging, intraoperative imaging, or any combination thereof. In some examples, the sample may be an in vivo sample, an in situ sample, an ex vivo sample, or an intraoperative sample. In another example, the sample may be an organ, an organ substructure, a tissue, or a cell. In some examples, the sample may be autofluorescent or exhibit autofluorescence. In one example, the autofluorescence may include an ocular fluorophore, tryptophan, or a protein present in a tumor or malignant tumor. In some examples, the method may be used to visualize vascular flow or vascular patency.

[0126] In further embodiments, the abnormal tissue, cancer, tumor, vasculature, or structure may include a blood vessel, lymphatic, vasculature, neurovasculature, or CNS structure. In some examples, the imaging may be angiography, arteriography, lymphography, or cholangiography. In one example, the imaging may include detecting a vascular abnormality, a vascular malformation, a vascular lesion, an organ or organ substructure, a cancer, or an affected region, tissue, structure, or cell. In a further example, the vascular abnormality, vascular malformation, or vascular lesion may be an aneurysm, an arteriovenous malformation, a venous malformation, a lymphatic malformation, a capillary abnormality, a mixed vascular malformation, a spinal dural arteriovenous fistula, or a combination thereof.

[0127] In various aspects, the organ or organ substructure may be the brain, heart, lung, kidney, liver, breast, skin, or pancreas. In some embodiments, the method may include performing surgery on the subject. In one example, the surgery may include angioplasty, cardiovascular surgery, aneurysm repair, valve replacement, aneurysm surgery, arteriovenous or cavernous malformation surgery, venous malformation surgery, lymphatic malformation surgery, telangiectasia surgery, mixed vascular malformation surgery, or spinal dural arteriovenous fistula surgery, fistula surgery, repair or bypass, arterial bypass, organ transplant, plastic surgery, eye surgery, reproductive system surgery, stent insertion or replacement, plaque ablation, removal of a cancerous or diseased region, tissue, structure, or cell in the subject, or any combination thereof. In some examples, imaging may include imaging a vascular abnormality, cancerous or diseased region, tissue, structure, or cell in the subject after surgery. In some aspects, the method may include treating cancer in the subject.

[0128] In various aspects, methods include repair of intracranial CNS vascular defects, spinal CNS vascular defects, peripheral vascular defects, removal of abnormally vascularized tissue, eye imaging and repair, anastomosis, reconstructive or plastic surgery, plaque ablation or treatment or restenosis in atherosclerosis, repair or resection (including selective resection), preservation (including selective preservation), intraoperative identification and management (sometimes preservation, sometimes selective resection) of vital organs or structures such as nerves, kidneys, thyroid glands, parathyroid glands, liver segments or ureters, diagnosis and treatment of limb ischemia, or treatment of chronic wounds.

[0129] Adapting the LC assembly to the microscope In some embodiments, the imaging system herein is stereoscopic. In some embodiments, the imaging system herein is not stereoscopic. In some embodiments, the imaging system herein is a surgical microscope, a confocal microscope, a fluoroscope, an exoscope, an endoscope, or a surgical robot or an add-on to any of the above (such as an imaging head).

[0130] In some embodiments, at least one of the microscope, confocal microscope, fluoroscope, exoscope, surgical instrument, endoscope, or surgical robot is selected from the group consisting of a KINEVO system (e.g., KINEVO900), a QEVO system, a CONVIVO system, an OMPI PENTERO system (e.g., PENTERO900, PENTERO800), an INFRARED800 system, a FLOW800 system, a YELLOW560 system, a BLUE400 system, an OMPI LUMERIA system, an OMPI Vario system (e.g., OMPI Vario and OMPI Vario700), an OMPI Pico system, an OPMI Senra, an OPMI Movena, an OPMI 1 FC, an EXTARO300, a TREMON 3DHD system, a CIRRUS system (e.g., CIRRUS6000 and CIRRUS HD-OCT), a CLARUS system (e.g., CLARUS500 and CLARUS700), a PRIMUS200, a PLEX Elite9000, AngioPlex, VISUCAM524, VIUSCOUT100, ALTEVO800, (and any other surgical microscope, confocal microscope, fluoroscope, exoscope, endoscope, ophthalmoscope, retinal camera system, optical coherence tomography (OCT) system, and surgical robot system manufactured by Carl Zeiss A / G), PROVido system, ARVido system, GLOW800 system, Leica ARveo, Leica M530 system (e.g., Leica M530 OHX, Leica M530 OH6), Leica M720 system (e.g., Leica M720 OHX5), Leica M525 system (e.g., Leica M525 F50, Leica M525 F40, Leica M525 F20, Leica M525 OH4), Leica M844 system, Leica HD C100 system, Leica FL system (e.g., Leica FL560, Leica FL400, Leica FL800), Leica DIC500, Leica ULT500, Leica Rotating Beam Splitter, Leica M651 MSD, LIGHTENING, Leica TCS and SP8 systems (e.g., Leica TCS SP8, SP8FALCON, SP8 DIVE, Leica TCS SP8 STED, Leica TCS SP8 DLS, Leica TCS SP8 X, Leica TCS SP8 CARS, Leica TCS SPE), Leica HyD, Leica HCS A, Leica DCM8, Leica EnFocus, Leica Proveo 8, Leica Envisu C2300, Leica PROvido, and other surgical microscopes, confocal microscopes, fluoroscopes, exoscopes, endoscopes, ophthalmoscopes, retinal camera systems, OCT systems, and surgical robotic systems manufactured by Leica Microsystems or Leica Biosystems, Haag-Streit5-1000 system, Haag-Streit3-1000 system, Haag-Streit HI-R NEO900, Haag-Streit Allegra900, Haag-Streit Allegra90, Haag-Streit EIBOS2 and any other surgical microscope, confocal microscope, fluoroscope, exoscope, endoscope, and Haag-Streit surgical robot system, Intuitive Surgical da Vicinc surgical robot system and any other surgical microscope, confocal microscope, fluoroscope, exoscope, endoscope, ophthalmoscope, retinal camera system, OCT system, and Intuitive Surgical surgical robot system, Heidelberg Engineering Spectralis OCT system and any other surgical microscope, confocal microscope, fluoroscope, exoscope, endoscope, ophthalmoscope, retinal camera system, OCT system, and Heidelberg Engineering surgical robot system, Topcon 3D OCT2000, DRI OCT Triton, TRC system (e.g., TRC 50DX, TRC-NW8, TRC-NW8F, TRC-NW8F Plus, TRC-NW400), IMAGEnet Stingray system (e.g., Stingray, Stingray Pike, Stingray Nikon), IMAGEnet Pike system (e.g., Pike, PikeNikon), and any other surgical microscopes, confocal microscopes, fluoroscopes, exoscopes, endoscopes, ophthalmoscopes, retinal camera systems, OCT systems, and Topcon surgical robot systems, Canon CX-1, CR-2 AF, CR-2 PLUS AF, and any other surgical microscopes, confocal microscopes, fluoroscopes, exoscopes, endoscopes, ophthalmoscopes, retinal camera systems, OCT systems, and Canon surgical robot systems, Welch Allyn 3.5 V systems (e.g., 3.5V, 3.5V Autostep), CenterVue DRS, Insight, PanOptic, Retina Vue systems (e.g., Retina Vue100, Retina Vue700), Eelite, Binocular Indirect, PocketScope, Prestige coaxial-plus, and any other surgical microscopes, confocal microscopes, fluoroscopes, exoscopes, endoscopes, ophthalmoscopes, retinal camera systems, OCT systems, and Welch Allyn surgical robot systems, Metronic Includes the INVOS system and any other surgical microscope, confocal microscope, fluoroscope, exoscope, endoscope, ophthalmoscope, retinal camera system, OCT system, and surgical robotic system manufactured by Medtronic, Karl Storz ENDOCAMELEON, IMAGE1 system (e.g., IMAGE1 S, IMAGE1 S 3D, with or without the OPAL1 NIR imaging module), SILVER SCOPE series instruments (e.g., gastroscope, duodenoscope, colonoscope) and any other surgical microscope, confocal microscope, fluoroscope, exoscope, endoscope, ophthalmoscope, retinal camera system, OCT system, and surgical robotic system manufactured by Karl Storz, or any combination thereof.

