WO2022234484A1

WO2022234484A1 - Devices and methods for rapid channel-by-channel fluorescence microscopy

Info

Publication number: WO2022234484A1
Application number: PCT/IB2022/054127
Authority: WO
Inventors: Sebastian Chakrit Punyaratabandhu Bhakdi; John Lea; Jeremy UFF
Original assignee: X-Zell Biotech Pte Ltd
Priority date: 2021-05-04
Filing date: 2022-05-04
Publication date: 2022-11-10

Abstract

A fluorescence microscope can perform a method of rapid channel-by-channel fluorescence microscopy that comprises exciting at least n+1 emission wavelengths from a sample; capturing a first set of images in a first scan of the sample and a second set of images on a second scan of the sample wherein each image of a set is captured simultaneously and separately on a distinct image capture device; wherein at least a first image of the first set and at least a second image of the second set capture an identical selection of the emission wavelengths; calculating an alignment shift between at least the first and second images; applying the alignment shift to at least one image; and combining at least one image of the first set with at least one image of the second set to create an aligned composite image.

Description

DEVICES AND METHODS FOR RAPID CHANNEL-BY-CHANNEL FLUORESCENCE MICROSCOPY

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the priority benefit of U.S. Provisional Patent

Application No. 63/183,796, filed on May 4, 2021, the disclosure of which is incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

[0002] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

TECHNICAL FIELD

[0003] This disclosure relates generally to the field of microscopy, and more specifically to the field of fluorescence microscopy. Described herein are systems and methods for rapid channel-by-channel fluorescence microscopy.

BACKGROUND

[0004] Researchers and clinical pathologists increasingly rely upon multi-stain fluorescence microscopy experiments to visualize and categorize tissue morphology. By utilizing a plurality of stains that each bind or adhere to different tissue or cell portions, a detailed image of the sample can be generated that can reveal much regarding tissue and cell morphology and health. However, in order to resolve each of the unique stains, traditional fluorescence microscopes rely upon a convoluted system of filters or excitation strategies that make for lengthy and tedious image capture process. Because modem microscope technologies cannot reliably return a scanning excitation beam path to a specific point on the sample with sufficient accuracy, standard fluorescence microscopes must collect fluorescence image data for each emission wavelength of interest at every position (i.e., field of view) of the scan across the whole sample. In other words, when using eight different fluorescent tags, for example, modem microscope technologies must collect fluorescence image data for each of the eight different fluorescent tags at a first field of view (i.e., cycling through all the parameters (e.g., filters) needed for each fluorescent tag) before moving onto a second field of view. At the second field of view, the process is repeated for all eight fluorescent tags, and so on for each subsequent field of view of the sample. This cycling time rapidly adds up to the point where more time can be spent cycling the filters than collecting data, even for small samples, and can become almost prohibitively lengthy for larger samples or those featuring a great number of unique stains. For example, image acquisition can take only about 30 to 50 milliseconds while cycling filters on a filter wheel can take about 50 to upwards of 500 milliseconds depending on the mechanism. Because of these lengthy image acquisition times, use of existing scanning technologies at the point-of-view is unfathomable.

[0005] Their remains a present need for new devices and methods for improving the speed of multi-stain fluorescence microscopy image capture and data collection, which would greatly improve the efficiency of pathology labs in hospitals and other healthcare facilities as well as increase the accessibility of multi-stain experiments to research groups.

SUMMARY

[0006] There is a need for new and useful device and method for performing fluorescence microscopy. In some aspects, the disclosure includes for a method of performing rapid channel-by-channel fluorescence microscopy with a fluorescence microscope having a plurality of image capture devices comprising: exciting at least n+1 emission wavelengths from a sample, wherein n is equal to a number of image capture devices available, n being greater than 1; capturing a first set of images of the sample in a first scan of the sample wherein each image of the first set of images is captured simultaneously and separately on a distinct image capture device; capturing a second set of images of the sample in a second scan of the sample wherein each image of the second set of images is captured simultaneously and separately on a distinct image capture device; wherein at least a first image of the first set and at least a second image of the second set capture an identical selection of one or more of the at least n+1 emission wavelengths; calculating an alignment shift between at least the first and second images; applying the alignment shift to at least one image of the first set of images or to at least one image of the second set of images; and combining at least one image of the first set with at least one image of the second set to create an aligned composite image.

[0007] In some embodiments, the first set of images further comprises at least one image capturing a different selection of one or more of the at least n+1 emission wavelengths than all the images of the second set. In further embodiments, the second set of images further comprises at least one image capturing a different selection of one or more of the at least n+1 emission wavelengths than all the images of the first set. In some embodiments, n is 2. In further embodiments, the first set of images comprises two images. In additional embodiments, the second set of images comprises two images. In other embodiments, n is 3. In further embodiments, the first set of images comprises three images. In additional embodiments, the second set of images comprises three images. In still other embodiments, n is greater than 3. In further embodiments, the first set of images comprises more than three images. In additional embodiments, the second set of images comprises more than three images.

[0008] In some embodiments, combining comprises forming a Z-stack of the at least one image of the first set and the at least one image of the second set. In further embodiments, the first image and the second image are reference images. In other embodiments, each image capture device of the plurality of image capture devices are independently selected from the group consisting of: a camera, a portion of a camera’s CCD array, and a portion of a camera’s sCMOS chip. In further embodiments, no pair of images within the first set of images and no pair of images within the second set of images capture a selection of emission wavelengths with a crosstalk intensity greater than 5%.

[0009] In some aspects, the disclosure includes for a fluorescence microscope comprising: at least one light source; an excitation beam path that passes light from the at least one light source through a sample stage, wherein at least one of the excitation beam path and the sample stage is configured to move an area of the sample stage through the excitation beam path as a scan; an emission beam path that passes emitted light of a plurality of predetermined emission wavelengths towards at least a first and second image capture device, wherein the emission beam path comprises at least one beam splitter to separate a first and second subdivision of the plurality of emission wavelengths and wherein the at least one beam splitter directs the first subdivision towards the first image capture device and the second subdivision towards the second image capture device; and a processor and a memory storing machine-readable instructions that, when executable by the processor, cause the processor to perform a method comprising: capturing a first set of images of a sample in a first scan wherein each image of the first set of images is captured simultaneously and separately on one of the at least first or second image capture devices; capturing a second set of images of the sample in a second scan wherein each image of the second set of images is captured simultaneously and separately on one of the at least first or second image capture devices; wherein at least a first image of the first set and at least a second image of the second set capture an identical selection of one or more emission wavelengths of a total of at least n+1 emission wavelengths, wherein n is equal to a total number of image capture devices; calculating an alignment shift between at least the first and second images that capture an identical selection of one or more of the at least n+1 emission wavelengths; applying the alignment shift to at least one of the first set of images or the second set of images; combining at least one image of the first set with at least one image of the second set to create an aligned composite image.

[0010] In some embodiments, the first set of images further comprises at least one image capturing a different selection of one or more of the at least n+1 emission wavelengths than all the images of the second set. In further embodiments, the second set of images further comprises at least one image capturing a different selection of one or more of the at least n+1 emission wavelengths than all the images of the first set.

[0011] In some embodiments, at least one of the excitation beam path and the emission beam path comprises a filter turret configured to automatically add, remove, or replace a first filter with a second filter during a period of time between the first scan and the second scan. [0012] In some embodiments, the emission beam path further comprises a beam splitter turret configured to automatically add, remove, or replace a first beam splitter with a second beam splitter during a period of time between the first scan and the second scan. In additional embodiments, the at least one beam splitter of the emission path is a dichroic filter or a dichroic mirror.

[0013] In some embodiments, n is 2. In further embodiments, the first set of images comprises two images. In additional embodiments, the second set of images comprises two images. In some embodiments, n is 3. In further embodiments, the first set of images comprises three images. In additional embodiments, the second set of images comprises three images. In some embodiments, n is greater than 3. In further embodiments, the first set of images comprises more than three images. In additional embodiments, the second set of images comprises more than three images.

[0014] In some embodiments, combining comprises forming a Z-stack of the at least one image of the first set and the at least one image of the second set. In further embodiments, the first image and the second image are reference images. In additional embodiments, each image capture device of the plurality of image capture devices are independently selected from the group consisting of: a camera, a portion of a camera’s CCD array, and a portion of a camera’s sCMOS chip. In further embodiments, no pair of images within the first set of images and no pair of images within the second set of images capture a selection of emission wavelengths with a crosstalk intensity greater than 5%.

[0015] In some aspects, the disclosure herein includes for a fluorescence microscope comprising: at least one light source; an excitation beam path that passes light from the at least one light source through a sample stage, wherein at least one of the excitation beam path and the sample stage is configured to move an area of the sample stage through the excitation beam path as a scan; an emission beam path that passes emitted light of a plurality of predetermined emission wavelengths towards at least a first and second image capture device, wherein the emission beam path comprises at least one beam splitter to separate a first and second subdivision of the plurality of emission wavelengths and wherein the at least one beam splitter transmits the first subdivision towards the first image capture device and reflects the second subdivision towards the second image capture device; wherein light from the excitation beam path is reflected by a first, second, or third dichroic mirror onto the sample stage, and wherein emission wavelengths from the sample are transmitted through the first, second or third dichroic mirror; wherein the first dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 488, BV 421, BV 480, DRAQ5, and PerCP or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the second dichroic mirror reflects the excitation wavelengths of the fluorescent stains of PE and DRAQ5 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the third dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 594 and DRAQ5 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the first, second, and third dichroic mirror can be exchanged in an operable position; wherein the at least one beam splitter is a 660 nm long pass dichroic mirror; wherein a first filter wheel is filter is positioned between the first image capture device and the beam splitter wherein the first filter wheel comprises filters of 810/90 and 697/60; and wherein a second filter wheel is positioned between the second image capture device and the beam splitter wherein the second filter wheel comprises filters of 431/28, 480/30, 537/29, 572/23, and 630/28.

