High-throughput snapshot spectral encoding device for fluorescence spectral microscopy

Abstract

Systems and methods for multispectral or hyperspectral fluorescence imaging are provided. In one example, a spectral encoding device can be positioned in a detection optical path between a microscope's detection objective and an imaging sensor. In one example, the spectral encoding device includes a first dichroic mirror with a sine transmittance profile and a second dichroic mirror with a cosine transmittance profile. In addition to collecting transmitted light, reflected light from each dichroic mirror is collected and used for total intensity normalization and image analysis. TIFF2023517677000008.tif120170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 62 / 989,493, filed March 13, 2020, entitled HIGH THROUGHPUT SNAPSHOT SPECTRAL ENCODING DEVICE FOR FLUORESCENCE SPECTRAL MICROSCOPY, the contents of which are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No. W81XWH-16-1-0253 awarded by the Department of Defense. The government has certain rights in this invention.

[0003] Field FIELD OF THE INVENTION The present disclosure generally relates to systems and methods for spectral fluorescence imaging. [Background technology]

[0004] background The following description contains information that may be helpful in understanding the present invention. No admission is made that any of the information provided herein is or constitutes prior art to the claimed invention, or that any publication specifically or implicitly cited is prior art.

[0005] Spectral fluorescence imaging can overcome signal overcrowding in molecules, cells, and tissues. Multicolor acquisition by multi- or hyperspectral imaging exists in laser scanning or wide-field configurations. The spatial or spectral scanning mechanisms required in standard configurations limit imaging speed and efficiency. To improve temporal resolution, snapshot methods have been developed for wide-field microscopy. However, low light throughput remains an unresolved challenge for multicolor fluorescence microscopy. Summary of the Invention

[0006] overview Spectral fluorescence imaging (SFI) has gained popularity in the life sciences due to its multiplexing capabilities. In this technique, the acquisition dimension of each pixel in an image is extended to the spectral domain. This type of detection can be realized as a single-point scanning microscope by combining a dispersive grating and a detector array. However, point-scanning detection methods generally suffer from low optical efficiency, slow imaging speed, and high phototoxicity, making them difficult to apply to multiplexed live imaging of light-sensitive samples.

[0007] Another technique for fluorescence snapshot imaging is selective plane illumination microscopy (SPIM). SPIM utilizes multiple objective lenses arranged in orthogonal directions to decouple the excitation and detection paths, generating a thin light sheet that reduces unwanted excitation, photobleaching, and phototoxicity of fluorophores. Higher imaging efficiency leads to improved ability to perform long-term volumetric imaging of large samples with high resolution. However, the ability to multiplex fluorescence signals in snapshot live microscopy is limited by the complexity of acquiring 3D spectral datasets (x, y, wavelength) on a 2D camera sensor. Most SPIM systems sequentially acquire multiple fluorescence signals or optical sections using bandpass filters. Sequential imaging limits temporal resolution and increases phototoxicity due to the increased acquisition time and light dose for each additional color.

[0008] In some approaches, the combination of image mapping spectroscopy (IMS) and SPIM overcomes the temporal resolution challenge by capturing a spectral dataset using a single snapshot, but still compromises light throughput efficiency. This reduced efficiency, combined with the characteristically low intensity of the fluorescent signal, has limited the widespread use of snapshot techniques in fluorescence microscopy.

[0009] Spectral phasor analysis can be used to process multi- or hyperspectral fluorescence data sets. Spectral phasors use sine and cosine Fourier transforms to convert high-dimensional spectral information into a 2D phasor plane, effectively simplifying the complexity of high-dimensional multiplexing. Dimensionality reduction also facilitates noise reduction.

[0010] One exemplary approach for spectral phasor analysis is shown in U.S. Patent Application Publication No. 2020 / 0378830 by Gratton et al., in which images are acquired sequentially using two sine / cosine color filters. Signal multiplexing with phasor analysis is applied to the acquired data.

[0011] The present inventors have identified numerous drawbacks associated with the above-described filter-based scanning techniques. For example, sequential mechanical filter switching requires multiple exposures, limiting imaging speed and temporal resolution. Furthermore, imaging speed is challenging for video high-speed imaging applications, such as multicolor imaging of beating zebrafish embryonic hearts, or for large, tiled volumetric imaging, such as single-cell resolution sections of tissue. Furthermore, the use of sinusoidal transmission filters results in fluorescence signal loss due to absorption. As a result, a significant amount of information, which would be useful for signal multiplexing and potentially beneficial under high-speed or low SNR conditions, is lost.

[0012] Some of the above-identified shortcomings may be at least partially addressed by an imaging assembly that includes a first dichroic mirror and a second dichroic mirror, where the first dichroic mirror's first spectral transmittance and reflectance curves have sine wave profiles and the second dichroic mirror's second spectral transmittance and reflectance curves have cosine wave profiles. By utilizing the first dichroic mirror and the second dichroic mirror, spectral encoding is integrated into the acquisition process; further, the dichroic mirrors enable simultaneous collection of transmitted and reflected light, which provides spectral information that can be used for multiplexing that would be lost with filter-based approaches.

[0013] As an example, a spectral encoding device including two sinusoidal dichroic mirrors is used as the encoding device. The spectral encoding device projects multiple spectrally encoded channels onto a single camera sensor in a single exposure acquisition. For example, each dichroic mirror transmits a portion of the fluorescent signal from the sample, thus generating a spectrally encoded transmitted channel. The remaining portion of the fluorescent signal is reflected and also captured by the spectral encoding device, thus each dichroic mirror also generates a spectrally encoded reflected channel. Thus, when two dichroic mirrors are used, four channels are generated and captured.

[0014] The dichroic mirror optically converts the spectral information into sine and cosine Fourier coefficients. The reflected antisine and anticosine portions are also recycled, detected, and used as intensity normalization factors for the phasor components (e.g., sine and cosine intensity images). This approach increases light throughput (e.g., a more than 80% increase for five commonly available fluorophores), thereby enhancing detection efficiency and temporal resolution and reducing phototoxicity. Furthermore, mechanical filter switching is unnecessary, and all spectral information is simultaneously captured by the transmitted and reflected portions, which greatly improves temporal resolution and thereby facilitates frame multiplexing during in vivo imaging to capture cellular dynamics.

[0015] These and other advantages and features of the present specification will become readily apparent from the following Detailed Description, read alone or in conjunction with the accompanying drawings. It should be understood that the foregoing Summary is provided to introduce a selection of concepts in a simplified form that are further described in the Detailed Description. It is not intended to identify key or essential features of the claimed subject matter, the scope of which is defined solely by the claims that follow the Detailed Description. Moreover, the claimed subject matter is not limited to embodiments that eliminate any disadvantages noted above or in any part of this disclosure. [Brief explanation of the drawings]

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the present invention and, together with the detailed description, serve to explain and illustrate the principles of the invention. The drawings are intended to diagrammatically show major features of exemplary embodiments. The drawings are not intended to depict every feature of the actual embodiment or the relative dimensions of the depicted elements, and are not drawn to scale.

[0017] [Figure 1A] 1 illustrates an exemplary overview of a microscope including a spectral encoding device according to aspects of the present disclosure. [Figure 1B]2 shows a schematic diagram of a portion of the spectral encoding device of FIG. 1 according to an embodiment of the present disclosure. [Figure 2A] 1 shows a schematic diagram of optical components of a spectral encoding device according to an aspect of the present disclosure. [Figure 2B] 2B shows a schematic diagram of simplified optical components of the spectral encoding device of FIG. 2A according to an embodiment of the present disclosure. [Figure 2C] 2C and 2D show example graphs of transmittance and reflectance curves of sine and cosine dichroic mirrors implemented in a spectral encoding device according to embodiments of the present disclosure. [Figure 2D] See legend to Figure 2C. [Figure 2E] 1 shows the overall transmission efficiency of a spectral encoding device including a dichroic mirror compared to the transmission efficiency of a sinusoidal filter. [Figure 3A] 3A, 3B, and 3C show exemplary wide-field, light sheet, and confocal microscopy embodiments of a spectral encoding device according to aspects of the present disclosure. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 4] 1 illustrates a high-level block diagram of an exemplary image acquisition, image pre-processing, and analysis pipeline for imaging using a spectral encoding device, according to aspects of the present disclosure. [Figure 5] 1 illustrates an exemplary image during image pre-processing, according to aspects of the present disclosure. [Figure 6] 6A and 6B show an example phasor plot and the resulting unmixed image, respectively, according to an embodiment of the present disclosure. [Figure 7] 1 depicts a high-level flowchart illustrating an exemplary method for acquiring and generating multispectral or hyperspectral images utilizing a spectral encoding device, according to aspects of the present disclosure. [Figure 8]1 illustrates an exemplary tiled volumetric in-vivo imaging output utilizing a spectral encoding device, according to aspects of the present disclosure. [Figure 9] 1 illustrates exemplary images acquired during dynamic in vivo imaging utilizing a spectral encoding device, according to aspects of the present disclosure. [Figure 10] 1 shows exemplary images acquired during dynamic in vivo imaging of a live zebrafish heart, according to aspects of the present disclosure.

[0018] For ease of understanding and convenience, the same reference numerals and any acronyms throughout the figures identify elements or acts having the same or similar structure or function. To easily identify the description of any particular element or act, the most significant digit in the reference numeral refers to the number of the figure in which that element is first introduced. DETAILED DESCRIPTION OF THE INVENTION

[0019] Detailed Description All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3 rd ed., Revised, J. Wiley & Sons (New York, NY 2006) and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4 th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provides those of skill in the art with a general guide to many of the terms used in this application.

[0020] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

[0021] In some embodiments, properties such as dimensions, shapes, relative positions, etc., used to describe and claim particular embodiments of the present invention should be understood to be modified by the word "about." As used herein, the word "about," when used in conjunction with a referenced numerical designation, means up to ±5% of the referenced numerical designation, unless otherwise specified herein. For example, the phrase "about 50%" encompasses a range of 45% to 55%. In various embodiments, the word "about," when used in conjunction with a referenced numerical designation, can mean up to ±4%, 3%, 2%, or 1% of the referenced numerical designation, if specifically defined in the claims.

[0022] Next, various examples of the present invention will be described. The following detailed description provides specific details for purposes of explanation that enable a thorough understanding and implementation of these examples. However, those skilled in the art will understand that the present invention can be practiced without many of these details. Likewise, those skilled in the art will also understand that the present invention can include many other obvious features that are not described in detail herein. In addition, in order to avoid unnecessarily obscuring the relevant description, some well-known structures or functions may not be shown or described in detail hereinafter.

[0023] The terms used below, while used in connection with the detailed description of certain embodiments of the present invention, should be interpreted in their broadest reasonable sense. Indeed, certain terms may be emphasized below. However, any term intended to be interpreted in any limited sense is expressly and specifically defined as such in this "Detailed Description" section.