[0131] The systems herein can be combined or integrated with existing surgical microscopes, confocal microscopes, fluoroscopes, endoscopes, endoscopes, or surgical robots by adapting or co-accommodating (in whole or in part) additional equipment, including imaging systems, or by combining or integrating one or more aspects or components of the disclosed systems into existing systems. Such combinations can enhance image clarity, sharpen image boundaries, enhance video rates and real-time imaging, reduce shadowing, motion effects, and / or ghosting, among other features of the systems and methods disclosed herein, to take advantage of improvements, enhance imaging, increase image clarity, optimize imaging, and improve surgical workflow. Additionally, such combinations or integrations can utilize LC shutter assemblies, LC cells, polarizers, toggling first and second voltages of the liquid crystal between open and closed states, light cutoff criteria, or any other feature of the systems disclosed herein, or any combination thereof.

[0132] The systems herein have been successfully adapted to one or more surgical microscopes, confocal microscopes, fluoroscopes, exoscopes, endoscopes, or surgical robots using two mating optical mounts, mounting hardware, and optical gaskets used to create a light-tight interface between the instruments to allow light to pass from the applicable existing device to the system described herein. For example, the systems herein have been successfully adapted to at least one KINEVO system (e.g., KINEVO900), OMPI PENTERO system (e.g., PENTERO900, PENTERO800) using such an interface, and the cloud could potentially be successfully adapted to other Zeiss systems or the Leica FL800, among others, using such an interface.

[0133] While the foregoing description sets forth particular embodiments, aspects, and examples, the subject matter of the present invention extends beyond the specifically disclosed embodiments or aspects to other alternative embodiments, aspects, and / or uses, as well as modifications and equivalents thereof. Accordingly, the scope of the appended claims is not limited by any of the specific embodiments or aspects 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 order and are not necessarily limited to any particular disclosed order. Various acts may be described as multiple separate acts performed in sequence, in a manner that is helpful for understanding particular embodiments. However, the order of description should not be construed to imply that these acts are order-dependent. Additionally, the structures, systems, and / or devices described herein may be embodied as integrated or separate components.

Claims

1. 1. A liquid crystal assembly for use with an imaging system having a light source, said assembly comprising: a polarizer disposed in the path of light from the light source, the polarizer configured to have a light cutoff outside its polarization range; a liquid crystal disposed in the path of light transmitted through the polarizer; a drive voltage source configured to apply a first voltage and a second voltage to the liquid crystal; The liquid crystal assembly.

2. 10. The liquid crystal assembly of claim 1, wherein the first and second voltages establish high and low electric fields across the liquid crystal.

3. 3. The liquid crystal assembly of claim 1, wherein the first voltage and the second voltage toggle the liquid crystal between an open state and a closed state.

4. 4. The liquid crystal assembly of claim 1, wherein application of one of the first voltage or the second voltage toggles the liquid crystal to the open state.

5. 5. The liquid crystal assembly of claim 1, wherein application of one of the first voltage or the second voltage toggles the liquid crystal to the closed state.

6. A liquid crystal assembly according to any one of claims 1 to 5, wherein the polariser is configured to be transparent above the light cut-off.

7. 7. A liquid crystal assembly according to any one of claims 1 to 6, wherein the light cutoff comprises the longest wavelength of a spectral range, and the polarizer is configured to be ineffective and transparent above the longest wavelength.

8. 8. The liquid crystal assembly of claim 1, wherein the liquid crystal comprises a twisted nematic (TN) cell, a Pi cell, a thermotropic liquid crystal, and a lyotropic liquid crystal, a liquid crystal display (LCD), a liquid crystal with photoconductor properties, or a liquid crystal that undergoes unfolding and alignment under an electric field.