[0016] In some aspects, the disclosure herein includes for a fluorescence microscope comprising: at least one light source; an excitation beam path that passes light from the at least one light source through a sample stage, wherein at least one of the excitation beam path and the sample stage is configured to move an area of the sample stage through the excitation beam path as a scan; an emission beam path that passes emitted light of a plurality of predetermined emission wavelengths towards at least a first and second image capture device, wherein the emission beam path comprises at least one beam splitter to separate a first and second subdivision of the plurality of emission wavelengths and wherein the at least one beam splitter transmits the first subdivision towards the first image capture device and reflects the second subdivision towards the second image capture device; wherein light from the excitation beam path is reflected by a first, second, third, or fourth dichroic mirror onto the sample stage, and wherein emission wavelengths from the sample are transmitted through the first, second, third, or fourth dichroic mirror; wherein the first dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 488, BV 421, BV 480, DRAQ5, and PerCP or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the second dichroic mirror reflects the excitation wavelengths of the fluorescent stains of PE-Cy7 and BV 480 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the third dichroic mirror reflects the excitation wavelengths of the fluorescent stains of PE and DRAQ5 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the fourth dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 594 and DRAQ5 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the first, second, third, and fourth dichroic mirror can be exchanged in an operable position; wherein the at least one beam splitter is a 647 nm short pass dichroic mirror; wherein a first filter wheel is positioned between the first image capture device and the beam splitter, wherein the first filter wheel comprises filters of 431/28, 480/30, 537/29, 572/23, and 630/28; and wherein a second filter wheel is positioned between the second image capture device and the beam splitter wherein the second filter wheel comprises filters of 810/90 and 697/60.

[0017] In some aspects, the disclosure includes for a fluorescence microscope comprising: at least one light source; an excitation beam path that passes light from the at least one light source through a sample stage, wherein at least one of the excitation beam path and the sample stage is configured to move an area of the sample stage through the excitation beam path as a scan; an emission beam path that passes emitted light of a plurality of predetermined emission wavelengths towards at least a first, second, and third image capture device, wherein the emission beam path comprises at least a first and second beam splitter; wherein the first beam splitter separates a first and second subdivision of the plurality of emission wavelengths, and wherein the first beam splitter transmits the first subdivision towards the first image capture device and reflects the second subdivision towards the second image capture device; wherein the second beam splitter separates a third subdivision of the plurality of emission wavelengths from the first and second subdivision of plurality of emission wavelengths, and wherein the second beam splitter transmits the first and second subdivision towards the first beam splitter and reflects the third subdivision towards the third image capture device; wherein light from the excitation beam path is reflected by a first, second, or third dichroic mirror onto the sample stage, and wherein emission wavelengths from the sample are transmitted through the first, second, or third dichroic mirror, wherein the first dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 594, BV 480, and DRAQ5 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the second dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 488, DRAQ5, and BV 421 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the third dichroic mirror reflects the excitation wavelengths of the fluorescent stains of PE, PerCP, and BV 421 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the first, second, and third dichroic mirror can be exchanged in an operable position; wherein the first beam splitter is a 455 nm long pass dichroic mirror; wherein the second beam splitter is a 647 nm short pass dichroic mirror; wherein a first filter wheel is positioned between the first image capture device and the first beam splitter, wherein the first filter wheel comprises filters 537/29, 572/23, and 630/28; wherein a second filter wheel is positioned between the second image capture device and the first beam splitter wherein a third filter wheel is positioned between the third image capture device and the second splitter wherein the third filter wheel comprises filters of 810/90 and 697/60.

[0018] In some aspects, the disclosure herein includes for a fluorescence microscope comprising: at least one light source; an excitation beam path that passes light from the at least one light source through a sample stage, wherein at least one of the excitation beam path and the sample stage is configured to move an area of the sample stage through the excitation beam path as a scan; an emission beam path that passes emitted light of a plurality of predetermined emission wavelengths towards at least a first, second, and third image capture device, wherein the emission beam path comprises at least a first and second beam splitter; wherein the first beam splitter separates a first and second subdivision of the plurality of emission wavelengths, and wherein the first beam splitter transmits the first subdivision towards the first image capture device and reflects the second subdivision towards the second image capture device; wherein the second beam splitter separates a third subdivision of the plurality of emission wavelengths from the first and second subdivision of plurality of emission wavelengths, and wherein the second beam splitter transmits the first and second subdivision towards the first beam splitter and reflects the third subdivision towards the third image capture device; wherein light from the excitation beam path is reflected by a first, second, third, or fourth dichroic mirror onto the sample stage, and wherein emission wavelengths from the sample are transmitted through the first, second, third, or fourth dichroic mirror; wherein the first dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 594, BV 480, and DRAQ5 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the second dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 488, DRAQ5, and BV 421 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the third dichroic mirror reflects the excitation wavelengths of the fluorescent stains of PE, PerCP, and BV 421 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the fourth dichroic mirror reflects the excitation wavelengths of the fluorescent stains of PE-Cy7 and BV 480 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the first, second, third, and fourth dichroic mirror can be exchanged in an operable position; wherein the first beam splitter is a 455 nm long pass dichroic mirror; wherein the second beam splitter is a 647 nm short pass dichroic mirror; wherein a first filter wheel is positioned between the first image capture device and the first beam splitter, wherein the first filter wheel comprises filters 537/29, 572/23, and 630/28; wherein a second filter wheel is positioned between the second image capture device and the first beam splitter, and wherein a third filter wheel is positioned between the third image capture device and the second splitter wherein the third filter wheel comprises filters of 810/90 and 697/60.

BRIEF DESCRIPTION OF THE DRAWINGS [0019] The foregoing is a summary, and thus, necessarily limited in detail. The above- mentioned aspects, as well as other aspects, features, and advantages of the present technology are described below in connection with various embodiments, with reference made to the accompanying drawings. [0020] FIG. 1 illustrates a block diagram of a one embodiment of the device.

[0021] FIG. 2 illustrates a detailed block diagram of another embodiment the device.

[0022] FIG. 3 presents a photograph of one embodiment of the device.

[0023] FIG. 4 illustrates a flowchart depicting a method for performing rapid channel-by- channel fluorescence microscopy.

[0024] FIG. 5 illustrates a cartoon of the image alignment process.

[0025] FIG. 6 illustrates a flowchart depicting a method for calibrating at least two image capture devices of a microscope.

[0026] FIG. 7 depicts an embodiment of a calibration diagram.

[0027] FIG. 8 illustrates one example of a portion of the device having two image capture devices.

[0028] FIG. 9 illustrates a second example of a portion of the device having two image capture devices.

[0029] FIG. 10 illustrates an example of a portion of the device having three image capture devices.

[0030] FIG. 11 illustrates an example of a portion of the device having three image capture devices.

[0031] FIG. 12 illustrates a second example of a portion of the device having four image capture devices.

[0032] FIG. 13 illustrates a second example of a portion of the device having four image capture devices.

[0033] FIG. 14 illustrates an example of a light source arrangement for one embodiment of the device.

[0034] FIG. 15 illustrates an example of a laser focus system for use with the embodiments described herein.

[0035] FIG. 16 illustrates an example slide and focus map to be used with the embodiments described herein.

[0036] FIG. 17 illustrates an example of using an offset value for focusing on an image plane.

[0037] The illustrated embodiments are merely examples and are not intended to limit the disclosure. The schematics are drawn to illustrate features and concepts and are not necessarily drawn to scale. DETAILED DESCRIPTION

[0038] The foregoing is a summary, and thus, necessarily limited in detail. The above- mentioned aspects, as well as other aspects, features, and advantages of the present technology will now be described in connection with various embodiments. The inclusion of the following embodiments is not intended to limit the disclosure to these embodiments, but rather to enable any person skilled in the art to make and use the contemplated invention(s). Other embodiments may be utilized, and modifications may be made without departing from the spirit or scope of the subject matter presented herein. Aspects of the disclosure, as described and illustrated herein, can be arranged, combined, modified, and designed in a variety of different formulations, all of which are explicitly contemplated and form part of this disclosure.

[0039] Throughout the disclosure herein, various fluorescent stains will be referred to by their trade name. Examples include DRAQ5™, PerCP, PE, PE-Cy7, Brilliant Violet 421 ™, Brilliant Violet 480™ , Alexa Fluor 488™, Alexa Fluor 594™, and Alexa Fluor 800™. Some of these acronyms refer to brand names, e.g., AF stands for; BV stands for Brilliant Violet™. Others stand for the chemical name itself, e.g., PE stands for R-Phycoerythrin, and PerCP stands for peridinin-chlorophyll-protein complex. However, one of skill in the art will appreciate the common usage of these names to refer to the dyes as their generic RJPAC names can be quite long and unwieldy. Furthermore, one of skill in the art will appreciate that certain fluorescent dyes can be considered “equivalent dyes” of other dyes due to their immense similarity in excitation and emission spectra despite minor changes in chemical structure. For example, DyLight 488™ or Atto 488™ can be functionally interchanged with Alexa Fluor 488™ with no modification needed to the experimental set up of a fluorescent microscope. Therefore, whenever a given fluorescent dye is named herein as an example, it should be understood that one or more equivalent dyes can be used instead regardless of whether the disclosure herein recites “or an equivalent dye.” See Table 7 for a representative but incomplete list of equivalent dyes available at the time of filing.

[0040] As used herein, the term “wavelength” is intended to refer to both a specific, singular wavelength as well as a band of wavelengths up to about 150 nm in bandwidth. As appreciated by those of skill in the art, it can be convenient to discuss fluorescence excitation and emission wavelengths in the singular (e.g., the DRAQ5™ stain is excited at 647 nm and emits at 681 nm); however, in actual practice, there is an operational width to each of these peak values that is valuable and effective to employ. As described herein, various filters, dichroic mirrors, and other optical elements are employed to adequately transmit but isolate at least a portion of the width of each of these excitation and emission wavelength peaks, even when described in the singular. For example, when described herein that an image capture device captures the emission wavelength of DRAQ5™, it can be collecting light in the range of about 667 nm to about 727 nm depending on the particular arrangement of filters and other optical elements in the embodiment. Therefore, use of the term “wavelength” should not limit the disclosure to examples where indeed only a singular wavelength of light (i.e., having no or minimal bandwidth) is used.

[0041] Similarly, the term “selection of wavelengths,” as used herein, is intended to refer to a selection of one or more excitation or emission wavelengths, often denoted by the excitation or emission peak wavelength value and thus inherits the flexibility of the term “wavelength” as described above. For example, a “selection of wavelengths,” in certain embodiments, can be light of one or more singular wavelengths, each having no bandwidth. In other embodiments, a “selection of wavelengths” can be one or more wavelengths each having continuous bandwidth. In some embodiments, the bandwidths of the one or more wavelengths in a selection can overlap. In other embodiments, the bandwidths of wavelengths in a selection do not overlap or share a value. In further embodiments, a “selection of wavelengths” can be a mixture of one or more wavelengths having no bandwidth and one or more wavelengths having bandwidth which both can and cannot overlap across various embodiments. For example, when it is said herein that an image capture device captures a selection of wavelengths consisting of the DRAQ5™ emission wavelength, this can include both an embodiment of 681 nm only (i.e., having no bandwidth) and an embodiment that captures a bandwidth of wavelengths near, surrounding, and including 681 nm (e.g., about 667 nm to about 727 nm). As another example, if it is said that an image capture device captures a selection of wavelengths consisting of the BV 421 and BV 480 stains’ emission wavelengths in an embodiment, this can include both an embodiment of capturing 422 nm and 478 nm (the emission peaks of the BV 421 and BV 480, respectively) with no bandwidth as well as embodiments where one or both of the wavelengths includes bandwidth as described above.