[0024] Overview The present specification relates to systems and methods for multispectral and / or hyperspectral fluorescence imaging. In particular, the present specification relates to a spectral encoding device used to achieve high light throughput and optically encode fluorescence signals from biological samples into sine and cosine Fourier coefficients. In one example, the spectral encoding device includes a sine dichroic mirror and a cosine dichroic mirror, each of which generates an encoded transmitted light channel and an encoded reflected light channel. All four channels from the two dichroic mirrors are used for intensity normalization, which greatly improves light throughput. Furthermore, the dichroic mirrors have high transmittance, which improves the signal-to-noise ratio. The spectral encoding device may be integrated with or interfaced to any microscope system. A non-limiting example of a microscope system into which the spectral encoding device may be integrated is shown in FIG. 1A. A schematic diagram of the spectral encoding device is shown in FIG. 1B. Furthermore, exemplary optical components and arrangements of the spectral encoding device are shown in FIGS. 2A and 2B. Exemplary transmittance and reflectance profiles of the sine and cosine dichroic mirrors included in the spectral encoding device are shown in Figures 2C and 2D, respectively. Figure 2E illustrates the high light throughput efficiency achieved with the spectral encoding device for known fluorophores. Figures 3A-3C illustrate exemplary embodiments of the spectral encoding device with various microscope systems. Figures 4A and 4B schematically and illustratively illustrate high-level image acquisition, image preprocessing, and image analysis pipelines during imaging with a microscope system including a spectral encoding device. Figure 5 illustrates an exemplary image during image preprocessing. Figures 6A and 6B illustrate exemplary phasor plots and unmixed images after preprocessing. Figure 7 illustrates a high-level method for acquiring and generating multispectral or hyperspectral images with a spectral encoding device. Figures 8, 9, and 10 illustrate exemplary images acquired via the spectral encoding device during various in vivo imaging experiments.

[0025] Technical advantages of imaging assemblies including sine and cosine dichroic mirrors for spectral encoding include increased light throughput and encoding of high-resolution spectral information. Further technical advantages include improved temporal and spatial resolution due to short acquisition times, since no mechanical switching is required and spectral information is captured simultaneously. Furthermore, spectrally encoded imaging requires only a single snapshot acquisition to generate a multicolor spectral image. Further technical advantages include, but are not limited to, easy integration with existing research imaging devices, easy integration with existing medical imaging devices, easy modification to create a standalone imaging device (wide field of view), optically processed spectral data output, and simplified post-processing procedures. Taken together, the systems and methods described herein for spectrally encoding devices with one or more dichroic mirrors provide significant improvements in multispectral or hyperspectral microscopy.

[0026] Exemplary Microscope System FIG. 1 illustrates a high-level block diagram of an exemplary configuration of a light sheet microscope 100 used for optical imaging of a biological sample 134 (hereinafter referred to as sample 134 or specimen). Light sheet microscope 100 is illustrated as an exemplary microscope system in which a spectral encoding device (alternatively referred to herein as a spectral acquisition device) may be integrated or interfaced to encode spectral information from a fluorescent signal. It will be understood that the spectral encoding device may be adapted for use with any optical imaging system and its associated imaging sensor without departing from the scope of the present disclosure. For example, the spectral encoding system may be used with any wide-field microscope, confocal microscope, and / or various types of light sheet microscope systems.

[0027] The microscope 100 includes a dual illumination system including a first illumination system 110 and a second illumination system 112 for illuminating the sample from opposite directions. In particular, the sample 134 is illuminated from each side 101 and 102 such that a first light sheet 114 from the first illumination system 110 and a second light sheet 116 from the second illumination system 112 penetrate the sample to illuminate a section or slice (indicated by shading) of the sample 134. For example, the section may be a thin section (e.g., 5-6 μm wide) along the z-axis. Furthermore, the first excitation light sheet 114 and the second excitation light sheet 116 may illuminate the specimen 134 such that there is spatial and temporal overlap between the light sheets 114 and 116. For example, the first light sheet and the second light sheet may illuminate the same illumination plane (i.e., the plane illuminated by the light sheets) simultaneously and for the same duration. As used herein, a light sheet refers to a sheet of light generated by an illumination system that passes through and illuminates a plane of the sample. The light sheet is used to optically slice the sample.

[0028] The first and second illumination systems 112 and 114 may each include a light source 113 and 117, respectively, for generating the light used to form the corresponding light sheets 114 and 116. The light source may be based on at least the type of excitation provided for light sheet microscopy, e.g., linear or nonlinear excitation. When linear excitation is utilized, the signal intensity is proportional to the excitation light intensity. Exemplary embodiments of linear excitation include one-photon excited fluorescence, elastic light scattering, and inelastic light scattering (e.g., Raman or Brillouin). When nonlinear excitation is realized, the signal intensity is proportional to the square (or cube) of the excitation light intensity, and the sample interacts with two (or three) photons nearly simultaneously. Exemplary embodiments of nonlinear excitation include two-photon excited fluorescence, second harmonic generation, three-photon excited fluorescence, and other higher-order processes. The excitation light sheet may be generated by a simple cylindrical lens or by scanning a Gaussian beam generated through a low-NA objective lens with a galvanometer or resonant scanner.

[0029] In one example, light sources 113 and 117 may each be a laser light source emitting a narrow-band excitation wavelength (e.g., 405 nm, 488 nm, 561 nm, 635 nm, 960 nm, etc.). In some examples, the narrow-band excitation wavelength may be generated by a light-emitting diode (LED). In another example, light sources 113 and 117 may be broadband light sources (e.g., incandescent light sources, arc light sources, broadband LEDs, etc.) that generate a broad spectrum of light wavelengths. In yet another example, one or more portions of the excitation wavelengths may be outside the visible range. In one example, light sources 113 and 117 may be the same type of light source emitting the same excitation light wavelength (e.g., when each light source is a laser light source emitting a desired excitation wavelength, such as 488 nm). In another example, light sources 113 and 117 may be the same type of light source but emitting different excitation light wavelengths (e.g., when each light source is a laser light source but emitting different wavelengths, such as 488 nm and 560 nm). In yet another example, light sources 113 and 117 may be different (eg, a laser and an LED).

[0030] Each illumination system 110 and 112 further includes illumination optics 115 and 119 for generating the corresponding light sheets 114 and 116. In one example, the light sheets 114 and / or 116 may be statically formed. Accordingly, the illumination system may include one or more cylindrical lenses (not shown) and a low-numerical-aperture illumination objective (not shown) for focusing the light sheets onto an illumination plane within the sample 134. In another example, the light sheets 114 and / or 116 may be formed by rapidly scanning a focused illumination beam along the illumination plane. Accordingly, the illumination optics 115 and / or 119 may include one or more galvanometer mirrors (not shown) for generating the one or more light sheets. Furthermore, in some examples, the illumination optics 115 and / or 119 may include one or more beam-shaping optics, such as a spatial light modulator (SLM), a lens, a mirror, and / or a diffraction grating, for generating a desired beam profile at the illumination plane.

[0031] 1 shows two illumination systems, in some examples, a single illumination system may be utilized. In some examples, illumination systems 110 and / or 112 may be configured to generate multiple light sheets slightly rotated from each other for multi-directional illumination.

[0032] The light sheet microscope 100 includes a stage 130 that includes a sample holder 132 for mounting a specimen 134. The stage 130 may be a motorized stage that is movable along the z-axis. The stage 130 may be used to move the specimen 134 along the z-axis to adjust the positions of the light sheets 114 and 116 within the specimen 134. The movement of the stage 130 may be regulated by a controller 140. For example, the controller 140 may provide an actuation signal to the stage 130 to move the stage along the z-axis.

[0033] The light sheet microscope 100 includes an imaging objective 115 for receiving a fluorescent signal from the specimen 134. The imaging objective 115 has an optical axis that is substantially perpendicular to the optical axes of the illumination systems 110 and 112. As used herein, substantially perpendicular may account for errors in disposing the detection system relative to the illumination system and / or manufacturing errors. Furthermore, as used herein, a fluorescent signal refers to an emission signal from the specimen, e.g., specimen 134, resulting from excitation of one or more fluorophores in the specimen. For example, the specimen 134 may include one or more fluorescent labels (e.g., fluorescent labeling via a fluorescent labeling reagent, e.g., Alexa-488, FITC, etc., or a fluorescent protein, e.g., GFP for a green label or mCherry for a red label) that emit fluorescence corresponding to an emission wavelength when excited by a light source, i.e., in this example, the light sheets 114 and 116. In some examples, the detection system may detect natural proteins and / or molecules in the specimen 134 that emit light of a particular wavelength in response to excitation by the light sheets 114 and 116.Exemplary fluorescent labeling reagents include, but are not limited to, hydroxycoumarin, succinimidyl ester, aminocoumarin, methoxycoumarin, cascade blue, hydrazide, Pacific blue, maleimide, Pacific orange, Lucifer yellow, NBD, NBD-X, R-phycoerythrin (PE), PE-Cy5 conjugates (Cychrome, R670, Tri-Color, Quantum Red), PE-Cy7 conjugates, Red 613, PE-Texas Red, PerCP, peridinin chlorophyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, fluorescein isothiocyanate (FITC), BODIPY-FL, TRITC, X-rhodamine (XRITC), lissamine rhodamine B, Texas Red, allophycocyanin (APC), APC-Cy7 conjugates, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, or Cy7.

[0034] The light sheet microscope 100 further includes a detection system 120 including a spectral encoding device 122 and an imaging sensor 150. Fluorescence signals from the specimen 134 captured by the imaging objective 115 are sent to the imaging sensor 150 via the spectral encoding device 122. In particular, the fluorescence signals are encoded by the spectral encoding device 122, and the encoded fluorescence signals (also referred to as encoded emission signals) are captured by the imaging sensor 150. In one example, the imaging sensor 150 is a scientific complementary metal-oxide semiconductor sensor (sCMOS sensor). In other examples, depending on the application, the imaging sensor may be a charge-coupled device (CCD), an electron-multiplying charge-coupled device (EMCCD), or a photomultiplier tube (PMT). Furthermore, in one example, the fluorescence signals from the detection system 120 are captured by a single imaging sensor. In some examples, more than one imaging sensor may be used to simultaneously capture encoded emission signals from one or more channels of the spectral encoding device 122. Details of the spectral encoding device 122 are further described below with respect to Figures 1B, 2A-2E and 3A-3C.

[0035] The imaging sensor 250 may be communicatively coupled to the controller 140 (e.g., via a wired and / or wireless connection), and image data from the imaging sensor 250 may be processed via the controller 140 and displayed in real time or near real time via a display portion 162 of a user interface 160 communicatively coupled to the controller 140.

[0036] The controller 140 may include at least one processor (CPU) 144 and memory, such as read-only memory (ROM) 146 and / or random access memory (RAM) 142, including computer-readable media that may be operatively coupled to the processor. Accordingly, one or more of the ROM 146 and RAM 142 may include system instructions that, when executed by the processor, perform one or more of the operations described herein, e.g., the process flows of the subsequent figures. The processor 144 may receive one or more input signals from various sensory components and output one or more control signals to the various control components described herein via an input / output (I / O) interface 148. In some examples, one or more of the various components of the controller 144 may communicate via a data bus. While the present example illustrates an exemplary configuration of the controller 140, it will be understood that the controller 140 may be realized in other configurations.

[0037] Controller 140 may provide synchronized control of all opto-mechanical components within microscope 100. For example, controller 140 may rapidly perform optical alignment between the light sheet and the objective lens on specimen 134 and enable simultaneous image acquisition by multiple detectors (or cameras) within detection system 120.