9. 9. A liquid crystal assembly according to claim 1, wherein the second voltage induces a backflow in the liquid crystal.

10. A liquid crystal assembly according to any one of claims 1 to 9, wherein one of the first voltage or the second voltage is 0V.

11. 11. A liquid crystal assembly according to any one of claims 1 to 10, wherein the second voltage is a plurality of voltages including a bipolar square wave having an average value of 0V.

12. 12. A liquid crystal assembly according to claim 1, wherein the first voltage, or the second voltage, or both the first voltage and the second voltage comprise a plurality of voltages that create a bipolar square wave with an average value of 0V.

13. 13. A liquid crystal assembly according to any one of claims 11 to 12, wherein the bipolar square wave comprises an amplitude of + / -0.1 to + / -5 Vp-p, + / -3 to + / -30 Vp-p, + / -16 Vp-p, + / -24 Vp-p, or + / -30 Vp-p, or an amplitude between + / -16 V (32 Vp-p) and + / -30 V (60 Vp-p) inclusive, or an amplitude between + / -30 V (60 Vp-p) and + / -100 V (200 Vp-p) inclusive.

14. 14. A liquid crystal assembly according to claim 1, wherein the second voltage is a bipolar square wave having an amplitude of + / -0.1 to + / -5 Vp-p, + / -3 to + / -30 Vp-p, + / -16 Vp-p, + / -24 Vp-p, or + / -30 Vp-p, or an amplitude between + / -16 V (32 Vp-p) and + / -30 V (60 Vp-p) inclusive, or an amplitude between + / -30 V (60 Vp-p) and + / -100 V (200 Vp-p) inclusive.

15. A liquid crystal assembly according to any one of claims 11 to 14, wherein the bipolar square wave amplitude is modulated at the video capture rate of the imaging system.

16. A liquid crystal assembly according to any one of claims 13 to 15, wherein the amplitude of the bipolar square wave is modulated between 500 and 2000 Hz.

17. 17. A liquid crystal assembly according to any one of claims 1 to 16, wherein the drive voltage source toggles the liquid crystal between the first voltage and the second voltage for a period of 1 millisecond to 40 milliseconds inclusive, or 2 milliseconds to 60 milliseconds inclusive, 60 milliseconds to 100 milliseconds inclusive, and up to 300 milliseconds.

18. 18. The liquid crystal assembly of claim 1, wherein the drive voltage source toggles the liquid crystal between the first and second voltages at a video rate sampling period of between about 1 ms and 10 ms, between 2 ms and 20 ms, or 6 ms.

19. 19. A liquid crystal assembly according to any one of claims 1 to 18, wherein the liquid crystal is a twisted nematic (TN) liquid crystal.

20. A liquid crystal assembly according to any one of claims 1 to 19, wherein the liquid crystal is heated to a temperature in the range of 30°C to 50°C.

21. A liquid crystal assembly according to any preceding claim, wherein the liquid crystal is enclosed within a housing of the imaging system, and wherein the liquid crystal is heated by waste heat generated by other components of the imaging system.

22. A liquid crystal assembly according to any one of claims 4 to 21, wherein the rate of transition between the open and closed states is determined by the temperature of the liquid crystal and the voltage supply drive level.

23. 23. The liquid crystal assembly according to any one of claims 1 to 22, wherein the polarizer is an engineered dye polarizer.

24. A liquid crystal assembly according to any one of the preceding claims, wherein the polariser is configured to have a light cut-off in a range that is optimal for fluorescence imaging processes.

25. 25. A liquid crystal assembly according to any one of claims 1 to 24, wherein the polarizer is selected to lose effectiveness or cut off at 650 to 800 nm, or between 600 nm and about 800 nm, or between about 700 nm and about 800 nm, about 800 nm and about 950 nm, about 800 nm and about 880 nm, about 775 nm and about 795 nm, or about 785 nm.

26. 26. A liquid crystal assembly according to any one of claims 1 to 25, wherein the polarizer is selected to lose effectiveness or cut off at 700 nm, 725 nm, 750 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm, 805 nm, 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm, 895 nm, or 900 nm.

27. A liquid crystal assembly according to any one of claims 1 to 26, further comprising a beam splitter.

28. 28. The liquid crystal assembly of claim 27, wherein the beam splitter has a front surface and a rear surface.

29. 30. A liquid crystal assembly according to claim 28, wherein the front surface has a front surface coating and the rear surface has a rear surface coating.

30. 30. The liquid crystal assembly of claim 29, wherein the front coating has a higher P-polarized light reflection than the rear coating.

31. 31. The liquid crystal assembly of claim 30, wherein the front coating and the rear coating produce a P-polarized front to back reflectivity ratio of at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 12:1, at least 14:1, at least 16:1, at least 18:1, or at least 20:

1.

32. A liquid crystal assembly according to any one of claims 29 to 31, wherein S-polarized light reflections at the front and rear surfaces of the beam splitter are minimized by the front and rear coatings.

33. A liquid crystal assembly according to any one of the preceding claims, wherein the polarizer is configured to block or attenuate ghost or secondary reflections reflected from a beam splitter.

34. A liquid crystal assembly according to any one of claims 27 to 33, wherein the polarizer is configured to block S-polarized light from the beam splitter.

35. 35. The liquid crystal assembly of claims 27 to 34, wherein the polarizer is configured to allow P-polarized light to pass through the liquid crystal assembly to an imaging lens and / or camera when the liquid crystal is in the open state.

36. 36. The liquid crystal assembly of any one of claims 27 to 35, wherein the first polarizer allows a front to back reflectance ratio from the beam splitter of at least 2:1, 4:1, 6:1, 8:1, 10:1, 12:1, 15:1, 17:1, or 20:1 to pass through to an imaging lens and / or camera.