[0042] As used herein, the term “scan” refers to a complete collection of image data by a microscope across an entire region of interest of a sample that is greater than the operating field-of-view of the microscope and therefore necessitates a mechanical movement of the sample through the excitation beam path. In embodiments wherein a single field-of-view is coextensive with or larger than the region of interest, no scan is needed.

[0043] As used herein, the term “image data” refers to data collected by an image capture device for a given one or more emission wavelengths for a given field-of-view. In many embodiments, image data comprises the presence/absence of light striking the various detectors of the image capture device (e.g., a CCD pixel). In further embodiments, image data can further comprise light intensity per each detector. In other embodiments, image data can further comprise wavelength of incident light. During a scan of a sample, image data will be collected at every data collection position of the excitation beam path across the region of interest of the sample.

[0044] As used herein, the term “image” refers to a compilation of image data of one or more emission wavelength selections from each data collection position during a scan. For example, all image data captured of a singular emission wavelength during a single scan can be compiled into a fluorescence image of that singular emission wavelength. In various embodiments, a plurality of fluorescence images can be combined to form a composite fluorescence image that depicts a presentation of two or more emission wavelengths of the constituting individual images. In certain embodiments wherein there is no scan, the image data collected from the singular field-of-view is equivalent to the image.

[0045] Disclosed herein are systems and methods for rapid channel-by-channel fluorescence microscopy. As described above, the inability of modem technology to accurately return to any given point in a scan forces pathologists and researchers to collect image data for every emission wavelength of interest at every data collection position of the excitation beam path of a scan across the region of interest of the sample. For many users and facilities, especially those using a single camera, this then involves the tedious, even when automated, operation of cycling through the full gamut of filters and/or excitation light sources at every position that greatly extends the time needed to capture a full composite image depicting all the emission wavelengths of interest.

[0046] The disclosure herein obviates this long-standing methodology by employing a channel-by-channel approach featuring at least two image capture devices and image alignment technology. Instead of cycling filters, the systems and methods described herein employ at least one beam splitter and/or filter that allows for the simultaneous capture of two or more images of different emission wavelength selections with each image collected by a unique image capture device (i.e., on unique “channels”), during a single scan. Images collected simultaneously during a scan can be considered as constituting a set since their image capture data will spatially correlate (i.e., are spatially aligned) since they were taken at the same position of the excitation beam path. In some embodiments, however, the image capture devices can be first calibrated in order to compensate for slight mechanical misalignments as well as an inherent difference in scale of the images if the image capture devices are positioned at different beam path lengths in the microscope. In order to capture a fluorescence image involving more than two emission wavelength selections, the systems and methods herein return the sample to the scan’s starting position as best as modern technology will allow, and change any number of beam splitters, filters, and excitation wavelengths (in many cases, by automated mechanisms) such that on a second or additional scan of the sample at least one channel will capture an image having an identical selection of one or more emission wavelengths to an image captured in the first scan while any other channels of the second scan can capture new selections of emission wavelengths.

[0047] After the completion of the second scan, the systems and methods disclosed herein can have generated two sets of images with each set of images including an image of the same selection of one or more emission wavelengths. In some embodiments, these two images can be considered “reference images.” In many embodiments, the reference images will be practically identical except for a spatial offset in the xy-plane at one, more than one, or all of the sample positions during a scan due to inaccuracies of the mechanism that moves the sample relative to the excitation beam path as well as any other possible errors common to fluorescence microscopy. The spatial offset can vary in direction or magnitude at each sample position. This spatial offset for each relevant sample position can be calculated (e.g., by a processor of the system), and then that spatial offset (i.e., the set of corrections in the xy- plane for each position) can be applied to images of one of the sets in order to spatially align the images between sets. Finally, these aligned images can then be compiled or stacked to form a composite aligned fluorescence image.

[0048] Although the disclosure herein describes the execution of at least two scans of a region of interest of a sample, the lack of any optics cycling during a scan greatly reduces the time necessary to generate the complete composite image. In many embodiments, using an increasing number of image capture devices further accelerates the process. For example, capturing nine fluorescent stains with a 40x objective by the traditional method can take around two hundred and ninety minutes with about two hundred and forty minutes of that duration spent cycling filters. Embodiments of the dual image capture device setup as described herein can generate the same image in about sixty-four minutes with comparable image quality. Using a quadruple image capture device setup as described herein can further reduce the time about thirty minutes with comparable image quality.

SYSTEMS AND DEVICES

[0049] The device functions to capture fluorescence microscopy images. In some embodiments, the device functions to perform rapid channel-by-channel fluorescence microscopy. The device is used for microscopy, often medical imaging of tissues or cells, but can additionally or alternatively be used for any suitable applications, clinical, industrial, or otherwise. The device can be configured and/or adapted to function for any suitable situation requiring the collection of image data under a plurality of optical parameters from a sample or source.

[0050] FIG. 1 depicts a simple block diagram of one embodiment of the device 100. Generally, the device 100 can comprise a light source 102 that directs light via an excitation beam path 104 onto a sample stage 106 that secures a sample (not shown) that has been suitably stained with fluorescent stains. In various embodiments, the light source 102 can be one or more light producing elements (e.g., bulbs, LEDs, etc.) that can produce one or more wavelengths of light. In further embodiments, the excitation beam path 104 can comprise various optical elements (e.g., prisms, mirrors, filters, etc.) for the combining, focusing, and/or filtering of light generated by the light source 102. In other embodiments, the excitation beam path 104 can further comprise additional optical elements (e.g., a dichroic mirror) to redirect excitation light in a geometry suitable for efficient performance of fluorescence microscopy experiments (e.g., the example arrangement depicted in FIG. 2) as appreciated by those of skill in the art. In some embodiments, the light of excitation beam path 104 is “ON” for the entire duration of a scan (e.g., the beam is continuously shown through the sample.) In other embodiments, light of the excitation beam path 104 is pulsed. In these embodiments, the light of the excitation beam path 104 can be pulsed by flickering or cycling the light source 102, interrupting the excitation beam path 104 with a shutter, or with another mechanism achieving a similar effect. In some embodiments, the pulsing of the light of the excitation beam path 104 can occur at a rate of about once every 10 milliseconds (ms) to about once every 500 ms. In other embodiments, the pulsing of the light of the excitation beam path 104 can occur at a rate of about once every 10 ms to about once every 100 ms. [0051] The sample stage 106 in many embodiments can comprise mechanical actuators (not shown) for moving at least a portion of a sample across the excitation beam path 104. It is this physical motion that allows the device 100 to perform a scan of a region of interest of a sample, thereby assembling an image of the sample greater than a single operational field-of- view of the device 100. In some embodiments, these actuators can physically move the sample through the excitation beam path 104. In other embodiments, the excitation beam path 104 is moved relative to the sample. It is these mechanical actuators that prevent modern microscopes from accurately returning the beam path to the same position over the sample in many embodiments.

[0052] In some embodiments, the sample comprises a biological sample positioned on a slide or other substrate. The slide can comprise glass, synthetic polymers, or metal in various embodiments. In some embodiments, the slide can comprise a magnetic metal in order to facilitate its handling by actuators (e.g., those of the sample stage 106 or an automated slide handling system). In other embodiments, the slide can comprise a polished metal surface on the side facing the emission beam path 108. The use of a polished, and therefore reflective, metal surface can, in some embodiments, enhance the fluorescence measurement by improving contrast and can enable brightfield microscopy in certain microscope constructions (e.g., fluorescence microscopes) commonly unequipped to perform it.

[0053] In some embodiments, the device 100 may utilize one or more metal slides to improve microscope autofocusing with respect to a sample. Replacing a conventional glass slide with a metal slide ensures that the reflecting surface is at the sample surface (i.e., in the imaging plane), to provide an improved reflection of the laser beam over reflections provided from conventional glass slides. In some embodiments, the use of a metal slide (and/or metal polished slide surface) may enhance a fluorescence measurement obtained from a sample by improving contrast of the image of the sample. The improved contrast may be a direct result of the use of metal slides. In general, a reflection of light directed onto the metal slide from above (commonly known as “incident” or “reflected” light in the field of fluorescence microscopy) can also be used to enable brightfield microscopy when using fluorescence microscopes even when such microscopes are typically unequipped to perform brightfield microscopy.

[0054] In operation, light beam(s) may be directed onto a metal slide from above. The light beam(s) will be reflected from the slide surface, thus illuminating the sample. Such an illumination of the sample does not occur with conventional glass slides because conventional brightfield microscopy relies on transmitted light (i.e., light (beam(s)) that shine through a sample from below).

[0055] In some embodiments, the device 100 may utilize a metal slide array. A metal slide array may include a plurality of slide sections (e.g., portions). Each slide section may be configured to hold a separate sample (or sample portion). The device 100 may use an alignment method (e.g., method 400) to rapidly image multiple fluorescent molecules from each sample portion. The images may then be aligned according to a slide section order. For example, a first fluorescent image may include a first plurality of images, each corresponding to each of the plurality of slide sections, a second fluorescent image may include a second plurality of images, each corresponding to each of the plurality of slide sections, a third fluorescent image may include a third plurality of images, each corresponding to each of the plurality of slide sections, and so on. A processor and memory (e.g., processor 120 and memory 122) can store such image data. The processor can compile the image data into image sets aligned, for example, according to the slide section ordering. The alignment may be performed according to the method described in FIG. 4, for example, to align the image data into a composite image according to slide section ordering as described herein.

[0056] In some embodiments, the device 100 is a fluorescence microscope for use with focus maps. For example, a particular focus map may be accessed to assist device 100 in automatically focusing on portions of a sample. A focus map may include two or more focus points spread about a sample area. One or more focus points of the focus map may be used to align a sample within the sample area, as described in further detail with respect to FIG. 16 below. Examples of using a polished (e.g., reflective, metal, etc.) surface are described below with respect to FIGS. 15-17.