[0038] Controller 140 may perform image preprocessing according to instructions stored in non-transitory memory, such as ROM 146 and RAM 142. For example, controller 140 may perform one or more of image registration and stitching on raw images acquired via imaging sensor 150. Additionally, controller 140 may perform image analysis on the preprocessed images. For example, image analysis may be performed according to one or more of a hyperspectral phasor analysis protocol and a linear unmixing protocol, among other image analysis methods. Details of image preprocessing and image analysis are further described below in FIGS. 4A, 4B, 5, 6, and 7.

[0039] The spectral encoding device 122 may be placed in the detection optical path between the imaging objective 115 and the imaging sensor 150 of the light sheet microscope 100. Although this example shows a light sheet microscope, the detection system 120 may be placed in the detection optical path between the imaging objective and one or more imaging sensors of any microscope system, such as a wide-field microscope, a confocal microscope, etc.

[0040] Spectral encoding device 122 includes encoding optics 128 for generating one or more encoded optical channels. In particular, encoding device 122 may include one or more dichroic mirrors configured to output spectrally encoded light, which is then collected by imaging sensor 150. In particular, transmitted and reflected light from each of the one or more dichroic mirrors is collected by imaging sensor 150. Details of spectral encoding device 122 are described further below. Encoding optics 128 may further include one or more routing mirrors, one or more tube lenses, and / or one or more gimbal mirrors for routing, focusing, and / or angulating each optical channel from the one or more dichroic mirrors to a respective portion on imaging sensor 150.

[0041] The spectral encoding device 122 further includes one or more pre-filtering optics 124 and one or more relay optics 126. The one or more pre-filtering optics 124 include one or more long-pass and short-pass filters for filtering out-of-range signals from the fluorescence emission signal from the objective lens 115, where the out-of-range signals include signals outside the spectral range of the one or more dichroic mirrors. For example, the one or more pre-filtering optics can be one, two, three, four, or more long-pass filters and / or one, two, three, four, or more short-pass filters. In one example, the spectral range includes wavelengths in the visible spectrum of light. Furthermore, the one or more pre-filtering optics 124 can include one or more band-pass filters for filtering out one or more excitation wavelengths from the illumination source. For example, the number of band-pass filters can be based on the number of excitation wavelengths. That is, as the number of excitation wavelengths increases, the number of band-pass filters also increases.

[0042] The one or more relay optics 126 include one or more lenses and a field stop adjustable by an aperture for adjusting the field of view of the imaging sensor 150. Details of the spectral encoding device 122 are further described below with respect to Figures 1B, 2A-2C and 3A-3C.

[0043] Spectral Encoding Device 1B shows a schematic diagram of a portion of detection system 120 including spectral encoding device 122 and relay optics 126 and encoding optics 128 portions of imaging sensor 150. Relay optics 126 includes a first relay lens disposed within first relay lens housing 162 and a second relay lens 166 disposed within second relay lens housing 166. A field stop is disposed at an intermediate image plane 164 between the first and second relay lens pairs. Furthermore, intermediate image plane 164 is a plane at back focal length 163 of the first relay lens and back focal length 165 of the second relay lens.

[0044] Downstream from relay optics 126, encoding optics 128 is disposed in the direction of the optical path from the sample to the objective lens through relay optics 126. Encoding optics 128 includes one or more routing mirrors disposed within roof mirror cube 176, a beam splitter disposed within beam splitter cube 170, a sine dichroic mirror disposed within sine dichroic mirror cube 174, and a cosine dichroic mirror disposed within cosine dichroic mirror cube 172. In various embodiments, the sine and cosine dichroic mirrors are each 1 to 100 mm, e.g., 1 to 10 mm, 10 to 20, 20 to 30, 30 to 40, 40 to 50, or 50 mm or more, along one dimension of the cube shape.

[0045] The beam splitter equally divides the fluorescence signal from the objective lens (e.g., objective lens 115) and directs the fluorescence signal toward the sine and cosine dichroic mirrors (in some examples, at least one routing mirror may direct the signal from the beam splitter toward the appropriate dichroic mirror). The sine and cosine dichroic mirrors transmit a portion of the received fluorescence signal and reflect the remaining portion of the received fluorescence signal, respectively. The transmitted and reflected signals from the sine and cosine dichroic mirrors, respectively, are collected and utilized for image acquisition and processing. In this way, the amount of information lost is greatly minimized (approximately 1% loss). As a result, the signal-to-noise ratio (SNR) is greatly improved. Exemplary transmittance and reflectance curves for the sine and cosine mirrors are shown in FIG. 2C. The sine dichroic mirror has sinusoidal transmittance and reflectance curves, thereby generating a sine-encoded transmitted light channel and a sine-encoded reflected light channel. Similarly, the cosine dichroic mirrors have cosine transmittance and reflectance curves, thereby generating corresponding cosine-encoded transmitted and reflected light channels. In other words, the first dichroic mirror generates a first spectrally-encoded transmitted light portion and a first spectrally-encoded reflected light portion; the second dichroic mirror generates a second spectrally-encoded transmitted light portion and a second spectrally-encoded reflected light portion. Furthermore, the transmission and reflection efficiencies of the sine and cosine dichroic mirrors, respectively, are high (e.g., greater than 80%). As a result, the amount of signal intensity received from each of the four channels is high, which significantly improves the signal-to-noise ratio. Furthermore, the reflected light portions from the sine and cosine dichroic mirrors, respectively, are utilized to normalize the intensity (as further described below), which further improves the SNR.

[0046] The sine and cosine mirrors together generate four light channels (two transmitted and two reflected light portions) that are detected by different portions of imaging sensor 150 or by different imaging sensors. In one example, gimbaled mirrors 180 and 182 direct the transmitted and reflected light from each of the four channels toward a respective tube lens located in tube lens housing 184. Each of the four channels is then detected by a respective quadrant of the imaging sensor. An exemplary optical layout of a spectral encoding device showing the fluorescence signal light path is further detailed in FIG. 2A.

[0047] 2A, a schematic diagram illustrating an exemplary optical arrangement of a spectral encoding device 222 is shown. The spectral encoding device 222 may be an example of the spectral encoding device 122 described with respect to FIGS. 1A and 1B. Accordingly, the spectral encoding device 122 may be coupled to a microscope, such as microscope 100, and positioned within the infinity space of the microscope between the imaging objective of the microscope and an imaging sensor 250. The imaging sensor 250 may be an example of the imaging sensor 150 described with respect to FIGS. 1A and 1B.

[0048] The spectral encoding device 222 includes one or more pre-filtering optics 202. The pre-filtering optics 202 may be an example of the pre-filtering 124 described in FIG. 1A. The one or more pre-filtering optics 202 may include one or more of a long-pass filter, a short-pass filter, and one or more notch filters. The long-pass and short-pass filters may be configured to filter out signals outside the spectral range of the dichroic mirrors utilized in the spectral encoding device 222. As a non-limiting example, for a set of dichroic mirrors having a spectral range in the visible spectrum (e.g., 400 nm to 700 nm), a 380 nm long-pass filter and a 715 nm short-pass filter may be utilized to filter out photons outside the spectral range of the set of dichroic mirrors. Additionally, one or more notch filters may be configured to filter out light having excitation wavelengths of one or more excitation light sources. As a non-limiting example, a set of notch filters may be utilized to filter out excitation laser light centered at 488 nm, 561 nm, and / or 640 nm.

[0049] As shown, emission light (i.e., fluorescence signal or simply fluorescence) from the imaging objective (shown as input) passes through pre-filtering optics 202 and then through a relay optics section consisting of a pair of relay lenses 204 and 208 and a field stop 206. In one non-limiting example, the relay optics may be configured as a Keplerian telescope with first and second 50 mm diameter relay lenses and a field stop. Relay lens 204 generates an intermediate image plane at field stop 206. In one example, the field stop may be a ring-actuated iris diaphragm. The diaphragm may be adjusted to a size that maximizes the final image formed on imaging sensor 250 without overlap. Relay lens 208 then recollimates the light to infinity.

[0050] The encoding optics portion of the spectral encoding device 222, positioned in infinity, receives light from the relay optics portion. In particular, the fluorescence from the relay lens 208 is incident on a beam splitter (BS) 210. In one non-limiting example, the beam splitter 210 can be a 50 / 50 beam splitter that splits the fluorescence emission light equally into two orthogonal paths (one path is directed to a sine dichroic mirror DMs 220 and the other path is directed to a cosine dichroic mirror DMc 221). The sine and cosine dichroic mirrors 220 and 221, along with three routing mirrors (RMs) RM211, RM213, and RM215, generate four spectrally encoded, correlated light paths.

[0051] The four spectrally encoded light paths may include a sine-encoded transmitted light path including transmitted light from sine dichroic mirror (DM) DMs220, a sine-encoded reflected light path including reflected light from sine dichroic mirror DMs220, a cosine-encoded transmitted light path including transmitted light from cosine dichroic mirror DMc221, and a cosine-encoded reflected light path including reflected light from cosine dichroic mirror DMc221. The transmitted light paths are indicated by solid arrowheads, and the reflected light paths are indicated by dashed arrowheads. Additionally, gimbal mirrors (GM) GM229, GM223, GM225, and GM227 are used before tube lenses (TL) TL231, TL233, TL235, and TL237 to adjust the angle of the incident light from each channel relative to imaging sensor 250 so that the channel images can be formed in the correct quadrant of imaging sensor 250. As shown, for a four-channel generating spectral encoding device 222, imaging sensor 250 may be divided into four quadrants: SIN quadrant 240, which receives transmitted light from sine dichroic mirror DMs220; COS quadrant 242, which receives transmitted light from cosine dichroic mirror DMc221; A-SIN quadrant 246, which receives reflected light from sine dichroic mirror DMs220; and A-COS quadrant 248, which receives reflected light from cosine dichroic mirror DMc221. Thus, each quadrant receives a portion of the encoded light transmitted or reflected from a dichroic mirror. Thus, for a spectral encoding device including one or more dichroic mirrors and generating N channels (each dichroic mirror generating an encoded transmitted light channel and an encoded reflected light channel), the imaging sensor may be divided into N non-overlapping portions, each receiving encoded light from a corresponding channel. Alternatively, N imaging sensors may be used to image the N channels. In either case, N tube lenses may be used to focus the light from the dichroic mirrors and form an N-channel final image onto the imaging sensor.In some examples, the focal length of each tube lens can be the same. In one non-limiting example, the focal length of the tube lens can be 175 mm. In various embodiments, each tube lens can have a focal length of 1 to 250 mm, e.g., 1 to 25, 25 to 50, 5 to 75, 75 to 100, and 100 to 250 mm. Furthermore, to achieve the same magnification in all four channels, the tube lenses can have the same focal length. Thus, in the example shown in FIG. 2A, tube lenses TL231, 233, 235, and 237 can each have the same focal length.

[0052] 2B shows a simplified diagram of the optical assembly of the spectral encoding device 222. Like components are like numbered and descriptions of like numbered parts will not be repeated for the sake of brevity.

[0053] 2B, optical assembly 260 includes a microscope imaging objective 266 (i.e., a detection objective) having a focal length f1. The optical assembly further includes pre-filtering optics 202, a first relay lens 204 having a focal length f2, a field stop 206, and a second relay lens 208 having a focal length f3. Optical assembly 260 also includes an image splitting component 278, including two dichroic mirrors and a routing mirror, that splits and spectrally encodes the emission light into four channels. Optical assembly 260 also includes a tube lens 280 (which may be any of tube lenses 231, 233, 235, or 237) having a focal length f4 for each channel.