37. 37. The liquid crystal assembly of any one of claims 27 to 36, wherein the total reflectance from the beam splitter is about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, or about 3% or less.

38. A liquid crystal assembly according to any one of claims 1 to 37, further comprising two or more polarisers.

39. A liquid crystal assembly according to any one of claims 1 to 38, adapted for use in medical equipment including surgical microscopes, confocal microscopes, fluoroscopes, exoscopes, endoscopes or surgical robots.

40. 40. The liquid crystal assembly of claim 39, wherein the liquid crystal assembly is adapted to the medical device, optionally using an optical gasket.

41. The liquid crystal assembly according to claim 39 or 40, wherein the medical device is a KINEVO system (for example, KINEVO900), an OMPI PENTERO system (for example, PENTERO900, PENTERO800), or a Leica FL800 system.

42. A method for imaging radiation emitted by a fluorophore using a liquid crystal assembly according to any one of claims 1 to 41.

43. 1. A method for imaging emitted light by a fluorophore in an imaging system including a liquid crystal assembly, comprising: enabling or directing visible light toward the sample; directing excitation light at the sample; directing the emitted light and reflected visible light from the sample to the liquid crystal assembly, the liquid crystal assembly comprising: an optically engineered polarizer disposed in the path of the light from the sample, the polarizer configured to have an optical cutoff in the range of 600 nm to 900 nm; a liquid crystal disposed in the path of light transmitted through the polarizer; a drive voltage source configured to apply a first voltage and a second voltage to the liquid crystal, the first voltage and the second voltage to toggle the liquid crystal between an open state and a closed state; said directing comprising: directing the emitted light and the reflected visible light through the engineered polarizer, wherein the reflected visible light having wavelengths below the optical cutoff passes through the polarizer with approximately 50% attenuation and the emitted light having wavelengths above the optical cutoff passes through the polarizer with minimal attenuation; directing polarized visible light and unpolarized radiation at the liquid crystal; applying the first voltage to the liquid crystal to cause it to assume the open state during a first video rate sampling period; detecting the polarized light with an imaging sensor; applying the second voltage to the liquid crystal to cause it to assume the closed state and block the polarized light from passing to the image sensor; detecting the unpolarized radiation with the imaging sensor during the closed state for a second video rate sampling period; The method comprising:

44. 44. The method of claim 43, wherein the minimum attenuation of the emitted light having wavelengths above the optical cutoff is between 0% attenuation and 15% attenuation.

45. 45. The method of claim 43 or 44, wherein the reflected visible light having wavelengths below the optical cutoff passes through the polarizer with no more than about 50% attenuation.

46. The method of any one of claims 43 to 45, wherein the first video rate sampling period is between 1 ms and 10 ms, between 2 ms and 20 ms, or 6 ms.

47. A method according to any one of claims 43 to 46, wherein the second video rate sampling period is between 1 ms and 10 ms, between 2 ms and 20 ms, or 6 ms.

48. A method according to any one of claims 43 to 47, wherein the first and second voltages achieve high and low electric fields respectively across the liquid crystal.

49. The method of any one of claims 43 to 48, wherein the first voltage and the second voltage toggle the liquid crystal between an open state and a closed state.

50. 50. The method of any one of claims 43 to 49, wherein the first voltage toggles the liquid crystal to the open state and the second voltage toggles the liquid crystal to the closed state, or vice versa.

51. The method of any one of claims 43 to 50, wherein one of the first voltage or the second voltage is 0V.

52. A liquid crystal assembly according to any one of claims 43 to 51, wherein the second voltage is a plurality of voltages including a bipolar square wave having an average value of 0V.

53. 53. A liquid crystal assembly according to any one of claims 43 to 52, wherein the first voltage, or the second voltage, or both the first voltage and the second voltage comprise a plurality of voltages that create a bipolar square wave with an average value of 0V.

54. 54. The method of any one of claims 52 to 53, wherein the bipolar square wave has an amplitude of + / -0.1 to + / -5 Vp-p, + / -3 to + / -30 Vp-p, + / -16 Vp-p, + / -24 Vp-p, or + / -30 Vp-p, or an amplitude between (any value inclusive) + / -16 V (32 Vp-p) and + / -30 V (60 Vp-p), or an amplitude between (any value inclusive) + / -30 V (60 Vp-p) and + / -100 V (200 Vp-p).

55. 55. The method of any one of claims 52 to 54, wherein the second voltage is a bipolar square wave having an amplitude of + / -0.1 to + / -5 Vp-p, + / -3 to + / -30 Vp-p, + / -16 Vp-p, + / -24 Vp-p, or + / -30 Vp-p, or an amplitude between (any value inclusive) + / -16 V (32 Vp-p) and + / -30 V (60 Vp-p), or an amplitude between (any value inclusive) + / -30 V (60 Vp-p) and + / -100 V (200 Vp-p).

56. A method according to any one of claims 52 to 55, wherein the bipolar square wave amplitude is modulated at the video capture rate of the imaging system.

57. 57. The method of any one of claims 43 to 56, wherein the first voltage is a bipolar square wave having an amplitude of + / -0.1 to + / -5 Vp-p, + / -3 to + / -30 Vp-p, + / -16 Vp-p, + / -24 Vp-p, or + / -30 Vp-p, or an amplitude between (any value inclusive) + / -16 V (32 Vp-p) and + / -30 V (60 Vp-p), or an amplitude between (any value inclusive) + / -30 V (60 Vp-p) and + / -100 V (200 Vp-p).

58. 58. A method according to any one of claims 54 to 57, wherein the amplitude is modulated from 500 to 2000 Hz.

59. Heating the liquid crystal using waste heat from the imaging system.

59. The method of any one of claims 43 to 58, further comprising:

60. The method of any one of claims 43 to 59, wherein the polarizer is a dye-based polarizer.

61. 61. The method of any one of claims 43 to 60, wherein the polarizer is configured to have a light cutoff in a range optimal for fluorescence imaging processes.