[0057] Light from the excitation beam path 104, when passed through the sample, causes corresponding fluorophores to subsequently emit light through an emission beam path 108. In many embodiments, the emission beam path 108 comprises various optics (e.g., lenses, beam splitters, dichroic mirrors, filters, etc.) to first collect and focus emitted light from the sample and then to separate out the emission wavelengths of the emission beam path into distinct groups of selected wavelengths that are captured on unique image capture devices 110 and 112. In many embodiments, the distinct group each comprises only a single selected wavelength, as per the definition of term “wavelength” and “selection of wavelengths” as described above. For example, a fluorescence microscopy experiment can be arranged such that two wavelengths of light are emitted from the sample through the emission beam path 108 where an appropriate beam splitter directs one of the wavelengths towards a first image capture device 110 and the other to the second image capture device 112. It is in this manner that the first image capture device 110 can be understood to be capturing image data (and therefore an image over the course of scan) of a first selection of the one or more emission wavelengths while the second image capture device 112 is capturing that of a second selection of one or more emission wavelengths. In other embodiments, any number of image capture device 110 and 112 greater than or equal to two can be employed (e.g., see FIG. 2). [0058] In some embodiments, the image capture devices 110 and 112 are cameras, but in other embodiments, they can be other image sensors used in microscopy and related fields for collecting and resolving incoming light. In various embodiments, the image capture devices 110 and 112 can each be a charge-coupled device (CCD) array or a CMOS image sensor chip, including sCMOS chips. Furthermore, these technologies can be subdivided such that one CCD array or sCMOS chip can be arranged in an optical experiment such that light of a first selection of emission wavelengths strikes a first portion of the array while that of a second selection of emission wavelengths strikes a second portion. Thus, in certain embodiments, the image capture devices 110 and 112 can be at least a portion of a CCD array or a CMOS sensor. One of skill in the art will appreciate that alternative or future technologies for image capture can likely be employed without deviating from the scope of this disclosure.

[0059] In many embodiments, various components of the device 100 can be in communication with at least one processor 120 and memory 122 that can store machine- readable instructions, executable by the at least one processor 120 to facilitate the operation of the device 100 during an imaging operation. For example, the at least one processor 120 and memory 122 can coordinate the actuators to move the sample relative to the excitation beam path and/or to instruct any filter wheels to rotate between scans. The same or an additional processor 120 and memory 122 can further be responsible for the storage of image data, the compilation of image data into images, the storage of image sets, and the execution of a method to align and combine images into an aligned composite image as described herein.

[0060] FIG. 2 depicts another embodiment of a device 200 for performing rapid channel- by-channel fluorescence microscopy. In some embodiments, the device 200 can comprise a plurality of light sources 202a and 202b. Various light sources 202a and 202b can be selected for various embodiments of the device 200 without deviating from the scope of the disclosure. In some embodiments, one or more light sources 202a and 202b of the plurality of light sources can be one or more LEDs, lasers, or similar technology that emit a singular or narrow range of wavelengths of light. In other embodiments, one or more light sources 202a and 202b of the plurality of light sources can be a white light or incandescent bulb or similar technology that emits a broader spectrum of wavelengths. Examples of light sources 202a and 202b can include, but are not limited to, a high-pressure mercury arc lamp, a metal halide lamp, a broad-spectrum LED (e.g., a CoolLED PE-300 (white)), or a multitude of single wavelength LEDS (e.g., a CoolLED PE800 or PE4000). In many embodiments, each light source 202a and 202b of the plurality of light sources can be individually selected.

[0061] Each fluorescent stain applied to a sample will only fluoresce when struck with its corresponding excitation wavelength. In some embodiments, the various light sources 202a and 202b are selected in advance and swapped out ahead of each scan or imaging operation of the device 200 such that the appropriate spectrum of excitation wavelengths is present in the subsequent excitation beam path 208. In other embodiments, the device 200 can comprise an excitation wavelength selector 204a and 204b for some, none, or ah of the light sources 202a and 202b of the plurality of light sources. The excitation wavelength selectors 204a and 204b, in many embodiments, can reduce and narrowly select portions of the spectrum of light being passed into the excitation beam path 208. For example, a light source 202a can be a white light, but an excitation wavelength selector 204a can transmit only a subset of this light such as specific colors or wavelength ranges (e.g., less than 1 nm, about 1 nm, about 1 nm to about 2 nm, about 1 nm to about 5 nm, about 1 nm to about 25 nm, and about 1 nm to about 100 nm). In some embodiments, the excitation wavelength selectors 204a and 204b can be one or more optical filters, gratings, or prisms. In other embodiments, the excitation wavelength selectors 204a and 204b can be filter wheels.

[0062] Following the excitation wavelength selectors 204a and 204b, the light from the plurality of light sources 202a and 202b then can enter a beam combiner 206. In many embodiments, the beam combiner 206 can comprise one or more of a prism, lens, or mirror in order to converge the light arriving from the plurality of light sources 202a and 202b into a single beam that is then directed down the excitation beam path 208 towards a turret 212. In some embodiments, the turret 212 holds a sample stage 210. In many other embodiments, the sample stage 210 is positioned above or below the turret 212, and the turret 212 is adapted to allow the appropriate transmission of excitation and emission wavelengths onto or out from the sample on the sample stage 210. In some embodiments, such as the embodiment of FIG. 2, the turret 212 can comprise a dichroic mirror 214 that reflects the excitation beam at an angle onto the sample stage 210. The dichroic mirror 214 can be selected such that it reflects the light of the excitation beam bath 208 at an angle towards a sample on a sample stage 210 but allows the transmission of light from the emission beam path 216. This arrangement can contribute to the reduction of stray light from the excitation beam path 208 from reaching one or more of the image capture devices 218a...218n. In some embodiments, the excitation wavelength selectors 204a and 204b, the beam combiner 206, and/or the dichroic mirror 214 can all be considered part of the excitation beam path 208.

[0063] In some embodiments, a turret 212 houses the dichroic mirror 214 and the sample stage 210. In many embodiments, the sample stage 210 further comprises actuators (not shown) capable of physically moving the sample through the excitation beam path 208. These actuators, or similar components, are what allows the device 200 to perform a scanning motion as described herein. Modem technology for these actuators or similar components is generally incapable of consistently and accurately returning the sample stage to a specific position with pixel or sub-pixel accuracy, as utilized for correct alignment of multi-channel images. The disclosure herein overcomes these issues with its channel-by-channel apparatus and method.

[0064] With a sample having already been stained by a plurality of fluorescent stains in most embodiments, the light from the excitation beam path 208 then induces these stains to fluoresce, emitting light of their corresponding emission wavelengths. This light can then travel through an emission beam path 216 of the device 200. In many embodiments, the emission beam path 216 can comprise various optical elements, such as one or more lenses or objectives (not shown) in order to collect and focus the emitted light. As the light travels through the emission beam path 216, it can encounter at least one beam splitter 220a adapted to separate and redirect predetermined selections of emission wavelengths towards separate image capture devices 218a and 218b or different portions of one image capture device. In alternative embodiments, the beam splitter 220a can be a dichroic mirror, a semi-transparent mirror, a grating, or a prism.

[0065] For example, a sample can be stained such that it will emit a first and second emission wavelength down the emission beam path 216. When the emitted light arrives at the beam splitter 220a, a first selection of emission wavelengths comprising the first emission wavelength will travel onwards to a first image capture device 218a while the beam splitter 220a can redirect a second selection of emission wavelengths comprising the second emission wavelength towards a second image capture device 218b. In this manner, image data of the first and second emission wavelengths are captured simultaneously on distinct image capture devices (i.e., on different channels). During a scan of a region of interest of the sample, the image data of each wavelength selection can be compiled to form a set of images (i.e., one image for each wavelength selection) that will spatially correlate because of their simultaneous collection.

[0066] As another example, a sample can be stained such that it will emit a first, second, and third emission wavelength down the emission beam path 216. When the emitted light arrives at the beam splitter 220a, a first selection of emission wavelengths comprising the first emission wavelength will travel onwards to a first image capture device 218a while the beam splitter 220a can redirect a second selection of emission wavelengths comprising the second and third emission wavelength towards a second image capture device 218b. In many embodiments, the second image capture device 218 will be unable to distinguish between light of the second and third wavelengths, so their individual contributions may be unresolved. However, the sum of their fluorescence combined can be confidently recorded. In this manner, image data of the first and second emission wavelength selections are captured simultaneously on distinct image capture devices. During a scan of a region of interest of the sample, the image data of each emission wavelength selection can be compiled to form a set of images (i.e., one image for each wavelength selection) that will spatially correlate because of their simultaneous collection.

[0067] Various embodiments of the device 200 can include more than two image capture devices 218a and 218b, and these embodiments can therefore employ additional beam splitters 220n as necessary to separate out further selections of emission wavelengths. For example, a sample can be stained such that it will emit a first, second, and third emission wavelength down the emission beam path 216. When the emitted light arrives at a first beam splitter 220n, a third selection of the emission wavelengths comprising the third emission wavelength is redirected towards a third image capture device 218n while the first and second emission wavelengths continue on until they encounter a second beam splitter 220a. The second beam splitter 220a then directs a first selection of emission wavelengths comprising the first emission wavelength towards a first image capture device 218a while directing a second selection of emission wavelengths comprising the second emission wavelength towards a second image capture device 218b. In this manner, image data of the first, second, and third emission wavelength selections are captured simultaneously on distinct image capture devices. During a scan of a region of interest of the sample, the image data of each emission wavelength selection can be compiled to form a set of images (i.e., one image for each wavelength selection) that will spatially correlate because of their simultaneous collection.

[0068] In further embodiments, the device 200 can comprise four, five, or more image capture devices 218a...218n and any number of additional beam splitters 220a...220n to adequately redirect the desired selections of emission wavelengths towards distinct image capture devices 218a...218n. Various examples of embodiments featuring a plurality of image capture devices 218a...218n and corresponding beam splitters 220a...220n are included herein in the below Examples section. Furthermore, in some embodiments, each image capture device 218a-218n can be preceded by an emission filter 222a...222n that selects for a predetermined emission wavelength selection in order reduce the quantity of stray light of other wavelengths from reaching the corresponding image capture device 218a...218n. In some embodiments, the emission filters 222a...222n can be individually selected from an optical filter, grating, or prism. In some embodiments, the emission filters 222a.. 222n are wheel filters.

[0069] In many embodiments, when assembling an acquisition strategy (i.e., the sequence of stains to capture across multiple scans with multiple image capture devices), it can be important to avoid capturing “adjacent” stains on different image capture devices in a single scan to minimize “crosstalk” between stains when undesired. For example, although various filters can be employed to minimize the crosstalk, the inherent overlap of the emission spectra of BV 421 and BV 480 means that some emission light from BV 421 will be captured by the image capture device intended for BV 480, and vice versa, if excited and captured on the same scan. The intensity of the crosstalk between two stains in a single scan is further dependent upon the one or more excitation wavelengths utilized for the given scan. For example, if an excitation wavelength is used that strongly excites both stains, the crosstalk will be more intense (i.e., a smaller difference between the intensity of the intended and unintended stains on a given image) than an example wherein the excitation wavelength strongly excites one and weakly excites the other. Because of this effect, crosstalk should be considered for a given set of stains on a case-by-case basis of one or more excitation wavelengths.