[0054] The effective magnification M can be calculated by equation (1). TIFF0007719789000001.tif8128

[0055] To achieve the same magnification among the four channels, all tubes may have the same focal length. Additionally, the distance D (denoted by 286) between relay lenses 204 and 208 may be adjusted according to equation (2). D=BFL1+BFL2(2) where BFL1 (denoted by 282) and BFL2 (denoted by 284) are the manufacturer-provided back focal lengths for relay lenses 204 and 208, respectively. Additionally, a clear diameter and clear aperture may be utilized to accommodate the maximum ray angle while minimizing vignetting. For example, an estimation may be performed using the back aperture size and maximum field of view of the detection objective.

[0056] The spectral encoding device 222 optically generates the first-order Fourier coefficients G(k) and S(k), which can be represented in a 2D plane called the phasor plane. The spectral phasor of an N-channel hyper- or multi-spectral vector is represented by equations (3, 4). TIFF0007719789000002.tif23128 where G and S are the real and imaginary coefficients at the k harmonic. n represents the wavelength of the nth spectral channel. I(λ n ) denotes the intensity value of the nth channel. Δλ is the wavelength bandwidth of a single channel. The denominators in equations (3 and 4) represent the integral of the intensity values of all N channels and are normalization factors to eliminate the effects of different intensity levels.

[0057] The spectral encoding device 222 optically performs phasor encoding and calculation during acquisition. As described above, two sinusoidal dichroic mirrors convolve the fluorescence emission spectrum by transmitting and reflecting it. The "SIN" and "COS" transmission channels shown in FIG. 2A represent the nominator portion in equations (3, 4). The "A-SIN" and "A-COS" reflection channels are used to estimate the total intensity along with "SIN" and "COS" for denominator normalization. G and S are calculated according to equations (5, 6). TIFF0007719789000003.tif45128In formula, C ideal and S idealand represent the nominators in equations (3, 4), respectively. C is the COS channel, S is the SIN channel, AS is the A-SIN channel, and AC is the A-COS channel. I is the intensity value used for normalization, which accounts for half of the total intensity detected in the four channels. C ideal and S ideal can be calculated from equations (8, 9) by applying normalization to the transmittance response profiles of the cosine and sine dichroic mirrors: c and c s are the center values of the cosine and sine transmittance profiles, respectively. c and a s are the amplitudes of the cosine and sine transmittance profiles, respectively. In one non-limiting example, for a given set of cosine and sine dichroic mirrors, the center values and amplitudes are c =0.52, c s =0.51, a c =0.44 and a s =0.40.

[0058] 2C and 2D are exemplary graphs showing exemplary transmittance and reflectance profiles of an exemplary sine dichroic mirror, such as sine dichroic mirror DMs 220 of FIG. 2A, and an exemplary cosine dichroic mirror, such as cosine dichroic mirror DMc 221 of FIG. 2A, respectively. The graphs show transmittance / reflectance percentage (i.e., the percentage of light transmitted through / reflected from the dichroic mirror) on the y-axis and a wavelength range of 400 nm to 700 nm on the x-axis.

[0059] In particular, trace 290 shows the transmittance curve for a sine dichroic mirror, trace 291 shows the reflectance curve for a sine dichroic mirror, trace 292 shows the transmittance curve for a cosine dichroic mirror, and trace 293 shows the reflectance curve for a cosine dichroic mirror. As shown, the spectral transmittance of the sine dichroic mirror closely resembles the shape of a sine function (FIG. 2C) and a cosine function (FIG. 2D), with maximum transmittance peaking at 95.8% for the sine and 91.1% for the cosine.

[0060] As explained further below, the reflected portion of the light signal is also detected and used for intensity normalization. The high transmittance percentage of the sine and cosine dichroic mirrors, combined with the recycling and detection of the reflected light portion by the sine and cosine dichroic mirrors, increases light throughput to over 80% for commonly used fluorophores, thereby increasing detection efficiency while reducing phototoxicity.

[0061] Figure 2E shows the total transmission efficiency of a spectral encoding device including a sine dichroic mirror, e.g., sine dichroic mirror DMs220, and a cosine dichroic mirror, e.g., cosine dichroic mirror DMc221, compared to two sinusoidal transmission filters. Specifically, the transmission efficiencies calculated using two sinusoidal filters (first row, sequential filters) and the spectral encoding device (second row) for five commonly used fluorophores, namely, cyan fluorescent protein (CFP), enhanced green fluorescent protein (eGFP), enhanced yellow fluorescent protein (eYFP), mCherry, and iRFP670, are shown. The spectral encoding device including sine and cosine dichroic mirrors allows for higher light throughput by utilizing the reflected portion in addition to the transmitted portion for acquisition and normalization, as evidenced by the higher total transmission efficiency when the spectral encoding device is used.

[0062] The transmission efficiency was estimated by considering realistic losses at each optical surface. For the two-filter approach, the optical system includes two sinusoidal filters followed by a tube lens, and is estimated using a four-sided achromatic doublet. The transmission profile is estimated by one period of ideal sine and cosine functions with a center value of 0.5 and an amplitude of 0.5.

[0063] For each fluorophore tested with the spectral encoding device, the total transmission efficiency is over 80%, and when sequential filters are applied, the efficiency drops to 28.3% (for mCherry).

[0064] Referring now to Figures 3A-3C, these figures show schematic diagrams of a spectral encoding device, e.g., spectral encoding device 122 of Figures 1A and 1B or spectral encoding device 222 of Figure 2A, implemented in a wide-field microscope, a light sheet microscope (only the detection portion is shown), and a confocal microscope (only the portion after the second pinhole is shown), respectively.

[0065] Specifically, Figure 3A shows a biological sample 301 illuminated by an illumination system 304 in a wide-field microscope system 300. Fluorescence signals from the sample 301 are directed by an excitation dichroic mirror 306 toward a beam splitter 310 of a spectral encoding device 322. Although not shown, the spectral encoding device 322 may include pre-filtering and relay optics as described above. Sine and cosine dichroic mirrors 312 and 314 each receive equal amounts of the fluorescence signal. The sine and cosine dichroic mirrors 312 and 314 generate four spectrally encoded optical channels (two transmitted and two reflected), which are then directed by routing mirrors 311, 313, 315, and 317 to tube lenses 331, 333, 335, and 337, respectively. Each tube lens 331, 333, 335, and 337 focuses light from each channel into a respective non-overlapping quadrant of imaging sensor 350. Imaging sensor 350 may be, for example, an example of imaging sensor 250 of FIG. 2A.

[0066] Next, Figure 3B shows a spectral encoding device 332 integrated with a light sheet microscope system 330, which includes a detection objective lens 303. The detection portion of the light sheet microscope system 330 is shown. The light sheet microscope system 330 may be an example of the light sheet microscope 100 of Figure 1A. Similar to Figure 3A, sine and cosine dichroic mirrors 312 and 314 generate four encoded channels, which are imaged by an imaging sensor 350.

[0067] 3C shows a portion 390 of the confocal microscope after the second pinhole 372. The fluorescent signal from the objective lens passes through a collimator lens 370 before being sent to a beam splitter 310. Furthermore, each of the four channels generated by sine and cosine mirrors 312 and 314 is detected by a separate photomultiplier tube (PMT).

[0068] It will be understood that the spectral encoding device or spectral encoding portion including at least one sine dichroic mirror and at least one cosine dichroic mirror may be positioned in infinity space between the objective lens of the microscope and one or more imaging sensors of the microscope.

[0069] Acquisition, image pre-processing and analysis pipeline A block diagram of an exemplary acquisition, image pre-processing, and analysis pipeline is shown in FIG. 4A. An exemplary implementation of the acquisition, image pre-processing, and analysis pipeline is shown for a biological sample in FIG. 4B. At 402, image acquisition is performed using a single snapshot acquisition (shown at 404) via a microscope, e.g., microscope 100 of FIG. 1 or any confocal, light sheet, or wide-field microscope. During acquisition, an integrated spectral encoding device coupled to the microscope, e.g., device 122, 222, or 322, generates four channels: sine (encoded transmission channel from the sine dichroic mirror), cosine sine (encoded transmission channel from the cosine dichroic mirror), antisine sine (encoded reflection channel from the sine dichroic mirror), and anticosine 9 sine (encoded reflection channel from the sine dichroic mirror). Each channel image is captured in a corresponding quadrant on an imaging sensor, e.g., imaging sensor 150, 250, or 350. In this manner, raw images from each channel are acquired via the spectral encoding device coupled to the microscope.

[0070] Next, at 406, preprocessing image alignment is applied to the raw images to properly align the raw images from the four encoded channels. In one example, the four channels are aligned using a non-rigid warping based image registration method. Using the brightfield image, control points for one reference channel and three alignment channels can be manually selected and exported (e.g., by using Fiji's BigWarp plugin function, which provides visualization of aligned images to assess the quality of the control points). These control points can serve as the basis for the alignment transformation of the four channels. The parameters of this transformation only need to be calculated once and do not need to be changed until the optical system is realigned or changed. The alignment is performed by loading the previously exported raw images and control points into a MATLAB function. The aligned image is then saved as a four-channel OME-TIFF phasor cube, with each layer corresponding to a sinusoid, cosine, a-sinusoid, and a-cosine.

[0071] The maximum field of view (FOV) of each tile is a hexagon with a predetermined diameter (e.g., 120 μm). For larger FOVs, image tiling may be applied during the acquisition stage, and image stitching (410) may be applied after image registration. For example, image stitching is required for mosaic synthesis acquisition. In one non-limiting example, stitching software (e.g., Imaris Stitcher 9.6 (Bitplane, Switzerland)) may be used for stitching. Other stitching protocols may also be used and are within the scope of this disclosure. In some examples, tiles may be manually positioned to improve the accuracy of the stitching results.

[0072] Image analysis is then performed at 412. In one example, hyperspectral phasor analysis may be performed (414) by converting the four channel images into phasor coefficients G and S, followed by phasor plane analysis. This involves computing the phasor G and S values by fast 2D matrix pixel-wise multiplication of the four SHy-Cam channels. The spectral signal is then denoised by filtering the two Fourier components. Representation of the encoded spectral signal as a phasor plot allows for graphical selection of regions of interest for exploring the multi-color data set. This analysis is performed using the HySP - Hyperspectral Phasor software. 16 It is executed using

[0073] Alternatively, perform phasor-encoded pixel-wise linear unmixing by processing the four-channel image in the spectral range (400 nm - 700 nm in this example) as spectrally correlated channels. Linear unmixing (LU) can be applied directly to the encoded four channels to provide the relative contributions of the fluorophores in each image pixel.

[0074] Taken together, the four channels from the spectral encoding device are aligned to generate a cube of data with dimensions (x, y, channel), where the channels are sine, cosine, antisine, and anticosine. The mosaicked image is then stitched into a larger FOV volume and then unmixed using various techniques.