62. 62. The method of any one of claims 43 to 61, wherein the polarizer in the liquid crystal assembly is configured to have a light cutoff in the range of about 600 nm to about 850 nm, or about 700 nm to about 850 nm, about 800 nm to about 950 nm, about 800 nm to about 880 nm, about 775 nm to about 795 nm, or about 785 nm.

63. 63. The method of any one of claims 43 to 62, wherein the polarizer in the liquid crystal assembly is selected to lose effectiveness or cut off at 700 nm, 725 nm, 750 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm, 805 nm, 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm, 895 nm, or 900 nm.

64. The method of any one of claims 43 to 63, wherein the polarizer in the liquid crystal assembly further comprises two or more polarizers.

65. 65. The method of any one of claims 43 to 64, wherein the excitation light is engineered to enhance the signal to noise ratio (SNR) of the NIR image such that the excitation light produces controlled energy excitation where the peak intensity and duration of the excitation light is below a safety threshold.

66. 66. The method of any one of claims 43 to 65, further comprising directing the emitted light and the reflected visible light towards a beam splitter before directing the emitted light and the reflected light towards the liquid crystal assembly.

67. 67. The method of claim 66, wherein the beam splitter has a front surface and a rear surface.

68. 68. The method of claim 67, wherein the front surface has a front surface coating and the rear surface has a rear surface coating.

69. 69. The method of claim 68, wherein the front surface coating has a higher P-polarized reflection than the back surface coating.

70. 70. The method of claim 69, wherein the front and back coatings produce a P-polarized front to back reflectivity ratio of at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 12:1, at least 14:1, at least 16:1, at least 18:1, or at least 20:

1.

71. 71. The method of any one of claims 68 to 70, wherein S-polarized reflections at the front and rear surfaces of the beamsplitter are minimized by the front and rear coatings.

72. A method according to any one of claims 43 to 71, wherein the polariser is configured to block or attenuate ghost or secondary reflections reflected from a beamsplitter.

73. A method according to any one of claims 66 to 72, wherein the polariser is configured to block S-polarised light from the beam splitter.

74. 74. A liquid crystal assembly according to any one of claims 66 to 73, wherein the polarizer is configured to allow P-polarized light to pass through the liquid crystal assembly to an imaging lens or camera when the liquid crystal is in the open state.

75. 75. The method of any one of claims 66 to 74, wherein the polarizer allows a front to back reflectance ratio from the beamsplitter of at least 2:1, 4:1, 6:1, 8:1, 10:1, 12:1, 15:1, 17:1, or 20:1 to pass through to the imaging lens and camera.

76. 76. The method of any one of claims 66-75, wherein the total reflectance from the beamsplitter is about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, or about 3% or less.

77. A liquid crystal assembly including any of the features described, either individually or in combination with any other feature, in any configuration disclosed herein.

78. A method for imaging emitted light emitted by a fluorophore in an imaging system comprising a liquid crystal assembly including any of the features described, either individually or in combination with any other feature, in any configuration disclosed herein.

79. 1. A liquid crystal assembly for use with an imaging system having a light source for providing excitation light, said assembly comprising: a first polarizer disposed in the path of light from the light source, the first polarizer configured to have a light cutoff outside its polarization range; a liquid crystal disposed in the path of light transmitted through the first polarizer; a second polarizer disposed in the path of light transmitted through the liquid crystal, the second polarizer configured to have a light cutoff outside its polarization range; a drive voltage source configured to apply a first voltage and a second voltage to the liquid crystal, the first voltage and the second voltage to toggle the liquid crystal between an open state and a closed state; The liquid crystal assembly.

80. 80. The liquid crystal assembly of claim 79, wherein the first polarizer and the second polarizer are cross-polarized.

81. 81. A liquid crystal assembly according to claim 79 or claim 80, wherein the liquid crystal is in the open state when one of the first voltage or the second voltage is applied across the liquid crystal.

82. A liquid crystal assembly according to any one of claims 79 to 81, wherein one of the first voltage or the second voltage is 0V.

83. A liquid crystal assembly according to any one of claims 79 to 82, wherein the liquid crystal is in the closed state when one of the first voltage or the second voltage is applied to the liquid crystal.

84. A liquid crystal assembly according to any one of claims 79 to 83, wherein the second voltage is a bipolar square wave.

85. 85. The liquid crystal assembly of claim 84, wherein the bipolar square wave has an amplitude of + / -0.1 to + / -5 Vp-p, + / -3 to + / -30 Vp-p, + / -16 Vp-p, + / -24 Vp-p, or + / -30 Vp-p.

86. The liquid crystal assembly an imaging lens in the path of light transmitted through the second polarizer; a camera in the path of light transmitted through the imaging lens; 86. A liquid crystal assembly according to any one of claims 79 to 85, comprising:

87. 87. The liquid crystal assembly of claim 86, wherein when the liquid crystal is in the open state, the liquid crystal rotates the polarization of polarized visible light transmitted through the first polarizer to allow the polarized visible light to pass through the second polarizer to the imaging lens with no more than about 50% attenuation.

88. 88. The liquid crystal assembly of claim 87, wherein when the liquid crystal is in the closed state, the liquid crystal does not rotate the polarization of the polarized visible light, and the polarized visible light transmitted through the first polarizer is blocked by the second polarizer.

89. 89. A liquid crystal assembly according to any one of claims 79 to 88, wherein the light cut-off ranges of the first polarizer and the second polarizer are where the polarizer extinction ratio is insufficient or minimal.

90. 90. The liquid crystal assembly of any one of claims 86 to 89, wherein light outside the light cutoff ranges of the first polarizer and the second polarizer passes through the first polarizer and the second polarizer with no or minimal attenuation to reach the imaging lens.

91. 91. A liquid crystal assembly according to claim 90, wherein the minimum attenuation is 15% or less.

92. 92. The liquid crystal assembly of any one of claims 79 to 91, wherein the light cutoff range of the first polarizer is from about 700 nm to about 800 nm, or about 700 nm, 725 nm, 750 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm, 805 nm, 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm, 895 nm, or 900 nm.