[0070] When unintended, crosstalk of emission light from one stain into the image intended for another can result in inexact or inaccurate images that interfere with confident analysis. Therefore, when constructing an acquisition strategy and/or a fluorescent microscope as described herein, one can improve accuracy of the images by capturing only stains that have sufficient spectral distance (i.e., minimal to no overlap in emission spectra) or a relative intensity of about 5% or less for a given one or more excitation wavelengths in the same scan in some embodiments. In other embodiments, one can improve accuracy by capturing only stains that have sufficient spectral distance or a relative intensity of about 3% or less for a given one or more excitation wavelengths in the same scan. In still other embodiments, one can improve accuracy by capturing only stains that have a sufficient spectral distance or a relative intensity of about 5% to about 10%, about 0% to about 5%, about 2% to about 6%, about 3% to about 6%, etc. for a given one or more excitation wavelengths in the same scan. [0071] Exceptions to this strategy can occur when one stain of an adjacent stain pair has an emission spectrum region sufficiently bright and separate from the overlapping region. For example, DRAQ5™ and AF 594 do overlap in emission spectra, however, DRAQ5™ has a very long emission “tail” reaching into the infrared such that a separate filter (e.g., an 810/90 filter, see filter notation as described below) can isolate this tail spectrum alone on an image capture device. Although the image of the tail of DRAQ5™ will likely be of less intensity compared to other image capture devices capturing spectra closer to an emission peak value, modem image capture devices can sufficiently capture enough light to generate an image sufficient for most applications.

[0072] As described above for FIG. 1, various components of the device 200 of FIG. 2 can be in communication with one or more processors and memories storing machine-readable instructions executable by the processor for the coordination and operation of various components of the device 200 during an imaging operation. The at least one processor and memory can further be responsible for the storage of image data, the compilation of image data into images, the storage of image sets, and the execution of a method to align and combine images into an aligned composite image as described herein.

[0073] In some embodiments, the device 200 may utilize one or more metal slides to improve microscope autofocusing with respect to a sample. Replacing a conventional glass slide with a metal slide ensures that the reflecting surface is at the sample surface (i.e., in the imaging plane), to provide an improved reflection of the laser beam over reflections provided from conventional glass slides. In some embodiments, the use of a metal slide (and/or metal polished slide surface) may enhance a fluorescence measurement obtained from a sample by improving contrast of the image of the sample. The improved contrast may be a direct result of the use of metal slides. In general, a reflection of light directed onto the metal slide from above (commonly known as “incident” or “reflected” light in the field of fluorescence microscopy) can also be used to enable brightfield microscopy when using fluorescence microscopes even when such microscopes are typically unequipped to perform brightfield microscopy.

[0074] In operation, light beam(s) may be directed onto a metal slide from above. The light beam(s) will be reflected from the slide surface, thus illuminating the sample. Such an illumination of the sample does not occur with conventional glass slides because conventional brightfield microscopy relies on transmitted light (i.e., light (beam(s)) that shine through a sample from below).

[0075] In some embodiments, the device 200 may utilize a metal slide array. A metal slide array may include a plurality of slide sections (e.g., portions). Each slide section may be configured to hold a separate sample (or sample portion). The device 200 may use an alignment method (e.g., method 400) to rapidly image multiple fluorescent molecules from each sample portion. The images may then be aligned according to a slide section order. For example, a first fluorescent image may include a first plurality of images, each corresponding to each of the plurality of slide sections, a second fluorescent image may include a second plurality of images, each corresponding to each of the plurality of slide sections, a third fluorescent image may include a third plurality of images, each corresponding to each of the plurality of slide sections, and so on. A processor and memory (e.g., processor 120 and memory 122) can store such image data. The processor can compile the image data into image sets aligned, for example, according to the slide section ordering. The alignment may be performed according to the method described in FIG. 4, for example, to align the image data into a composite image according to slide section ordering as described herein.

[0076] In some embodiments, the device 200 is a fluorescence microscope for use with focus maps. For example, a particular focus map may be accessed to assist device 200 in automatically focusing on portions of a sample. A focus map may include two or more focus points spread about a sample area. One or more focus points of the focus map may be used to align a sample within the sample area, as described in further detail with respect to FIG. 16 below.

[0077] FIG. 3 depicts a photograph of one embodiment of the device 300 for rapid channel- by-channel fluorescence microscopy. The device 300 comprises a first light source 302 and second light source (not shown) that each pass through a respective excitation wavelength selector 304a and 304b that meet at a beam combiner 306 that directs the excitation beam path towards a turret 308 that in turn directs the excitation beam path onto a sample (not shown). In the embodiment of FIG. 3, the device is arranged such that the emission beam path is orthogonal to the excitation beam path. Light emitted by the sample then moves upwards until it strikes the beam splitter 310 that directs a first selection of emission wavelengths through a first emission filter 312a to a first image capture device 314a, while sending a second selection of emission wavelengths through a second emission filter 312b towards a second image capture device 314b. In this embodiment, the excitation wavelength selectors 304a and 304b and the emission filters 312a and 312b are filter wheels.

METHODS

[0078] As shown in FIG. 4, a method 400 for performing rapid channel-by-channel fluorescence microscopy of one embodiment includes exciting at least n+1 emission wavelengths from a sample in block S402, capturing a first set of images in a first scan in block S404, capturing a second set of images in a second scan wherein at least a first image of the first set and a second image of the second set capture an identical selection of one or more emission wavelengths in block S406, calculating an alignment shift between at least the first and second image in block S408, applying the alignment shift to at least one image of the first set or to at least one image of the second set in block S410, and combining at least one image of the first set with at least one image of the second set to create an aligned composite image in block S412. In some embodiments, the method functions to perform rapid channel-by-channel fluorescence microscopy. The method is used for microscopy, often medical imaging of tissues or cells, but can additionally or alternatively be used for any suitable applications, clinical, industrial, or otherwise. The method can be configured and/or adapted to function for any suitable situation requiring the collection of image data under a plurality of optical parameters from a sample or source.

[0079] In many embodiments, the method 400 includes exciting at least n+1 emission wavelengths from a sample, such that n is equal to a number of image capture devices available on a fluorescence microscopy being employed in the method, n being greater than 1 at block S402. The disclosure herein utilizes at least two image capture devices and is particularly advantageous in situations wherein the total number of emission wavelengths of interest exceeds that of available image capture devices. In various embodiments, the at least n+1 emission wavelengths need not all be excited simultaneously. As described herein, in some embodiments, a subset of the n+1 emission wavelengths are excited and collected during a first scan while additional and different subsets are excited and collected in subsequent scans. In other embodiments, all of the n+1 emission wavelengths are excited simultaneously but are collected in various subsets across the plurality of scans.

[0080] At block S404, the method 400 includes capturing a first set of images of the sample in a first scan. In many embodiments, each image of the first set of images is captured simultaneously and separately on a distinct image capture device, and in many embodiments, each image will capture a distinct selection of the n+1 emission wavelengths than each other image of its set. For example, the first set of images can include three images wherein the three images capture a first, second, and third emission wavelength only and respectively. [0081] At block S406, the method includes capturing a second set of images of the sample in a second scan, such that at least a first image of the first set and a second image of the second set capture an identical selection of one or more of the n+1 emission wavelengths. For example, a first set of images can comprise three images where the images capture a selection of a first, second, and third emission wavelength only and separately, and a second set of images can comprise three images where the images capture a first, fourth, and fifth emission wavelength only and separately. In this example, both the first and second set of images include an image that captures the same selection of the emission wavelengths. In some embodiments, these two images can be referred to as “reference images.” Despite these two images capturing the same selection of the emission wavelengths, they are unlikely to be identical. Because they are taken on separate scans of the region of interest of the sample, the fluorescence microscope was unlikely to be able to return to the exact starting point of the first scan to begin the second. Therefore, in many embodiments, there is likely to be a substantial shift in the xy -plane of the first and second reference image at one, more than one, or all of the sample positions of a scan in addition to other minor artifacts or distortions that might occur due to an imperfect motion of the sample stage.

[0082] At block S408, the method 408 includes calculating an alignment shift between the first and second image at each relevant sample position during a scan. As stated above, the spatial offset can vary in direction or magnitude at each sample position, and the “alignment shift” or “xy-shift” discussed herein can, in some embodiments, be considered to mean the collection of spatial offsets for each relevant sample position. In many embodiments, a variety of image registration and alignment software can be employed on a processor accessing machine-readable instructions to complete this block. These programs are capable of generating spatial measurement data between pixels or voxels of images and then performing various operations on the data sets, sometimes including rotational or translation transformations, in order to calculate an optimal “fit” of one image onto another. Examples of software programs include elastix. Due to the size of the data sets, complexity of calculation, rigorous accuracy requirements, and high probability for conflicting visual artifacts between the first and second image, calculating this xy-shift can be prohibitively difficult for the unaided mind.

[0083] At block S410, the method 400 includes applying the alignment shift to at least one image of the first set of images or to at least one image of the second set of images. Because images taken simultaneously during a scan correlate spatially, the alignment shift calculated between the first and second reference images is applicable to other images of their sets. In many embodiments, the alignment shift is applied to one of the sets of images to align both sets.

[0084] At block S412, the method 400 includes combining at least one image of the first set with the at least one image of the second set to create an aligned composite image. In many embodiments, at least one, if not all, of the images from the first or second set can be images appropriately aligned in block S410. In some embodiments, the aligned composite image can include at least one of the first or second reference images. In this manner, the emission wavelength selection used by the method 400 as the reference image can still be an emission wavelength selection of interest and need not be an extra acquisition solely for the purpose of aligning images of other wavelength selections. In some embodiments, the images used as reference images can capture the emission of the DRAQ5™ stain, a far-red dye that adheres to DNA and reliably produces a strong signal that is both convenient for use as a reference and scientifically relevant to many tissue or cell imaging experiments. In many embodiments, the aligned composite image can be considered a z-stack.

[0085] In some embodiments involving more than two scans, blocks S408 and S410 can be repeated for multiple pairs of reference images. For example, an embodiment of the method 400 can include acquiring, across three scans, a first set of images comprising an image of a first wavelength selection and an image of a second wavelength selection, a second set of images comprising an image of the first wavelength selection and an image of a third wavelength selection, and a third set of images comprising an image of the first wavelength selection and an image of a fourth wavelength selection. In this example, the images of the first wavelength selection from the first and second sets can be used to align the images of the second and third wavelength selections, while the images of the first wavelength selection from the first and third sets can be used to align images of the second and fourth wavelength selections. With the appropriate alignment shifts applied, the aligned composite image produced in block S412 can include an image of all four wavelength selections.