[0075] FIG. 5A shows an example of image preprocessing of raw data from four channels. In 502, cropping of the four channels from a brightfield image is performed in MATLAB once per alignment. The image shows the raw camera data with the four channels distributed across four quadrants. Next, in image 504, (b) shows a screenshot of the “COS” channel (e.g., from the “BigWarp” plugin in Fiji) used as a reference channel. The reference channel contains manually selected control points. Also in 504, (c) shows a screenshot of the “ASIN” channel, one of the three translation channels. (d) shows an overlay image of the “COS” and “ASIN” channels before alignment, showing misalignment. (e) shows an overlay image of the “COS” and “ASIN” channels after alignment, showing proper alignment. Furthermore, 506 shows screenshots of the nine image tiles before stitching. Finally, 508 is a screenshot of the stitched image after rotation and crop correction.

[0076] Correct alignment and alignment of the four channels may be required once per optical alignment. As described above, image alignment is applied to the raw image before data analysis. Manual cropping is applied to the target image, and the cropping box coordinates are exported and saved for future automatic alignment. A split channel is loaded to manually position control points between one reference channel and three translation channels. The control points are then saved for further use. A 10-blade adjustable iris is then used as a field stop to limit the field of view. These vertices are used as control points. The imaging target used to capture the alignment dataset is one of a biological sample due to its large number of features. In this example, a zebrafish larva is imaged. During target image acquisition, a laser excites fluorescent signals coming from a single area, preferably one with a distinct shape across the entire field of view. Simultaneously, brightfield illumination is activated to capture other textures useful for control points. The exemplary target texture in Figure 5A includes kdrk:mCherry, labeled vasculature, and the surface texture of the zebrafish larva. The control points only need to be updated when misalignment or physical changes occur.

[0077] In the case of mosaic synthesis acquisition, stitching of multiple tiles may be performed after alignment. Since the image cube contains four channels (ASIN, ACOS, SIN, and COS), alignment is performed on one channel that shows the most distinct features, and the other channels are used for visual confirmation of approximate alignment. After tile alignment (506), stitching is performed.

[0078] 6A and 6B show an example image analysis using a phasor plot and the resulting unmixed image, respectively. In one example, spectral analysis and multiplexing can be achieved by applying a region of interest (ROI) to the phasor plane. Alternatively, linear unmixing (LU) can be applied to the image acquired via the spectral encoding device as a pixel-wise spectral analysis method for automated ratiometric unmixing results.

[0079] Figure 6A shows a phasor plot of a spectrally encoded device image. The sample zebrafish embryo, Tg(krt4:lyn-EGFP; kdrl:mCherry; lyz:TagRFP), contains three transgenic fluorescent proteins. A polygon ROI selection on the phasor plane selects corresponding pixels in the original image, enabling selective unmixing of signatures. The phasor plot is a 2D histogram representing the population distribution of phasor coefficients. Higher peaks in the phasor plane distribution correspond to more frequent spectral signatures across all pixels. Each independent spectral signature corresponds to a cluster with large values on the phasor plane. However, for spatially sparse fluorescent signatures, the clusters do not have strong clarity. If various signatures have large differences in spatial sparsity, the phasor plot may be viewed on a logarithmic scale for easier visualization.

[0080] A selected region on the phasor plot highlights the corresponding spatial region in the original data in real time as a saturated color for reference (Figure 6B, image 606). Additional overlap is added within the selected ROI where signal spatial overlap occurs.

[0081] In some instances, linear unmixing (LU) may be applied to the preprocessed spectrally encoded device image. This requires a set of four-channel reference spectra—i.e., spectra of pure fluorescence signatures—measured under the same imaging conditions used in the final experiment. This step accounts for experimental nonlinearities in intensity at different camera exposures, gains, and laser excitation powers. These reference spectra are provided as a 4 x n array (n is the number of signatures), and a pixel-wise linearly constrained least-squares problem is solved using the n reference spectra and the preprocessed image in the shape of four channels (ASIN, ACOS, SIN, and COS). The result is the relative contribution of the various signatures in each pixel.

[0082] 7 shows a high-level flowchart illustrating an exemplary method 700 of acquiring and generating hyperspectral or multispectral images via a spectral encoding device, such as those described in FIGS. 1A, 1B, 2A-2E, and 3A-3C. Method 700 may be implemented by a controller, such as controller 140, according to instructions stored in a non-transitory memory, such as memory 146.

[0083] At 702, method 700 includes acquiring a fluorescence image through a single-exposure acquisition using a spectral encoding device coupled between the objective lens and the imaging sensor. During acquisition, the fluorescence signal from the biological sample may be optically split and encoded by one or more dichroic mirrors having periodic transmittance waveforms (e.g., sinusoidal) and periodic reflectance waveforms (e.g., sinusoidal). For example, each dichroic mirror generates an encoded transmitted light channel and an encoded reflected light channel.

[0084] At 704, the method 700 includes normalizing each channel according to the total intensity detected in all channels, including the reflected and transmitted intensities for each channel.

[0085] At 706, the method 700 includes pre-processing the raw images acquired in all channels. Depending on the field of view, the pre-processing may include performing channel alignment 708 and mosaic synthesis 710. Exemplary pre-processing and mosaic synthesis are described with respect to Figures 5, 6A, and 6B.

[0086] Next, at 712, method 700 includes performing image analysis, which may be phasor analysis (step 714) or linear unmixing (step 716). Method 700 further includes displaying the unmixed image. Phasor analysis using images generated from a spectral encoding device can distinguish between different species emitting at different wavelengths. An example of imaging multiple fluorescent signatures is provided below. For example, a four-channel image may be used to obtain several fluorescent signatures, where the number of fluorescent signatures is less than, equal to, or greater than four. For example, the number of fluorescent signals may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. [Example]

[0087] 8 shows exemplary tiled volumetric in vivo imaging using a light sheet microscope integrated with a spectral encoding device, e.g., encoding device 122, 222, or 322. 802 is a maximum intensity projection image showing five signatures unmixed using Phasor Hybrid Unmixing, acquired at the trunk region of a 4 dpf transgenic zebrafish embryo with a 200 ms exposure time. When zoomed in, the signals in box 803 correspond to cyan—autofluorescence in 804, green—Tg(krt4:GFP) in 806, yellow—Tg(lyz:TagRFP) in 808, magenta—Tg(kdrl:mCherry) in 810, and purple—ubi:H2B-iRFP670 in 812.

[0088] Figure 9 shows dynamic in vivo imaging using SPIM-SHy-Cam. Images 902–908 show a zebrafish tail clip wound healing (dashed line) acquired as sequential volumetric time-lapse images with a 50 ms exposure time. The embryo is labeled with membrane (green) Tg(krt4:GFP), nuclei (purple) ubi:H2B-iRFP670, and neutrophils (yellow) Tg(lyz:TagRFP). Neutrophil migration into the wound can be observed in high-resolution 3D with respect to the tissue and tracked over time. The beating heart (dashed circle) of the same embryo (910–916) was captured at 20 frames per second with a 50 ms exposure time. Neutrophils flowing through the cardiac compartment can be tracked.

[0089] Image acquisition and sample preparation The spectral coding device prototype was installed in Micro-manager. 23 A home-made light sheet microscope was fitted with the software used for acquisition and stage control. The instrument is equipped with five laser lines and a PCO Edge 5.5 camera (PCO, GmbH) with a resolution of 2560 × 2160 pixels. During the testing phase of the spectral encoding device prototype, three laser lines were used to effectively excite the fluorescent signal. During image acquisition, 2 × 2 binning was applied to improve the signal-to-noise ratio (SNR).

[0090] For multicolor in vivo imaging studies, zebrafish embryos that showed specific expression of transgenic fluorescent proteins were collected and grown in low-salt embryo medium according to established procedures until the appropriate imaging stage (4 dpf). Prior to imaging, embryos were immersed in a 1% low-melting-point agarose solution (prepared with 30% Danieau solution) and drawn into a glass capillary tube (5-000-1025, Drummond Wiretrol) with a stainless steel plunger. After the agarose solidified (1–2 min) at room temperature (21–23 °C), the capillary tube was transferred to an imaging chamber filled with Danieau solution, and the agarose containing the embryo was extruded from a micropipette to gain optical access. To prevent embryo movement, 0.075% tricaine was added to both the agarose solution and the imaging chamber filled with Danieau solution. During imaging, the imaging chamber temperature was set and maintained at 28.5 °C.

[0091] Image preprocessing A two-step preprocessing step is applied to the SHy-Cam images. The first step is registration. A non-rigid warping-based image registration method is used to align the four channels. Using bright-field images, control points for one reference channel and three alignment channels are manually selected and exported using Fiji's BigWarp plugin. BigWarp provides visualization of the aligned image to assess the quality of the control points. These control points serve as the basis for the alignment transformation of the four spectrally encoded device channels. The parameters of this transformation only need to be calculated once and do not need to be changed until the optical system is realigned or modified. Registration is performed by loading the raw images and control points, which have been previously exported, into a MATLAB function. The aligned image is saved as a four-channel OME-TIFF phasor cube, with each layer corresponding to a sinusoid, cosine, a-sinusoid, and a-cosine.

[0092] The second step is image stitching for mosaic composite acquisition (Fig. 5). Imaris Stitcher 9.6 (Bitplane, Switzerland) was used as the stitching software due to its easy-to-understand interactive user interface. Due to the lack of positional metadata for each individual 4-channel phasor cube, tiles were manually positioned to improve the accuracy of the stitching results. The final results were saved in ims format.

[0093] Geometric Unmixing on the Phasor Plane Hyperspectral Phasors software was used for fluorescence signature unmixing and analysis on the 2D phasor plane. Regions of interest (ROIs) were applied to the phasor plane to separate multiple signatures (Figure 6A). Individual signatures were identified by imaging the sample with a single fluorescence. Separation locations were identified by visualizing the phasors on a logarithmic count scale and identifying local minima.

[0094] Ratiometric Spectral Unmixing Spectral linear unmixing may be applied directly to the four-channel spectrally encoded device intensity image before conversion to the G and S spectral phasor coefficients. LU is treated here as a constrained linear least squares (CLS) problem, which can be expressed by Equation (10). TIFF0007719789000004.tif9159 where n is the number of fluorescence signatures. 4×n is a 4xn matrix of reference spectra, where each column of the matrix contains a reference 4-channel spectrum of a pure spectral signature captured using the spectral encoding device under the same imaging conditions as the experimental sample, maintaining the same laser power, exposure time, and gain, in which all signatures are present. n is the optimal solution for the contribution vector of n different signatures. 4 = [AC AS SC]T is the four-channel spectral vector corresponding to each pixel from the image. nis the nD identity matrix. n is an nD unity matrix. n and 1 n is an nD all-0 and all-1 vector. Two constraints are used to better define the problem. The first constraint ensures that the sum of all contributions equals 1. The second constraint limits the range of contributions to 0 to 1.

[0095] Zebrafish strains Strains were raised and maintained according to standard literature practices and in accordance with the Guide for the Care and Use of Laboratory Animals provided by the University of Southern California. Fish samples were part of an IACUC-approved protocol (Permit Number: 12007 USC). The krt4:lyn-egfp and krtt1c19e:lyn-tdtomato transgenic strains were a gift from Thomas J. Carney (A*STAR, Singapore). The Kdrk:mCherry transgenic strain was a gift from Ching-Ling Lien (Children's Hospital Los Angeles). The TgBAC(sox10:BirA-mCherry) ox104a strain was used.