93. 93. A liquid crystal assembly according to any one of claims 79 to 92, wherein the light cutoff range of the second polarizer is from about 700 nm to about 800 nm, or about 700 nm, 725 nm, 750 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm, 805 nm, 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm, 895 nm, or 900 nm.

94. 94. A liquid crystal assembly according to any one of claims 79 to 93, wherein the light cut-off ranges of the first polarizer and the second polarizer are greater than the longest wavelength of the visible light spectrum.

95. A liquid crystal assembly according to any one of claims 79 to 94, wherein the liquid crystal assembly is configured to view visible light when the liquid crystal is in the open state.

96. A liquid crystal assembly according to any one of claims 79 to 95, wherein the liquid crystal assembly is configured to block visible light when the liquid crystal is in the closed state.

97. 97. The liquid crystal assembly of claim 96, wherein the blocked visible light allows fluorescence imaging from a fluorophore.

98. 98. The liquid crystal assembly of claim 97, wherein the fluorophore provides the emitted light emitted by the fluorophore.

99. 99. A liquid crystal assembly according to claim 97 or 98, wherein the excitation light is white light, NIR light, IR light, or any other type of excitation light.

100. A liquid crystal assembly according to any one of claims 79 to 99, wherein the excitation light is near-infrared light provided by a laser diode.

101. A liquid crystal assembly according to any one of claims 79 to 100, further comprising a beam splitter.

102. 102. The liquid crystal assembly of claim 101, wherein the beam splitter has a front surface and a rear surface.

103. 103. A liquid crystal assembly according to claim 102, wherein the front surface has a front surface coating and the rear surface has a rear surface coating.

104. 104. The liquid crystal assembly of claim 103, wherein the front coating has a higher P-polarized light reflection than the rear coating.

105. 105. A liquid crystal assembly according to claim 103 or 104, wherein the front coating and the rear coating produce a P-polarized front to back reflectivity ratio of at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 12:1, at least 14:1, at least 16:1, at least 18:1, or at least 20:

1.

106. A liquid crystal assembly according to any one of claims 103 to 105, wherein S-polarized light reflections at the front and rear surfaces of the beam splitter are minimized by the front and rear coatings.

107. A liquid crystal assembly according to any one of claims 79 to 106, wherein the first polarizer is configured to block or attenuate ghost or secondary reflections reflected from a beam splitter.

108. A liquid crystal assembly according to any one of claims 101 to 107, wherein the first polarizer is configured to block S-polarized light from the beam splitter.

109. A liquid crystal assembly as described in claims 101 to 108, wherein the first polarizer and the second polarizer are configured to allow P-polarized light to pass through the liquid crystal assembly to an imaging lens or camera when the liquid crystal is in the open state.

110. 110. The liquid crystal assembly of any one of claims 101 to 109, wherein the first polarizer allows a front to back reflectance ratio from the beam splitter of at least 2:1, 4:1, 6:1, 8:1, 10:1, 12:1, 15:1, 17:1, or 20:1 to pass through to the imaging lens and camera.

111. 111. The liquid crystal assembly of any one of claims 101 to 110, wherein the total reflectance from the beam splitter is about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, or about 3% or less.

112. 87. The liquid crystal assembly of claim 86, wherein the imaging lens and the camera are configured to view fluorescence images using a video rate sampling period of 1 ms to 10 ms, 2 ms to 20 ms, or 6 ms.

113. 113. The liquid crystal assembly of claim 112, wherein the fluorescent image has reduced motion blur.

114. 1. A method for imaging emitted light by a fluorophore in an imaging system including a liquid crystal assembly, comprising: enabling or directing visible light toward the sample; directing excitation light at the sample; directing the emitted light and the reflected visible light to the liquid crystal assembly, the liquid crystal assembly comprising: a first engineered polarizer in the path of light from the sample, the first engineered polarizer configured to have an optical cutoff in the range of 600 nm to 900 nm, or 800 nm to 880 nm; a liquid crystal disposed in the path of light transmitted through the first engineered polarizer; a second engineered polarizer in the path of light transmitted through the liquid crystal, the second engineered polarizer configured to have a light cutoff in the range of 600 nm to 900 nm, or 800 nm to 880 nm, and the first engineered polarizer and the second engineered polarizer being cross-polarized; a drive voltage source configured to apply a first voltage and a second voltage to the liquid crystal, the first voltage and the second voltage to toggle the liquid crystal between an open state and a closed state; said directing comprising: directing the emitted light and the reflected visible light through the first engineered polarizer, the first engineered polarizer polarizing the reflected visible light below the light cutoff range to produce polarized reflected visible light; directing the emitted light and the polarized reflected visible light through the liquid crystal; applying the first voltage to the liquid crystal to cause it to assume the open state during a first video rate sampling period; using the liquid crystal in the open state to rotate the polarization of the polarized reflected visible light; directing the emitted light and the polarized reflected visible light through the second engineered polarizer, the second engineered polarizer allowing the polarized reflected visible light to pass through the second engineered polarizer; detecting, with an imaging sensor, a first portion of the polarized reflected visible light that passes through the second engineered polarizer during the open state; applying the second voltage to the liquid crystal to cause it to assume the closed state during a second video rate sampling period; passing the emitted light and the polarized reflected visible light through the liquid crystal without rotating the emitted light and the polarized reflected visible light; blocking the polarized reflected visible light with the second engineered polarizer; detecting a portion of the emitted light with the imaging sensor during the closed state; The method comprising:

115. 115. The method of claim 114, wherein the method further comprises using the first polarizer to block secondary reflections of the reflected visible light.

116. 116. The method of claim 114 or 115, wherein the excitation light is provided to the sample via a laser diode.