[0086] In some embodiments involving more than two scans, blocks S408 and S410 can be repeated for multiple pairs of reference images that can be different selections of wavelengths between the pairs. For example, an embodiment of the method 400 can include acquiring, across three scans, a first set of images comprising an image of a first wavelength selection and an image of a second wavelength selection, a second set of images comprising an image of the first wavelength selection and a third wavelength selection, and a third set of images comprising the third wavelength selection and a fourth wavelength selection. In this example, the images of the first wavelength selection from the first and second sets can be used to align the images of the second and third wavelength selections. Similarly, the images of the third wavelength selection of the second and third sets can be used to align the image of the fourth wavelength selection with that of the third wavelength selection. Because an image of the third wavelength selection was taken simultaneously with that of the first wavelength selection and can be aligned with that of the second wavelength selection, the image of the fourth wavelength selection can be accordingly aligned with all the other images and included in the aligned composite image. In this example, two different pairs of images having two different wavelength selections between two different sets can be considered and utilized as “reference images” for the method 400.

[0087] In order to avoid crosstalk between stains of a given scan as described above, the method 400 can make careful selection of which emission wavelengths to excite for a given set of images. In some embodiments, no pair of images within the first set of images and no pair of images within the second set of images capture a selection of emission wavelengths with a crosstalk intensity greater than 5% as described herein. In other embodiments, no pair of images within the first set of images and no pair of images within the second set of images capture a selection of emission wavelengths with a crosstalk intensity greater than 3% as described herein. In other embodiments, no pair of images within the first set of images and no pair of images within the second set of images capture a selection of emission wavelengths with a crosstalk intensity greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 6%, greater than about 7%, etc., as described herein. [0088] FIG. 5 depicts a cartoon illustrating certain portions of an embodiment of a method for performing rapid channel-by-channel fluorescence microscopy (e.g., the embodiment of FIG. 4). A first set of images 502 and a second set of images 504 are taken of a region of interest of a sample during a first and second scan. Each set of images includes one image 506a and 506b that has captured an identical selection of the emission wavelengths to that of an image in the other set (e.g., an image of the fluorescence of the DRAQ5™ stain). These images 506a and 506b can be considered reference images in some embodiments. Due to the inaccuracies of most fluorescence microscopes, the images 506a and 506b will be nearly identical except for a shift in the xy-plane at one, more than one, or all of the sample positions during the scan. The spatial offset can vary in direction and/or magnitude at each relevant sample position during the scan. This alignment shift (e.g., the collection of spatial offsets for each relevant sample position, in some embodiments) can be calculated as described herein and subsequently applied to one or more of the images of one of the sets (e.g., the first set 502) to generate an “aligned” set of images 502’. The aligned set of images 502’ can then be stacked (with or without one of the reference images 506a and 506b) in order to create an aligned composite image.

[0089] FIG. 6 depicts one embodiment of a method for calibrating at least two image capture devices of a microscope (e.g., a fluorescence microscope configured for channel -by- channel fluorescence microscopy as described herein). In many embodiments, when assembling a fluorescence microscope with multiple image capture devices (e.g., the microscope of FIG. 2), physical limitations with the exactitude of the equipment and installation procedures prevent a perfect alignment of each image capture device relative to the beam path. These subtle deviations from a perfect corresponding alignment can result in systematic errors during imaging operations of the microscope wherein light from a singular sample position will be recorded simultaneously on each image capture device but in non- analogous pixels (or the equivalent thereof) of each image capture device.

[0090] For example, two identical image capture devices can be installed in a microscope in an orientation that both have a “top left corner” pixel. These two top left comer pixels can be considered “analogous pixels.” If the two image capture devices of this example could be perfectly aligned relative to the beam path, a given region of the beam path should strike both of these analogous pixels exactly. However, due to the limiting imprecisions of the manufactured parts, it is not uncommon, in many embodiments, for a given region of the beam path to strike nearby but non-analogous pixels. For example, the given region of the beam path could strike the top left corner pixel of one image capture device, but on the other, it may land a single-digit number of pixels elsewhere (e.g., four pixels to the right and two pixels down).

[0091] Additionally, in embodiments wherein the at least two image capture devices are positioned at different lengths along the beam path, scalar issues of the captured images can arise wherein the image from one image capture device would appear more “zoomed in” or “zoomed out” in comparison to images from other image capture devices. Therefore, to facilitate the collection of accurate image data with channel-by-channel fluorescence microscopy as described herein, it can be valuable, in some embodiments, to first calibrate the at least two image capture devices (i.e., measure and account for this systematic error in their physical arrangement) before the collection of images of samples and in addition to the alignment described above in FIGS. 4 and 5. In many embodiments, this calibration method, as described in FIG. 6, can be performed once and the calibration used for any number of imaging operations until the physical arrangement of the image capture devices or other optics is changed or disturbed.

[0092] In some embodiments, the method 600 for calibrating a microscope that comprises at least two image capture devices includes providing a calibration diagram to a microscope comprising at least two image capture devices in block S602, capturing a unique image of the calibration diagram with the at least two image capture devices simultaneously in block S604, locating on each unique image two endpoints of each terminal spline in block S606, calculating the midpoint of each terminal spline to generate at least one calibration coordinate for each unique image in block S608, calculating at least one transformation to equate the at least one calibration coordinates of the unique images in block S610, and calibrating the at least two image capture devices with the at least one transformation in block S612.

[0093] In block S602, the method 600 includes providing a calibration diagram to a microscope comprising at least two image capture devices. In some embodiments, the calibration diagram can be the diagram 700 of FIG. 7 or a diagram presenting a similar geometry of splines 702 of equivalent thickness arranged on two orthogonal axes. The splines 702 extend orthogonally from their axis in the plane of the diagram 700. The ends or termini of each axis is marked with a terminal spline 704a-704d having a length between two spline endpoints 706a and 706b. The diagram 700 can be printed on a variety of materials as appreciated by those of skill in the art and of sufficient contrast that the space between the splines 702 can be resolved on the image capture devices when illuminated. [0094] In block S604, the method 600 includes capturing a unique image of the calibration diagram 700 with the at least two image capture devices simultaneously. One unique image is taken for each image capture device. For example, if the given microscope features two image capture devices, each image capture devices records its own image, resulting in a total of two unique images. In many embodiments, at least one processor of the microscope operates the at least two image capture devices to capture an image of the calibration diagram 700. In many embodiments, no movement of the sample stage (and therefore the calibration diagram 700) is needed to collect sufficient image data representing the entire calibration diagram 700.

[0095] In block S606, the method 600 includes locating on each unique image the two endpoints or termini 706a and 706b of each terminal spline 704a-704d. In many embodiments, a processor (the same or a different processor of block S604) running machine-readable instructions parses the image data of each unique image to identify pixels having sufficient contrast to neighboring pixels that indicate the existence or non-existence of a spline. By systematically analyzing across the entire plane of the calibration image, the method 600 (and/or the processor in some embodiments) can identify the locations (in units of pixels or distance) of the terminal splines 704a-704d. Subsequently, the endpoints or termini 706a and 706b of each terminal spline 704a-704d can be located by a sudden change in contrast when traversing along the length of the corresponding terminal spline 704a-704d. [0096] In block S608, the method 600 includes calculating the midpoint 708 of each terminal spline 704a-704d to generate at least one calibration coordinate for each unique image. Once the location of each endpoint or terminus 706a and 706b is determined, the midpoint 708 between them can be readily calculated (e.g., by a processor). The midpoint 708 (marked in units of pixels or distance) of each terminal spline 704a-704d can then be identified as at least one calibration coordinate. In some embodiments, such as the embodiment of FIG. 7, the midpoint 708 of each terminal spline 704a-704d can be interpreted as a separate calibration coordinate. Therefore, in this example, each unique image of the calibration diagram 700 will have four calibration coordinates.

[0097] Each calibration coordinate of an image of the calibration diagram 700 will have an analogous calibration coordinate in all other unique images of the calibration diagram 700 taken simultaneously by the at least two image capture devices. In many embodiments, analogous calibration coordinates are derived from the same feature of the calibration diagram 700. For example, all calibration coordinates derived from the midpoint 708 of the terminal spline 704a of a first axis can be considered analogous, whereas a calibration coordinate derived from the midpoint 708 of a terminal spline 704a of a first axis and a calibration coordinate derived from the midpoint 708 of a terminal spline 704b of a second axis cannot be considered analogous calibration coordinates.

[0098] In block S610, the method 600 includes calculating at least one transformation to equate the at least one calibration coordinates of the unique images. In some embodiments, a processor performing machine-readable instructions calculates a difference (e.g., in terms of distance) between the locations (e.g., in terms of pixels) of the at least one analogous calibration coordinates between the unique images. From this difference, the processor then calculates at least one transformation that, when applied to at least one calibration coordinate of one unique image, results in the calibration coordinate to have the same location as an analogous calibration coordinate of another unique image.

[0099] Transformations can include at least one of a translation, a rotation, or a scale. A translation moves at least one calibration coordinate in either dimension of the xy-plane of the calibration diagram 700. A rotation moves at least one calibration coordinate about a defined center-of-rotation point (often in the middle of the calibration diagram 700 but can be elsewhere other embodiments) from fixed distance. A scale moves at least one calibration coordinate towards or away from a defined center point (in many embodiments, a point equivalent to a center-of-rotation point for any rotation transformations). In many embodiments when there is a plurality of calibration coordinates, any transformation operates on all calibration coordinates equally.

[00100] In block S612, the method 600 includes calibrating the at least two image capture devices with the at least one transformation. The processor can store the at least one transformation to a memory associated with the microscope such that whenever image data of a sample is taken by the microscope (such as during an imaging operation like the embodiment shown in FIG. 4), the processor can apply the at least one transformation to the image data of at least one of the image capture devices, resulting in much more accurate and aligned images of the sample than would be achieved without the calibration method described herein. In some embodiments, the method 600 utilizes device 200 (FIG. 2), which may employ metal slides, metal slide arrays, and/or focus maps to capture and align images. [00101] The systems and methods of the preferred embodiment and variations thereof can be embodied and/or implemented at least in part as a machine configured to receive a computer- readable medium storing computer-readable instructions. The instructions are preferably executed by computer-executable components preferably integrated with the system and one or more portions of the processor on the microscope and/or computing device. The computer- readable medium can be stored on any suitable computer-readable media such as RAMs, ROMs, flash memory, EEPROMs, optical devices (e.g., CD or DVD), hard drives, floppy drives, or any suitable device. The computer-executable component is preferably a general or application-specific processor, but any suitable dedicated hardware or hardware/firmware combination can alternatively or additionally execute the instructions.