[0096] The mpv17a9 / a9;mitfaw2 / w2 (Casper) strain was purchased from the Zebrafish International Resource Center (ZIRC), and the csf1rj4e1 / j4e1 (Panther) strain was a gift from David Parichy (University of Virginia). Casper was crossed with Panther to generate triple heterozygous mpv17a9 / +;mitfaw2 / +;csf1rj4e1 / + F1 generation fish, which were subsequently inbred to generate F2 generation fish with 27 combinations of mutation status for these genes. Because the csf1rj4e1 phenotype was not evident in the F2 adults with the casper phenotype, these fish were outcrossed with panther fish, and the zygosity of the csf1rj4e1 mutation was determined by fluorescence microscopy based on the frequency of xanthophore-positive larvae (heterozygotes and homozygotes produced 50% and 0% of xanthophore-positive larvae, respectively). The casper;csf1rj4e1 / j4e1 strain was viable and fertile; the casper;csf1rj4e1 / j4e1 strain or the casper;csf1rj4e1 / + strain was outcrossed with other fluorescent transgenic strains over several generations to obtain fish carrying multiple transgenes on the casper background, with or without xanthophore.

[0097] Tg(PGK1:H2B-chFP) 32The coding sequences of human histone 2b region (H2B) and the fluorescent protein iRFP670 were amplified from the vector using primers #1 and #2, and from the vector piRFP670-N1 (Addgene #45457) using primers #3 and #4. The PCR products were fused to generate an H2B-iRFP670 fusion fragment, which was then cloned into pDONR221 (Thermo Fisher Scientific). A MultiSite Gateway reaction was then performed using the Tol2kit vector according to the manufacturer's instructions. pENTR5'_ubi (Addgene #27320), pDONR221-H2B-iRFP670, and pDONR P2R-P3-WPRE were assembled into pDestTol2pA2 (Tol2kit #394). The resulting pDestTol2-ubi:H2B-iRFP670 was co-injected with tol2 mRNA into one-cell stage Caspase zebrafish embryos. The injected F0s were fostered and screened for founders. Positive F1s grown to breeding age were subjected to Splinklette PCR analysis to determine the genomic integration site. Lines showing single-copy integration within an unannotated region determined by the Ensembl Zebrafish GRCz11 database were selected and outcrossed with the other transgenic and mutant lines described above for imaging experiments. TIFF0007719789000005.tif56128

[0098] Figure 9 shows an exemplary multicolor snapshot image of a live zebrafish heart. A 4-day-old zebrafish genetically labeled for epithelium (green), vasculature (magenta), and nucleus (red) was imaged at 33 frames per second during snapshot multispectral fluorescence imaging.

[0099] In one embodiment, described herein is an imaging assembly including two specially designed dichroic mirrors (DMs), where the first DM has a sinusoidal transmittance curve and one harmonic of an anti-sinusoidal reflectance curve, and the second DM has a cosine transmittance curve and one harmonic of an anti-cosine reflectance curve. In various embodiments, each of the two DMs is a DM cube shape. In various embodiments, the DM cube is 1-100 mm along one dimension of the cube shape, e.g., 1-10 mm, 10-20 mm, 20-30 mm, 30-40 mm, 40-50 mm, or 50 mm or more. In various embodiments, the assembly includes a beamsplitter. In various embodiments, the assembly includes a 50 / 50 beamsplitter cube. In various embodiments, the assembly includes one or more routing mirrors. In various embodiments, the assembly includes four routing mirrors. In various embodiments, the assembly includes one or more tube lenses. In various embodiments, the tube lens comprises a focal length of 1-250 mm, e.g., 1-25, 25-50, 5-75, 75-100, and 100-250 mm. In various embodiments, the assembly comprises one or more relay lenses. In various embodiments, the assembly comprises a sensor. In various embodiments, the assembly comprises an iris. In various embodiments, the assembly comprises a roof mirror cube. In various embodiments, the assembly comprises two or more gimbaled mirrors.

[0100] Also described herein is a method for using the aforementioned imaging assembly. In various embodiments, the method includes a continuous Fourier transform (FT). In various embodiments, the FT is a normalized sine and cosine Fourier transform performed at one specific harmonic. In various embodiments, the FT includes: TIFF0007719789000006.tif19128N: Number of spectral channels i: Spectral channel k: harmonic number, usually 1 or 2

[0101] In various embodiments, normalization is achieved by utilizing the total intensity of the image. Spectral imaging relies on color information combined with spatial information to resolve biological properties. It is a trade-off triangle whose vertices are spectral resolution, temporal resolution, and spatial resolution.

[0102] The high-throughput spectral-encoding device described herein can encode high-resolution spectral information, has high optical throughput, has minimal compromises in temporal and spatial resolution, and can acquire spectral information in a single image (snapshot acquisition). Furthermore, the high-throughput spectral-encoding device has synergistic effects, including easy integration with existing research imaging devices, easy integration with existing medical imaging devices, easy modification to become a standalone imaging device (wide field of view), and output of optically processed spectral data, which simplifies post-processing procedures.

[0103] Hyperspectral Phasor (HySP) is a Fourier transform (FT)-based computational post-processing method for hyperspectral / spectral image data. It converts high-dimensional spectral data into a 2D vector consisting of G and S coefficients (1), where G and S are the real and imaginary parts of the first- or second-harmonic Fourier coefficients of the original spectral vector, respectively.

[0104] By transforming the HySP computation into an optical device that can be integrated into the infinity space of existing imaging methods, continuous Fourier transform-based spectral encoding can be achieved.

[0105] Image spectral information is encoded by two dichroic mirrors (DMs): the first DM has a sinusoidal transmittance curve and one harmonic of an anti-sinusoidal reflectance curve within the spectral range of interest, and the second DM has a cosine transmittance curve and one harmonic of an anti-cosine reflectance curve within the spectral range of interest.

[0106] The light collected by the detector after passing through the two DMs contains spectrally encoded information that can be viewed as first-harmonic continuous Fourier coefficients.

[0107] The antisine and anticosine encoded reflected light is also collected by the sensor and combined with the transmitted light to recover the total emitted signal, which is then used for normalization.

[0108] For WFM and LSFM applications, only one camera is needed to acquire the spectrally encoded image, whereas for CFM applications, four PMTs are needed to acquire the encoded spectral signal.

[0109] In WFM and LSFM applications, specialized routing mirror arrays and tube lens arrays are used to route the four split optical paths and form images on the same camera sensor.

[0110] Case 1: Hyperspectral snapshot imaging of low-signal samples Current snapshot hyperspectral techniques utilize multiple color filters arranged on the sensor in a 4x4 or 5x5 square pattern, an extension of the 2x2 pixel Bayer filter (RGGB) commonly used in cell phone cameras. These filters are designed to reject all light except for a spectral band equivalent to 1 / 16 (for 4x4) or 1 / 25 (for 5x5) of the total spectral range captured. Limitations:

[0111] In this process, 15 / 16 (for a 4x4 pattern) or 24 / 25 (for a 5x5 pattern) of the light is rejected and lost. This translates to a 93.7% and 96% loss in light collection, respectively. Such low efficiency makes it extremely difficult to use this type of snapshot hyperspectral camera with fluorescent samples, since such signals are typically characterized by a low signal-to-noise ratio.

[0112] The spectral encoding devices described herein suffer from an estimated 10% light collection loss, providing up to 9.6 times lower light loss and up to 22 times higher signal acquisition efficiency. The increased efficiency enables hyperspectral imaging of fluorescent signals.

[0113] The final resolution of the image is 4 or 5 times lower than the resolution of the camera sensor. For example, a 2000 x 2000 pixel camera will only produce a 500 x 500 pixel image at 4 x 4 resolution, and a 400 x 400 pixel image at 5 x 5 resolution. The spectral encoding device herein produces an image with half the number of pixels of the camera sensor, doubling the resolution of the final image.

[0114] Case 2: High-speed multiplexed fluorescence imaging Imaging and separating multiple fluorescent dyes or proteins within 2D or 3D samples has been challenging and has been limited by two factors: i) the absence of sensitive snapshot spectral imagers, and ii) spectral overlap (spectral similarity) of fluorescent signals, which leads to information spillover between channels. Standard multicolor samples are typically imaged using a sensitive camera paired with a set of sequentially switched light-emission filters. For a three-color fluorescent sample, this requires the acquisition of three images and switching between three filters. Spectral overlap has limited how many and what types of fluorescent dyes can be used in a sample. Typical dyes are sufficiently spectrally separated to minimize spectral overlap (e.g., blue, green, and red fluorescence). Often, the sample moves during filter switching, requiring image realignment. With a 30ms exposure per image, even using a very high-end, expensive, fast filter changer that changes filters every 30ms, a 3-color image takes 30ms * 3 colors + 30ms * 3 filters = 180ms, i.e., a frame rate (fps) of 5.5 (for 3 colors). A 4-color frame takes over 60ms, dropping the fps to 4.2.

[0115] The spectral encoding device described herein solves both of the above problems by providing highly sensitive, spectrally resolved images. With no moving parts, only one image needs to be acquired. The absence of spectral bandpass filters overcomes the overlap and spillover issues, allowing for rapid acquisition of three-color or higher-color images. Using the same 30 ms exposure as in the example above, a three-color frame takes only 30 ms (33 fps), and a four-color frame also takes only 30 ms (33 fps), enabling acquisition that is 2*n times faster than standard (n is the number of fluorophores being imaged). For example, a three-color fluorescent zebrafish heart is acquired at 33 fps, which is six times faster than using bandpass sequential filters.

[0116] Case 3: Large sample / high-throughput multiplexed imaging Imaging samples with multiple spectrally overlapping labels requires extending the imaging to the spectral dimension to acquire a spectral cube containing (x, y, wavelength) dimensions. Current instruments capable of performing this type of imaging are point- or line-scan spectrally resolved confocal fluorescence microscopes. Point scanning acquires one point at a time and spectrally spreads the signal across a line of detectors to access the spectral dimension. The point is then raster-scanned through a matrix of positions to acquire the image. Line scanners spread the information across a 2D camera sensor (one axis is the spatial dimension (line) and the other is wavelength). For a 2048 × 2048 × 32 spectral cube, a point-scan spectral confocal microscope would require 1.5 μs per pixel, including raster scanning time. This translates to 6.3 seconds per spectral cube. A line-scan fluorescence imaging system reported in [DOI: 10.1038 / ncomms8990] can collect 1500 lines per second. A spectral cube of the same size (2048 x 2048 x 32) takes 1.36 seconds to acquire. The proposed spectral encoding device described herein takes only 30 milliseconds, which is 210 times faster than a point scan and 45 times faster than a line scan.

[0117] While this specification contains details of many specific embodiments, these should not be construed as limitations on the scope of any invention or what may be claimed, but rather as descriptions of features specific to particular embodiments of a particular invention. Also, certain features described herein with respect to separate embodiments may also be implemented in combination in a single embodiment. Conversely, various features described with respect to a single embodiment may also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, while features may be described above as acting in a particular combination and initially claimed as such, one or more features from a claimed combination may in some cases be separated from the combination, and the claimed combination may also relate to a subcombination or a variation of the subcombination.