117. turning off the laser diode to cease providing excitation light; providing the second voltage to the liquid crystal to cause it to assume the closed state during a third video rate sampling period; detecting a dark background image of the sample; subtracting the dark background image of the sample from the detected portion of the emitted light to generate a fluorescence image; 117. The method of claim 116, further comprising:

118. 118. The method of any one of claims 114 to 117, wherein the first video rate sampling period is between about 2 ms and about 10 ms.

119. A method according to any one of claims 114 to 118, wherein the second video rate sampling period is between about 8 ms and about 16 ms.

120. 118. The method of claim 117, wherein the third video rate sampling period is from about 8 ms to about 16 ms.

121. 121. The method of any one of claims 114 to 120, wherein one of the first voltage or the second voltage is about 0V.

122. A method according to any one of claims 114 to 121, wherein the second voltage is a bipolar square wave.

123. 123. The method of claim 122, wherein the bipolar square wave has an amplitude of + / -0.1 to + / -5 Vp-p, + / -3 to + / -30 Vp-p, + / -16 Vp-p, + / -24 Vp-p, or + / -30 Vp-p, or an amplitude between (any value inclusive) + / -16 V (32 Vp-p) and + / -30 V (60 Vp-p), or an amplitude between (any value inclusive) + / -30 V (60 Vp-p) and + / -100 V (200 Vp-p).

124. 118. The method of claim 117, wherein the fluorescence image has reduced motion blur.

125. The method of any one of claims 114 to 124, wherein the second and third video rate sampling periods allow post-processing with digital gain.

126. 126. The method of any one of claims 114 to 125, the method further comprising orienting the first engineered polarizer to block undesired polarized light.

127. 118. The method of claim 117, wherein the second and third video-rate sampling periods enhance the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of the fluorescence image.

128. 128. The method of claim 127, wherein the enhanced CNR enables a faster frame rate.

129. 129. The method of any one of claims 114 to 128, wherein the method provides a real-time display of the reflected visible light and fluorescent images.

130. 130. The method of any one of claims 114 to 129, further comprising directing the emitted light and the reflected light towards a beam splitter before directing the emitted light and the reflected light towards the liquid crystal assembly.

131. 131. The method of claim 130, wherein the beam splitter has a front surface and a rear surface.

132. 132. The method of claim 131, wherein the front surface has a front surface coating and the rear surface has a rear surface coating.

133. 133. The method of claim 132, wherein the front surface coating has a higher P-polarized reflection than the back surface coating.

134. 134. The method of claim 132 or 133, wherein the front and back coatings produce a P-polarized front to back reflectivity ratio of at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 12:1, at least 14:1, at least 16:1, at least 18:1, or at least 20:

1.

135. A method according to any one of claims 132 to 134, wherein S-polarized light reflections at the front and rear surfaces of the beamsplitter are minimized by the front and rear coatings.

136. 136. The method of any one of claims 114 to 135, wherein the first polarizer is configured to block or attenuate ghost or secondary reflections reflected from a beam splitter.

137. A method according to any one of claims 130 to 136, wherein the first polarizer is configured to block S-polarised light from the beam splitter.

138. 138. The method of any one of claims 130 to 137, wherein the first polarizer and the second polarizer are configured to allow P-polarized light to pass through the liquid crystal assembly to an imaging lens and / or camera when the liquid crystal is in the open state.

139. 139. The method of any one of claims 130-138, wherein the first polarizer allows a front to back reflectance ratio from the beamsplitter of at least 2:1, 4:1, 6:1, 8:1, 10:1, 12:1, 15:1, 17:1, or 20:1 to pass through to the imaging lens and camera.

140. 140. The method of any one of claims 130-139, wherein the total reflectance from the beamsplitter is about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, or about 3% or less.

141. 106. A method of imaging abnormal tissue, cancer, tumor, vasculature, or structure in a sample from a subject, the method comprising generating an image of the vasculature or structure by imaging fluorescence using an imaging system according to any one of claims 1 to 42 or claims 79 to 106.

142. 141. A method of imaging abnormal tissue, cancer, tumor, vasculature, or structure in a sample from a subject according to the method of any one of claims 43-78 or 107-140, comprising imaging fluorescence using an imaging system including a liquid crystal assembly to generate an image of the abnormal tissue, cancer, tumor, vasculature, or structure.

143. 143. The method of claim 141 or 142, wherein the imaged fluorescence is autofluorescence, a contrast or imaging agent, a chemical agent, a radiolabeled agent, a radiosensitizer, a photosensitizer, a fluorophore, a therapeutic agent, an imaging agent, a diagnostic agent, a protein, a peptide, a nanoparticle, or a small molecule, or any combination thereof, or any combination thereof.

144. 144. The method of any one of claims 141 to 143, wherein the imaged fluorescent agent is autofluorescence, a contrast or imaging agent, a chemical agent, a radiolabeled agent, a radiosensitizer, a photosensitizer, a fluorophore, a therapeutic agent, an imaging agent, a diagnostic agent, a protein, a peptide, a nanoparticle, or a small molecule, or any combination thereof.

145. 145. The method of any one of claims 141 to 144, wherein the method further comprises administering a contrast or imaging agent to the subject.

146. 106. A method of imaging abnormal tissue, cancer, tumor, vasculature, or structure in a fluorophore from a subject using the imaging system of any one of claims 1-42 or 79-106, comprising: (a) administering a contrast or imaging agent to the subject; (b) imaging the contrast or imaging agent using an imaging system to generate an image of the abnormal tissue, cancer, tumor, vasculature, or structure; The method comprising:

147. 140. A method of imaging abnormal tissue, cancer, tumor, vasculature, or structure within a fluorophore from a subject according to the method of any one of claims 43-78 or 107-140, comprising: (a) administering a contrast or imaging agent to the subject; (b) imaging the contrast or imaging agent using an imaging system to generate an image of the abnormal tissue, cancer, tumor, vasculature, or structure; The method further comprises:

148. 148. The method of claim 146 or 147, wherein the contrast or imaging agent comprises a dye, a fluorophore, a fluorescent biotin compound, a luminescent compound, a chemiluminescent compound, or any combination thereof.