EXAMPLES

Example 1 Two Image Capture Devices, Seven Stains, Six Scans

[00102] FIG. 8 depicts an embodiment of a portion of the device for rapid channel-by- channel fluorescence microscopy having two image capture devices and arranged for capturing the emission wavelengths of seven stains across six scans during an imaging procedure. In some embodiments, the image capture devices have been calibrated according to the method of FIG. 6. Each image capture device in FIG. 8 is labeled with the stain whose emission wavelength the image capture device is configured to capture in at least one scan. In this example, Image Capture Device 1 is configured to capture the emission wavelength of the DRAQ5™ or PerCP stains in various scans, while Image Capture Device 2 is configured to capture the emission wavelengths of BV 421, BV 480, AF 488, PE, or AF 594 stains across various scans. AF stands for AlexaFluor; BV stands for Brilliant Violet; PE stands for R-Phycoerythrin, and PerCP stands for peridinin-chlorophyll-protein complex. In front of each image capture device is a first or second filter or filter wheel that helps select for the intended wavelength (e.g., an emission filter of FIG. 2). In these examples, filters are denoted by the center wavelength in nanometers (nm) followed by the bandwidth also in nm. For example, the 697/60 filter depicted in Example 1 as part of the first filter wheel in front of Image Capture Device 1 will transmit light of 667 to 727 nm and will block all other wavelengths. The filters of the filter wheels are listed in a corresponding sequential order to the stains of the adjacent image capture device. For example, filter 431/28 of the second filter wheel should be in the operable position when a scan collects an image of BV 421 on Image Capture Device 2. An exception to this notation involves DRAQ5™ stain and the filters of the first filter wheel. When a scan collects an image of DRAQ5™ on Image Capture Device 1 and an image of AF 594 on Image Capture Device 2, the filter 810/90 should be used for Image Capture Device 1 in order to avoid crosstalk between the two stains. In all other scans involving DRAQ5™, the filter 697/60 can be used to acquire a brighter image of DRAQ5™ The filter 697/60 is generally used for scans of PerCP in many embodiments. In order to appropriately split the emission wavelengths towards Image Capture Device 1 and 2, a 660 nm long-pass dichroic mirror can be used as a beam splitter for all scans.

[00103] The turret of Example 1 holds three different dichroic mirrors to be used for various scans during the imaging procedure. Each dichroic mirror is listed above the corresponding emission wavelengths for which it is adapted (also listed in TABLE 1 below). These dichroic mirrors can be swapped automatically or by hand between scans in various embodiments. The stains listed below the line dividing the list of corresponding emission wavelengths for each dichroic mirror in FIG. 8 denotes which stains of which images can be used as “reference images” for the imaging operation of the given example. For example, a 612 nm long-pass dichroic mirror can be used during a collection of AF 594 and DRAQ5™. D1 and D2 are both multiband pass dichroic mirrors that will reflect the corresponding incoming excitation light but transmit the appropriate emission light towards the image capture devices along the emission beam path as depicted for the stains listed in their row as shown in Table 1. The various dichroic mirrors listed throughout Tables 1 though 6 should be interpreted to have similar functionality according to the tables in which they appear. More specifically, D1 is a multi-edge dichroic mirror 412/460/520/660 wherein the numbers recited are the approximate wavelengths (nm) it reflects, and D2 is a multiedge dichroic mirror 560/660. 612 lp is a 612 nm long-pass mirror that reflects shorter wavelengths. The various dichroic mirrors and/or beam splitters recited in the various examples herein are commercially available either prepackaged or for custom order. Not shown in FIG. 6 of Example 1 are the one or more excitation light sources, excitation filters, or any excitation beam path optical components. For the purposes of FIG. 8, it can be assumed that the appropriate excitation wavelength is arriving at the dichroic mirror of the turret.

[00104] For efficient collection of the images used to generate an aligned composite fluorescent image of the seven stains in six scans, the following acquisition strategy can be employed. Alternate acquisition strategies or sequences can be used in other embodiments, but may use additional or alternative switching of filters, dichroic mirrors, beam splitters, and/or other optical components other than what is described here.

[00105] In a first scan, the device, using the D1 dichroic in the turret and the 431/28 filter of the filter wheel acquires an image of DRAQ5™ on Image Capture Device 1 and an image of BY 421 on Image Capture Device 2. Then, having cycled the filter wheel in front of Image Capture device 2 to 480/30, the device captures an image of DRAQ5™ on Image Capture Device 1 and an image of B V 480 on Image Capture Device 2 on a second scan. On a third scan, the device captures images of DRAQ5™ and AF 488 after cycling the filter wheel accordingly. On a fourth scan, the device captures an image of PerCP on Image Capture Device 1 and an image of BV 480 on Image Capture Device 2. Next, the dichroic of the turret must be exchanged for D2 in addition to the filter wheel cycling in order to capture an image of DRAQ5™ on Image Capture Device 1 simultaneously with an image of PE on Image Capture Device 2. Finally, the dichroic mirror of the turret is exchanged again for the 612 nm long-pass mirror, the filter wheel of ICD 1 changes to 810/90 to avoid crosstalk, and an image of DRAQ5™ and AF 594 can be taken on Image Capture Device 1 and 2, respectively. TABLE 1 summarizes the acquisition strategy.

TABLE 1

[00106] Following the imaging procedure, the device, or a processor in communication with the device, can then construct an aligned composite fluorescent image of two or more of the images of DRAQ5™, PerCP, BV 421, BV 480, AF 488, PE, and AF 594 as described herein since images of BV 421, BV 480, AF 488, PE, and AF 594 were all taken simultaneously with an image of DRAQ5™. Thus, the DRAQ5™ images can be used as a “reference images” to align the other images. An image of PerCP can be aligned to and combined with the other images because it was simultaneously taken with an image of BV 480. Because another image of B V 480 was taken simultaneously with an image of DRAQ5™, the PerCP image can be aligned to that of DRAQ5™. Thus, the BV 480 images can be considered “reference images” for the alignment of the PerCP image. In some embodiments, the example described above may employ metal slides, metal slide arrays, and/or focus maps to capture and align images, as described in detail herein. Example 2 Two Image Capture Devices, Eight Stains, Seven Scans

[00107] FIG. 9 depicts an embodiment of a portion of the device for rapid channel-by- channel fluorescence microscopy having two image capture devices and arranged for capturing the emission wavelengths of seven stains across six scans during an imaging procedure. In some embodiments, the image capture devices have been calibrated according to the method of FIG. 6. The format and notation of FIG. 9 is analogous to that of FIG. 8 above. However, the beam splitter of the emission beam path is a 647 short-pass dichroic mirror. PE-Cy7 stands for PE-Cyanine7, another fluorescent stain. D3 is a multiedge dichroic beamsplitter 460/760. Considering the filter exception described above in Example 1, the filter 810/90 of the filter wheel before Image Capture Device 2 can also be used for both DRAQ5™ and PE-Cy7. An acquisition strategy for the operation of Example 2 of FIG. 9 for generating an aligned composite image is of the eight stains is summarized below in Table 2.

TABLE 2

[00108] Following the imaging procedure, the device, or a processor in communication with the device, can then construct an aligned composite fluorescent image of two or more of the images of DRAQ5™, PerCP, PE-Cy7, BV 421, BV 480, AF 488, PE, and AF 594 as described herein since images of BV 421, BV 480, AF 488, PE, and AF 594 were all taken simultaneously with an image of DRAQ5™. Thus, the DRAQ5™ images can be used as a “reference images” to align the other images. Images of PerCP and PE-Cy7 can be aligned to and combined with the other images because it was simultaneously taken with an image of BV 480. Because another image of BV 480 was taken simultaneously with an image of DRAQ5™, the PerCP and PE-Cy7 images can be aligned to that of DRAQ5™. Thus, the BV 480 images can be considered “reference images” for the alignment of the PerCP and PE-Cy7 images to the images of the other sets. In some embodiments, the example described above may employ metal slides, metal slide arrays, and/or focus maps to capture and align images, as described in detail herein.

Example 3 Three Image Capture Devices, Seven Stains, Three Scans [00109] FIG. 10 depicts an embodiment of a portion of the device for rapid channel -by- channel fluorescence microscopy having three image capture devices and arranged for capturing the emission wavelengths of seven stains across three scans during an imaging procedure. In some embodiments, the image capture devices have been calibrated according to the method of FIG. 6. The format and notation of FIG. 10 is analogous to that of FIG. 8 above. However, FIG. 8 includes an additional image capture device and corresponding filter and beam splitter. The two beam splitters of the emission beam path are a 647 nm short-pass dichroic mirror that separates out light towards Image Capture Device 3, while a 455 nm long-pass dichroic mirror separates out the remaining light for Image Capture Device 1 and 2. The turret of FIG. 8 includes two custom dichroic mirrors. D4 is a multiedge dichroic mirror 460/612 capable of reflecting the excitation wavelengths of BV 480, AF 594, and DRAQ5™ while transmitting their emission wavelengths. D5 is a multiedge dichroic mirror 412/560/660. An acquisition strategy for the operation of Example 3 of FIG. 10 for generating an aligned composite image is of the eight stains is summarized below in Table 3. ICD stands for “Image Capture Device.”

TABLE 3

[00110] Following the imaging procedure, the device, or a processor in communication with the device, can then construct an aligned composite fluorescent image of two or more of the images of DRAQ5™, PerCP, BV 421, BV 480, AF 488, PE, and AF 594 as described herein since images of BV 421, BV 480, AF 488, and AF 594 were all taken simultaneously with an image of DRAQ5™. Thus, the DRAQ5™ images can be used as a “reference images” to align the other images. Images of PerCP and PE can be aligned to and combined with the other images because it was simultaneously taken with an image of BV 421. Because another image of BV 421 was taken simultaneously with an image of DRAQ5™, the PerCP and PE images can be aligned to that of DRAQ5™. Thus, the BV 421 images can be considered “reference images” for the alignment of the PerCP and PE images to the images of the other sets. In some embodiments, the example described above may employ metal slides, metal slide arrays, and/or focus maps to capture and align images, as described in detail herein.