[0118] Similarly, while the figures may depict operations in a particular order, this should not be understood as requiring such operations to be performed in the particular order or sequence depicted, or that all of the depicted operations be performed, to achieve desired results. In certain situations, multitasking and parallel processing may be advantageous. Moreover, the separation of various system components in the above embodiments should not be understood as requiring such separation in all embodiments, and it should be understood that the described program components and systems may generally be integrated into a single software product or packaged into multiple software products.

[0119] Computer and Hardware Embodiments of the Disclosure It should be understood at the outset that the disclosure herein may be implemented by any type of hardware and / or software, including pre-programmed general-purpose computing devices. For example, the system may be implemented using a server, personal computer, portable computer, thin client, or any suitable device. The disclosure and / or its components may be a single device at a single location, or multiple devices at single or multiple locations connected via any communication medium, e.g., electrical cable, fiber optic cable, or wirelessly, using any suitable communication protocol.

[0120] It should also be noted that the present disclosure is shown and described herein as having multiple modules that perform specific functions. It should be understood that these modules are illustrated generally based on their functionality for clarity only and do not necessarily represent specific hardware or software. In this regard, these modules may be hardware and / or software implemented to substantially perform the specific functions described. Moreover, modules may be combined within the scope of the present disclosure or divided into further modules based on desired specific functions. Therefore, the present disclosure should not be construed as limiting the present invention, but should be understood as merely illustrating one exemplary embodiment thereof.

[0121] A computing system may include clients and servers. Clients and servers are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other. In some embodiments, a server sends data (e.g., HTML pages) to a client device (e.g., to display the data to and receive user input from a user interacting with the client device). Data generated at the client device (e.g., the result of a user interaction) can be received by the server from the client device.

[0122] Embodiments of the subject matter described herein can be implemented as a computing system that includes a back-end component, e.g., a data server, or includes a middleware component, e.g., an application server, or includes a front-end component, e.g., a client computer having a graphical user interface or web browser through which a user can interact with embodiments of the subject matter described herein, or includes any combination of one or more such back-end, middleware, or front-end components. The components of the system can be interconnected by any form or medium of digital data communication, e.g., a communications network. Examples of communications networks include a local area network ("LAN"), a wide area network ("WAN"), an internetwork (e.g., the Internet), and a peer-to-peer network (e.g., an ad-hoc peer-to-peer network).

[0123] Embodiments of the subject matter and operations described herein can be implemented in digital electronic circuitry, or in computer software, firmware, or hardware, including the structures disclosed herein and their structural equivalents, or one or more combinations thereof. Embodiments of the subject matter described herein can be implemented as one or more computer programs, i.e., one or more modules of computer program instructions encoded on a computer storage medium, for execution by or to control the operation of a data processing apparatus. Alternatively or additionally, the program instructions can be encoded on an artificially generated propagated signal, e.g., a machine-generated electrical, optical, or electromagnetic signal generated to encode information for transmission to an appropriate receiver apparatus for execution by the data processing apparatus. A computer storage medium can be, or be included in, a computer-readable storage device, a computer-readable storage substrate, a random or serial access memory array or device, or one or more combinations thereof. Moreover, while a computer storage medium is not a propagating signal, a computer storage medium can be a source or destination of computer program instructions encoded in an artificially generated propagated signal. A computer storage medium may also be, or be included in, one or more separate physical components or media (eg, multiple CDs, disks or other storage devices).

[0124] The operations described herein may be implemented as operations performed by a "controller" on data stored in one or more computer-readable storage devices or received from other sources.

[0125] The term "controller" encompasses all kinds of apparatus, devices, and machines for processing data, including programmable processors, computers, systems on a chip, or multiple systems, or combinations of the above. An apparatus can include special-purpose logic circuitry, such as a field programmable gate array (FPGA) or an application-specific integrated circuit (ASIC). In addition to hardware, an apparatus can also include code that creates an execution environment for a subject computer program, such as code that configures processor firmware, a protocol stack, a database management system, an operating system, a cross-platform runtime environment, a virtual machine, or one or more combinations thereof. The apparatus and execution environment can implement a variety of different computing model infrastructures, such as web services, distributed computing, and grid computing infrastructures.

[0126] A computer program (also known as a program, software, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, declarative or procedural languages, and can be deployed in any form, for example, as a stand-alone program or as a module, component, subroutine, object, or other unit suitable for use in a computing environment. A computer program may, but need not, correspond to a file in a file system. A program can be stored in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files storing one or more modules, subprograms, or portions of code), or in a portion of a file that holds other programs or data (e.g., one or more scripts stored in a markup language document). A computer program can be deployed to run on one computer or on multiple computers located at one site or distributed across multiple sites and interconnected by a communications network.

[0127] The processes and logic flows described herein may be performed by one or more programmable processors executing one or more computer programs to perform operations by manipulating input data and generating output. The processes and logic flows may also be performed by, or devices may be realized as, special purpose logic circuitry, such as a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC).

[0128] Processors suitable for executing a computer program include, by way of example, general-purpose and special-purpose microprocessors, and any one or more processors of any kind of digital computer. Generally, a processor receives instructions and data from a read-only memory or a random-access memory, or both. The essential elements of a computer are a processor for performing operations in accordance with the instructions and one or more memory devices for storing instructions and data. Typically, a computer also includes one or more mass storage devices, such as magnetic, magneto-optical, or optical disks, for storing data, or is operatively coupled to receive data from and transmit data to them. However, a computer need not have such devices. Moreover, a computer can be embedded in another device, such as a mobile phone, a personal digital assistant (PDA), a mobile audio or video player, a game console, a global positioning system (GPS) receiver, or a portable storage device (e.g., a universal serial bus (USB) flash device), to name a few. Suitable devices for storing computer program instructions and data include all forms of non-volatile memory, media, and memory devices, such as, by way of illustration, semiconductor memory devices, e.g., EPROM, EEPROM, and flash memory devices; magnetic disks, e.g., internal hard disks or removable disks; magneto-optical disks; and CD-ROM and DVD-ROM disks. The processor and memory can be supplemented by, or incorporated in, special purpose logic circuitry.

[0129] In some aspects of the present invention, software is provided for performing operations associated with the formulas and calculations provided herein. The software for executing the instructions provided herein may be stored on a non-transitory computer-readable medium, and when executed on a processor or controller, the software performs some or all of the steps of the present invention.

[0130] Selected aspects While the above detailed description and appended claims disclose several embodiments of the present invention, other alternative aspects of the present invention are disclosed in further embodiments below. Aspect 1. a first dichroic mirror; and The second dichroic mirror and Including, the first spectral transmittance curve and the first spectral reflectance curve of the first dichroic mirror have sinusoidal profiles; the second spectral transmittance curve and the second spectral reflectance curve of the second dichroic mirror have cosine wave profiles; Imaging assembly. Aspect 2. 2. The imaging assembly of embodiment 1, wherein a first dichroic mirror generates a first spectrally-encoded transmitted light portion and a first spectrally-encoded reflected light portion; a second dichroic mirror generates a second spectrally-encoded transmitted light portion and a second spectrally-encoded reflected light portion; and the first spectrally-encoded transmitted light portion, the first spectrally-encoded reflected light, the second spectrally-encoded transmitted light, and the second spectrally-encoded reflected light are detected at an imaging sensor. Aspect 3. The imaging assembly of embodiment 2, wherein the imaging sensor is a cMOS sensor. Aspect 4. 2. The imaging assembly of embodiment 1, wherein a first dichroic mirror generates a first spectrally-encoded transmitted light portion and a first spectrally-encoded reflected light portion; a second dichroic mirror generates a second spectrally-encoded transmitted light portion and a second spectrally-encoded reflected light portion; the first spectrally-encoded transmitted light portion is detected via a first detector, the first spectrally-encoded reflected light is detected via a second detector, the second spectrally-encoded transmitted light is detected via a third detector, and the second spectrally-encoded reflected light is detected via a fourth detector. Aspect 5. 5. The imaging assembly of embodiment 4, wherein the first, second, third, and fourth detectors are photomultiplier tubes. Aspect 6. 2. The imaging assembly of embodiment 1, wherein the first and second dichroic mirrors each receive a fluorescent signal from an imaging objective of a microscope. Aspect 7. 2. The imaging assembly of embodiment 1, wherein the microscope is one of a light sheet microscope, a wide-field fluorescence microscope, or a confocal microscope. Aspect 8. 2. The imaging assembly of embodiment 1, further comprising: at least one first routing mirror positioned to receive the first spectrally-encoded transmitted light or the first spectrally-encoded reflected light from the first dichroic mirror; and at least one second routing mirror positioned to receive the second spectrally-encoded transmitted light or the second spectrally-encoded reflected light portion from the second dichroic mirror, wherein the first and second dichroic mirrors and the at least one first and second routing mirrors generate four spectrally-encoded light portions, the four spectrally-encoded light portions comprising the first spectrally-encoded transmitted light, the first spectrally-encoded reflected light, the second spectrally-encoded transmitted light, and the second spectrally-encoded reflected light. Aspect 9. 3. The imaging assembly of embodiment 2, further comprising four tube lenses, each tube lens positioned to receive one of the four spectrally encoded light portions and focus a corresponding spectrally encoded light portion onto the imaging sensor. Aspect 10. 10. The imaging assembly of embodiment 9, further comprising four adjusting mirrors, each arranged to adjust a corresponding angle of each of the spectrally encoded light portions relative to the imaging sensor so that each of the spectrally encoded light portions is imaged in a different quadrant of the imaging sensor. Aspect 11. 2. The imaging assembly of embodiment 1, further comprising a beam splitter positioned to receive the fluorescent signal from the microscope imaging objective, the beam splitter configured to equally split the fluorescent signal into a first fluorescent signal and a second fluorescent signal; and wherein the first fluorescent signal is directed toward a first dichroic mirror and the second fluorescent signal is directed toward a second dichroic mirror. Aspect 12. 12. The imaging assembly of embodiment 11, further comprising one or more additional routing mirrors for directing one or more of the first and second fluorescent signals toward one or more of the first and second dichroic mirrors, respectively. Aspect 13. 12. The imaging assembly of embodiment 11, further comprising one or more relay lenses disposed between the imaging objective and the beam splitter. Aspect 14. 2. The imaging assembly of embodiment 1, further comprising one or more pre-filtering optics, the one or more pre-filtering optics configured to filter out signals outside the spectral ranges of the first and second dichroic mirrors. Aspect 15. 13. The imaging assembly of embodiment 12, further comprising one or more pre-filtering optics configured to filter out signals outside the spectral ranges of the first and second dichroic mirrors, and / or one or more filters configured to filter out wavelengths corresponding to one or more excitation light sources that illuminate the sample being imaged through the imaging objective. Aspect 16. The imaging assembly of embodiment 1, wherein the first and second dichroic mirrors each have a spectral range of 400 nm to 700 nm. Aspect 17. an encoding portion configured to encode the emission light received from the imaging objective of the microscope into a plurality of encoded light channels; Including, the encoding portion includes at least two dichroic mirrors; each of the at least two dichroic mirrors generates coded light having a periodic waveform; Spectral encoding assembly for integration with microscopes. Aspect 18. 20. The assembly of embodiment 17, wherein the spectral encoding assembly is disposed within infinity space of a microscope and between an imaging objective lens and an imaging sensor of the microscope. Aspect 19. 18. The assembly of embodiment 17, wherein the encoding portion further comprises at least one beam splitter for directing the emitted light equally toward at least two dichroic mirrors. Aspect 20. 20. The assembly of embodiment 17, wherein the microscope is either a light sheet microscope, a wide-field fluorescence microscope, or a confocal microscope. Aspect 21. 19. The assembly of embodiment 18, further comprising one tube lens corresponding to each encoded light channel, the one tube lens focusing light from each channel onto a separate portion of the imaging sensor. Aspect 22. 22. The assembly of embodiment 21, wherein the encoding portion includes one or more tuning mirrors for directing each of the multiple encoded light channels from each of the tube lenses toward a separate portion of the imaging sensor. Aspect 23. 22. The assembly of embodiment 21, wherein the imaging sensor is a cMOS sensor. Aspect 24. a pair of relay lenses and a ring-operated iris diaphragm at an intermediate image plane between the pair of relay lenses; an opening amount of the ring-actuated iris diaphragm based on an imaging area on the imaging sensor such that respective images from each of the plurality of channels do not overlap; Assembly of embodiment 21. Aspect 25. 18. The assembly of embodiment 17, further comprising one or more pre-filtering optics between the imaging objective and the encoding portion configured to filter out emission signals outside the spectral range of the at least two dichroic mirrors, and / or one or more filters configured to filter out wavelengths corresponding to one or more excitation light sources that illuminate the sample being imaged through the imaging objective. Aspect 26. 18. The assembly of embodiment 17, further comprising relay optics between the imaging objective and the encoding portion. Aspect 27. 18. The assembly of embodiment 17, wherein the at least two dichroic mirrors each have a spectral range that includes wavelengths in the visible spectrum. Aspect 28. 20. The assembly of embodiment 18, wherein the imaging sensor is communicatively coupled to the imaging processor. Aspect 29. an imaging objective lens for acquiring a fluorescent signal from the sample; an imaging sensor; a spectral encoding device disposed between the imaging objective and the imaging sensor; Including, the spectral encoding device includes a first dichroic mirror and a second dichroic mirror; the spectral encoding device generates, via the first and second dichroic mirrors, four encoded optical channels, each having a periodic waveform; Imaging system. Aspect 30. 30. The imaging system of embodiment 29, wherein the four encoded light channels include a first transmitted light channel and a first reflected light channel from a first dichroic mirror, and a second transmitted light channel and a second reflected light channel from a second dichroic mirror. Aspect 31. The method further includes a controller including executable instructions stored in a non-transitory memory, the instructions, when executed, causing the controller to: determining a normalized intensity by calculating the integral of the intensity values of the four encoded optical channels; acquiring, for each of the four encoded optical channels, a corresponding channel image via an imaging sensor; normalizing each corresponding channel image according to the normalized intensity; The imaging system of embodiment 30. Aspect 32. The controller includes further executable instructions stored in a non-transitory memory that, when executed, cause the controller to: generating a hyperspectral or multispectral image according to each normalized channel image; 32. The imaging system of embodiment 31. Aspect 33. receiving a fluorescent signal from the biological sample with a spectral encoding device; generating at least two transmitted light channels and at least two reflected light channels via the spectral encoding device; and imaging the at least two transmitted light channels and the at least two reflected light channels with an imaging sensor. Including, the spectral encoding device includes at least two dichroic mirrors, each of the two dichroic mirrors having a transmittance and reflectance profile resembling a periodic wave form; Methods for spectral fluorescence imaging. Aspect 34. 34. The method of embodiment 33, further comprising the step of registering an image formed by each of the at least two transmitted light channels with an image formed by each of the at least two reflected light channels. Aspect 35. performing a phasor analysis on the aligned images to generate a phasor plot; and generating an unmixed image according to the phasor plot; 35. The method of embodiment 34, further comprising:

[0131] Conclusion The various methods and techniques described above provide several ways to implement the present invention. Of course, it should be understood that not all of the described objectives or advantages can necessarily be achieved in accordance with any particular embodiment described herein. Thus, for example, one skilled in the art will recognize that a method may be implemented in a manner that achieves or optimizes one advantage or group of advantages taught herein, but does not necessarily achieve other objectives or advantages taught or suggested herein. Various alternative embodiments are included herein. It should be understood that some embodiments specifically include one, another, or several features, other embodiments specifically exclude one, another, or several features, and still other embodiments mitigate certain features by including one, another, or several advantageous features.

[0132] Additionally, those skilled in the art will recognize the applicability of various features from various embodiments. Similarly, the various elements, features, and steps described above, as well as other known equivalents of each such element, feature, or step, can be used in various combinations by those skilled in the art to implement methods in accordance with the principles described herein. Some of the various elements, features, and steps are explicitly included in various embodiments, while others are explicitly excluded.

[0133] While the present application has been disclosed with respect to particular embodiments and examples, it will be understood by those skilled in the art that the embodiments of the present application extend beyond the specifically disclosed embodiments to other alternative embodiments and / or uses, and variations and equivalents thereof.

[0134] In some embodiments, the use of singular articles (“a,” “an,” and “the”) and similar designations in the context of describing particular embodiments of the present application (particularly in the context of some of the claims below) can be construed to encompass both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of all examples or exemplary language (e.g., “such as”) provided with respect to particular embodiments of the present application is intended merely to better describe the application and does not pose a limitation on the scope of the application as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0135] Certain aspects of the present application are described herein. Variations of these aspects will become apparent to those of skill in the art upon reading the foregoing description. Those of skill in the art will be able to employ such variations as appropriate, and it is understood that the present application can be practiced otherwise than as specifically described herein. Accordingly, many aspects of the present application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present application unless otherwise indicated herein or otherwise clearly contradicted by context.

[0136] Specific embodiments of the subject matter have been described. Other embodiments are within the scope of the following claims. In some cases, the actions recited in the claims can be performed in a different order and still achieve desirable results. Additionally, the processes depicted in the accompanying figures do not necessarily require the particular order or sequence shown to achieve desirable results.

[0137] All patents, patent applications, patent application publications, and other materials, such as books, specifications, publications, documents, articles, etc., referenced herein are incorporated by reference in their entirety for all purposes, except for any related prosecution history, which may be inconsistent with or conflict with this document, or which may have a limiting effect on the broadest scope of any patent claims now or hereafter related to this document. Illustratively, if there is an inconsistency or conflict between the description, definition, and / or usage of a term associated with any of the incorporated documents and the description, definition, and / or usage of that term associated with this document, the description, definition, and / or usage of that term in this document will control.

[0138] Finally, it should be understood that the aspects of the present application disclosed herein are illustrative of the principles of the aspects of the present application. Other variations that may be employed may fall within the scope of the present application. Thus, by way of example, and not of limitation, alternative forms of the aspects of the present application may be utilized in accordance with the teachings herein. Thus, the aspects of the present application are not limited to the precise embodiments shown and described.

Claims

1. A method of projecting a first spectrally encoded transmitted light portion and a first spectrally encoded reflected light portion, comprising: a second dichroic mirror that generates a second spectrally encoded transmitted light portion and a second spectrally encoded reflected light portion; Including, the first spectral transmittance curve and the first spectral reflectance curve of the first dichroic mirror have sinusoidal profiles; the second spectral transmittance curve and the second spectral reflectance curve of the second dichroic mirror have cosine wave profiles; the first spectrally encoded transmitted light portion, the first spectrally encoded reflected light, the second spectrally encoded transmitted light, and the second spectrally encoded reflected light are detected by at least one imaging sensor or at least one detector; Imaging assembly.

2. 10. The imaging assembly of claim 1, wherein the imaging sensor is a scientific complementary metal oxide semiconductor sensor (sCMOS sensor).

3. 10. The imaging assembly of claim 1, further comprising a fourth detector, wherein the second spectrally encoded reflected light is detected via the fourth detector, and wherein the first, second, third, and fourth detectors are photomultiplier tubes.

4. 10. The imaging assembly of claim 1, wherein each of the first and second dichroic mirrors receives a fluorescent signal from an imaging objective of a microscope.

5. 5. The imaging assembly of claim 4, wherein the microscope is one of a light sheet microscope, a wide-field fluorescence microscope, or a confocal microscope.

6. 10. The imaging assembly of claim 1, further comprising: at least one first routing mirror positioned to receive the first spectrally-encoded transmitted light or the first spectrally-encoded reflected light from the first dichroic mirror; and at least one second routing mirror positioned to receive the second spectrally-encoded transmitted light or the second spectrally-encoded reflected light portion from the second dichroic mirror, wherein the first and second dichroic mirrors and the at least one first and second routing mirror generate four spectrally-encoded light portions, the four spectrally-encoded light portions comprising the first spectrally-encoded transmitted light, the first spectrally-encoded reflected light, the second spectrally-encoded transmitted light, and the second spectrally-encoded reflected light.

7. 7. The imaging assembly of claim 6, further comprising four tube lenses, each tube lens positioned to receive one of the four spectrally encoded light portions and focus a corresponding spectrally encoded light portion onto the imaging sensor.

8. 8. The imaging assembly of claim 7, further comprising four adjusting mirrors, each arranged to adjust a corresponding angle of each of the spectrally encoded light portions relative to the imaging sensor so that each of the spectrally encoded light portions is imaged in a different quadrant of the imaging sensor.

9. A method of measuring a light beam from a first dichroic mirror, the first dichroic mirror having a first spectral transmittance curve and a first spectral reflectance curve each having a sinusoidal profile; a second dichroic mirror, wherein a second spectral transmittance curve and a second spectral reflectance curve of the second dichroic mirror have cosine wave profiles; and a beam splitter positioned to receive a fluorescent signal from an imaging objective of a microscope, the beam splitter configured to equally split the fluorescent signal into a first fluorescent signal and a second fluorescent signal; the first fluorescent signal being directed toward a first dichroic mirror and the second fluorescent signal being directed toward a second dichroic mirror; and an imaging assembly including:

10. 10. The imaging assembly of claim 9, further comprising one or more additional routing mirrors for directing one or more of the first and second fluorescent signals toward one or more of the first and second dichroic mirrors, respectively.

11. 10. The imaging assembly of claim 9, further comprising one or more relay lenses disposed between the imaging objective and the beam splitter.

12. A method of measuring a light beam from a first dichroic mirror, the first dichroic mirror having a first spectral transmittance curve and a first spectral reflectance curve having a sinusoidal profile; a second dichroic mirror, wherein a second spectral transmittance curve and a second spectral reflectance curve of the second dichroic mirror have cosine wave profiles; and one or more pre-filtering optics configured to filter out signals outside the spectral ranges of the first and second dichroic mirrors; and an imaging assembly including:

13. 11. The imaging assembly of claim 10, further comprising one or more pre-filtering optics configured to filter out signals outside the spectral range of the first and second dichroic mirrors, and / or one or more filters configured to filter out wavelengths corresponding to one or more excitation light sources that illuminate the sample being imaged through the imaging objective.

14. receiving a fluorescent signal from the biological sample with a spectral encoding device; generating at least two transmitted light channels and at least two reflected light channels via the spectral encoding device; and imaging the at least two transmitted light channels and the at least two reflected light channels with an imaging sensor. Including, the spectral encoding device includes at least two dichroic mirrors, each of the two dichroic mirrors having a transmittance and reflectance profile resembling a periodic wave form; Methods for spectral fluorescence imaging.

Images