149. The method of claims 146-148, wherein the contrast or imaging agent further comprises a protein, a peptide, an amino acid, a nucleotide, a polynucleotide, or any combination thereof.

150. The method of claims 146-149, wherein the contrast or imaging agent further comprises tozurelitide.

151. 151. The method of any one of claims 146 to 150, wherein the contrast or imaging agent absorbs at wavelengths from about 200 nm to about 900 nm.

152. The contrast or imaging agent may be selected from the group consisting of DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, or indocyanine green (ICG), and derivatives of any of the foregoing; fluorescein and fluorescein dyes (such as fluorescein isothiocyanate or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM), carbocyanines, merocyanines, styryl dyes, oxonol dyes, phylloerythrin, litrosin, eosin, rhodamine dyes (such as carboxytetramethyl fluorescein, ... chiral rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine (TMR), etc.), coumarin, coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA)), etc.), Oregon green dyes (e.g., Oregon green 488, Oregon green 500, Oregon green 514, etc.), Texas red, Texas red-X, spectrum red, spectrum green, cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY-5.5, etc.), ALEXA FLUOR dyes (e.g., ALEXA FLUOR350, ALEXA FLUOR488, ALEXA FLUOR532, ALEXA FLUOR546, ALEXA FLUOR568, ALEXA FLUOR594, ALEXA FLUOR633, ALEXA FLUOR660, ALEXA FLUOR680, etc.), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, etc.), IRDye (e.g., IRD40, IRD700, IRD800, etc.), 7-aminocoumarin, dialkylaminocoumarin reactive dyes, 6,The method of any one of claims 146 to 151, comprising an 8-difluoro-7-hydroxycoumarin fluorophore, a hydroxycoumarin derivative, an alkoxycoumarin derivative, a succinimidyl ester, a pyrene succinimidyl ester, a pyridyloxazole derivative, an aminonaphthalene dye, dansyl chloride, a dapoxyl dye, a dapoxyl sulfonyl chloride, an amine-reactive dapoxyl succinimidyl ester, a carboxylic acid-reactive dapoxyl (2-aminoethyl) sulfonamide), a bimane dye, a bimane mercaptoacetic acid, an NBD dye, QsY35, or any combination thereof.

153. 153. The method of any one of claims 146-152, wherein said administering comprises intravenous administration, intramuscular administration, subcutaneous administration, intraocular administration, intraarterial administration, intraperitoneal administration, intratumoral administration, intradermal administration, or any combination thereof.

154. 154. The method of any one of claims 146 to 153, wherein the imaging comprises tissue imaging, ex vivo imaging, intraoperative imaging, or any combination thereof.

155. 155. The method of any one of claims 146 to 154, wherein the sample is an in vivo sample, an in situ sample, an ex vivo sample, or an intraoperative sample.

156. 156. The method of any one of claims 146 to 155, wherein the sample is an organ, organ substructure, tissue, or cell.

157. 157. The method of any one of claims 146 to 156, wherein the sample is autofluorescent.

158. 158. The method of claim 157, wherein the autofluorescence of the sample comprises an ocular fluorophore, tryptophan, or protein present within a tumor or malignant tumor.

159. 159. The method of any one of claims 146 to 158, wherein the method is used to visualize vascular flow or vascular patency.

160. 160. The method of any one of claims 146-159, wherein the abnormal tissue, cancer, tumor, vasculature or structure comprises a blood vessel, lymphatic vasculature, neurovasculature, or CNS structure.

161. 161. The method of any one of claims 146 to 160, wherein the imaging is angiography, arteriography, lymphography, or cholangiography.

162. 162. The method of any one of claims 146 to 161, wherein said imaging comprises detecting vascular abnormalities, vascular malformations, vascular lesions, organs or organ substructures, cancer or diseased areas, tissues, structures or cells.

163. 163. The method of claim 162, wherein the vascular abnormality, vascular malformation, or vascular lesion is an aneurysm, arteriovenous malformation, cavernous malformation, venous malformation, lymphatic malformation, telangiectasia, mixed vascular malformation, spinal dural arteriovenous fistula, or a combination thereof.

164. 164. The method of any one of claims 146 to 163, wherein the organ or organ substructure is the brain, heart, lung, kidney, liver, or pancreas.

165. 165. The method of any one of claims 146 to 164, further comprising performing surgery on the subject.

166. 166. The method of any one of claims 146-165, wherein the surgery comprises angioplasty, cardiovascular surgery, aneurysm repair surgery, valve replacement surgery, aneurysm surgery, arteriovenous or cavernous malformation surgery, venous malformation surgery, lymphatic malformation surgery, telangiectasia surgery, mixed vascular malformation surgery, or spinal dural arteriovenous fistula surgery, fistula surgery, repair or bypass, arterial bypass, organ transplant, plastic surgery, eye surgery, reproductive system surgery, stent insertion or replacement, plaque ablation, removal of cancerous or diseased areas, tissues, structures or cells in a subject, or any combination thereof.

167. 167. The method of any one of claims 146-166, wherein said imaging comprises imaging vascular abnormalities, cancerous or diseased areas, tissues, structures, or cells of said subject after surgery.

168. 168. The method of any one of claims 146-167, further comprising treating the subject for cancer.

169. 169. The method of any one of claims 146 to 168, comprising repair of intracranial CNS vascular defects, spinal CNS vascular defects, peripheral vascular defects, removal of abnormally vascularized tissue, eye imaging and repair, anastomosis, reconstructive or plastic surgery, plaque ablation or treatment or restenosis in atherosclerosis, repair or resection (including selective resection), preservation (including selective preservation), intraoperative identification and management (sometimes preserving, sometimes selective resection) of vital organs or structures such as nerves, kidneys, thyroid glands, parathyroid glands, liver segments or ureters, diagnosis and treatment of limb ischemia, or treatment of chronic wounds.

Images