Example 4 Three Image Capture Devices, Eight Stains, Four Scans

[00111] FIG. 11 depicts an embodiment of a portion of the device for rapid channel-by- channel fluorescence microscopy having three image capture devices and arranged for capturing the emission wavelengths of seven stains across three scans during an imaging procedure. In some embodiments, the image capture devices have been calibrated according to the method of FIG. 6. The format and notation of FIG. 11 is analogous to that of FIG. 10 above. PE-Cy7 stands for PE-Cyanine7, another fluorescent stain. Considering the filter exception described above in Example 1, the filter 810/90 of the filter wheel before Image Capture Device 3 can also be used for both DRAQ5™ and PE-Cy7. An acquisition strategy for the operation of Example 4 of FIG. 11 for generating an aligned composite image is of the eight stains is summarized below in Table 4. N/A means that no image is captured on the given image capture device for the given scan

TABLE 4

[00112] Following the imaging procedure, the device, or a processor in communication with the device, can then construct an aligned composite fluorescent image of two or more of the images of DRAQ5™, PerCP, PE-Cy7, BV 421, BV 480, AF 488, PE, and AF 594 as described herein since images of BV 421, BV 480, AF 488, and AF 594 were all taken simultaneously with an image of DRAQ5™. Thus, the DRAQ5™ images can be used as a “reference images” to align the other images. Images of PerCP and PE can be aligned to and combined with other images because it was simultaneously taken with an image of BV 421. Because another image of BV 421 was taken simultaneously with an image of DRAQ5™, the PerCP and PE images can be aligned to that of DRAQ5™. Thus, the BV 421 images can be considered “reference images” for the alignment of the PerCP image. The image of PE-Cy7 can be aligned to and combined with the other images because it was taken simultaneously with an image of BV 480. Because another image of BV 480 was taken simultaneously with an image of DRAQ5™, the PE-Cy7 image can be aligned to that of DRAQ5™ and therefore, to the images of the other sets. In some embodiments, the example described above may employ metal slides, metal slide arrays, and/or focus maps to capture and align images, and/or laser autofocus systems, as described in detail herein.

Example 5 Four Image Capture Devices, Seven Stains, Two Scans: Version 1 [00113] FIG. 12 depicts an embodiment of a portion of the device for rapid channel-by- channel fluorescence microscopy having four image capture devices and arranged for capturing the emission wavelengths of seven stains across two scans during an imaging procedure. In some embodiments, the image capture devices have been calibrated according to the method of FIG. 6. The format and notation of FIG. 12 is analogous to that of FIG. 8 above. However, FIG. 12 includes two additional image capture devices and corresponding filter and beam splitter. The three beam splitters of the emission beam path are a 505 nm long-pass dichroic mirror that separates out light towards Image Capture Device 4, a 612 nm long-pass dichroic mirror separates out towards Image Capture Device 3, while a 760 nm long-pass dichroic mirror separates out the remaining light for Image Capture Device 1 and 2. The turret of FIG. 12 includes two custom dichroic mirrors. D6 is a multiedge dichroic mirror 412/520/660/760, and D7 is a multiedge dichroic mirror 460/612/660/760. An acquisition strategy for the operation of Example 5 of FIG. 12 for generating an aligned composite image is of the eight stains is summarized below in Table 5.

TABLE 5

[00114] Following the imaging procedure, the device, or a processor in communication with the device, can then construct an aligned composite fluorescent image of two or more of the images of DRAQ5™, PerCP, PE-Cy7, BV 421, BV 480, AF 488, and AF 594 as described herein since images of DRAQ5™, PerCP, BV 421, BV 480, AF 488, and AF 594 were all taken simultaneously with an image of PE-Cy7. Thus, the PE-Cy7 images can be used as a “reference images” to align the other images. In some embodiments, the example described above may employ metal slides, metal slide arrays, and/or focus maps to capture and align images, as described in detail herein.

Example 6 Four Image Capture Devices, Seven Stains, Two Scans: Version 2 [00115] FIG. 13 depicts an embodiment of a portion of the device for rapid channel -by- channel fluorescence microscopy having four image capture devices and arranged for capturing the emission wavelengths of seven stains across two scans during an imaging procedure. In some embodiments, the image capture devices have been calibrated according to the method of FIG. 6. The format and notation of FIG. 13 is analogous to that of FIG. 12 above. However, the three beam splitters of the emission beam path of FIG. 13 are a 647 nm short-pass dichroic mirror that separates out light towards Image Capture Device 4, a 455 nm long-pass dichroic mirror separates out towards Image Capture Device 3, while a 612 nm long-pass dichroic mirror separates out the remaining light for Image Capture Device 1 and 2. The turret of FIG. 13 includes two custom dichroic mirrors. D8 is a multiedge dichroic mirror 412/560/612/760, and D9 is a multiedge dichroic mirror 460/560/660/760. An acquisition strategy for the operation of Example 6 of FIG. 13 for generating an aligned composite image is of the eight stains is summarized below in Table 6.

TABLE 6

[00116] Following the imaging procedure, the device, or a processor in communication with the device, can then construct an aligned composite fluorescent image of two or more of the images of DRAQ5™, PE, BV 421, BV 480, AF 488, AF 594, and AF 800 as described herein since images of DRAQ5™, PerCP, BV 421, BV 480, AF 488, and AF 594 were all taken simultaneously with an image of AF 800. Thus, the AF 800 images can be used as a “reference images” to align the other images. In some embodiments, the example described above may employ metal slides, metal slide arrays, and/or focus maps to capture and align images, as described in detail herein.

Example 7 Light Sources

[00117] FIG. 14 depicts an embodiment of a portion of the device for rapid channel-by- channel fluorescence microscopy having two lights sources that are combined to form the excitation beam path via a beam combiner. The embodiment of FIG. 12 can be paired with any of the embodiments of the device that feature two image capture devices (e.g., Examples 1 and 2.)

[00118] In this embodiment, both Light Source 1 and Light Source 2 are non-LED widefield light sources. Within each box for each light source, Light Source 1 and Light Source 2 are labeled with a list of stains for which the light source is to excite via a corresponding excitation wavelength selector (e.g., a filter of a filter wheel) as notated in the filter notation of Example 1 above (e.g., for Light Source 1 to excite a DRAQ5™ stain, its corresponding excitation wavelength selector will need to have its 642/20 filter in position.) BV 480 is listed in both Light Source 1 and Light Source 2 so that BV 480 can be paired with DRAQ5™ in one scan and with PE-Cy7 in another (e.g., the acquisition strategy of Example 2). In many embodiments, any selection of the Light Source 1 can be transmitted with any selection from Light Source 2.

[00119] For embodiments involving more than two image capture devices and more than two excitation wavelengths, additional widefield light sources and corresponding excitation wavelength selectors can be added to Example 7. In other embodiments, one or more multi- LED light sources (e.g., a CoolLED PE800) can replace all or a subset of the widefield light sources. In many embodiments featuring multi-LED sources, each multi-LED source can be fitted with an excitation wavelength selector comprising a multiband pass filter that corresponds to an appropriate dichroic mirror of the turret of the device such that the excitation wavelength from the multi-LED source is transmitted by the excitation wavelength selector but reflected by the dichroic mirror of the turret onto the stained sample for a given scan. In some embodiments, the example described above may employ metal slides, metal slide arrays, and/or focus maps to capture and align images, as described in detail herein. Example 8 Equivalent Dyes

[00120] As described herein, certain dyes can be considered “equivalent” due to the immense similarity in excitation and emission spectrums. Table 7 below outlines a representative but incomplete list of examples. One of skill in the art will appreciate the following list is not exhaustive at the time of filing and that new equivalent dyes will likely be developed in the future. New or alternative equivalent dyes not listed here can readily be utilized by the devices and methods contained herein. Therefore, the following list of equivalent dyes should not be interpreted as limiting in any manner. Dyes listed across a row can be considered equivalent. In some embodiments, the example described above may employ metal slides, metal slide arrays, and/or focus maps to capture and align images, as described in detail herein.

TABLE 7

[00121] FIG. 15 illustrates an example of a laser focus system 1500 for use with the embodiments described herein. The system 1500 may be configured to capture images of a sample 1502 and/or emission wavelengths from stains applied to such samples. As shown, the system 1500 includes at least an image capture device 1504, a tube lens 1506, an objective lens 1508, a dichroic mirror 1510, an adjustable collimating lens 1512, a line sensor 1514, a beamsplitter 1516, and a laser diode source 1520.

[00122] The laser focus system 1500 may be utilized in a microscope to maintain focus of the sample such that the image capture device 1504 can capture an in-focus image of the sample 1502. The system 1500 may focus the sample by adjusting a distance (d) between the sample and the image capture device (e.g., image sensor, camera, etc.). The system 1500 represents an automatic system that uses laser beams to adjust the focus. For example, a laser beam (e.g., an incident beam) may be initiated by a processor and emitted from the laser diode 1520 through one or more beamsplitters (e.g., beamsplitter 1516) and onto the sample 1502 through one or more lenses, mirrors, etc. (e.g., adjustable collimating lens 1512, dichroic mirror 1510, objective lens 1508, and/or other intervening components in a beam path). The laser beam (e.g., a reflected beam) may be reflected back from the sample to pixels on the line sensor 1514. The line sensor 1514 may be configured to determine whether the beam representing the image is in focus. For example, if the beam is detected in a center portion of the line sensor 1514, then an image may be determined to be in focus. If the beam is detected left or right of center of the line sensor 1504, the image may be determined to be out of focus. If the image is determined to be out of focus, the system 1500 may adjust the distance (d) between the sample 1502 and image capture device 1504 by triggering (via an onboard processer) to move the stage holding the sample 1502 upward or downward.

[00123] The laser focus system 1500 may provide the advantage of performing focus measurements for each image captured. Thus, for each image of a sample collected, the system 1500 may engage the line sensor 1514 to focus the sample to ensure image quality. Focused images may be stored for later use. In some embodiments, the system 1500 is a fluorescence microscope for use with focus maps. For example, a particular focus map may be accessed to assist device 1500 in automatically focusing on portions of sample 1502. [00124] FIG. 16 illustrates an example slide and focus map to be used with the embodiments described herein. The focus map shown here includes five focus points (e.g., focus point 1602) that make up the focus map. The focus points are spread about a sample area 1604. The sample area 1604 includes a sample placed on a slide 1606. Here, the sample area 1604 may be placed on the slide 1606 and a coverslip may be placed on top of the sample area 1604 and may extend beyond the sample area. In some embodiments, the coverslip may be mounted on top of the sample and the focus point 1602 is centered in the sample area 1604. The focus point 1602 may represent a top portion of a pyramid shaped focus area with the four surrounding focus points (e.g., marked as X in FIG. 16) representing the base of the pyramid and the focus point 1602 representing the top of the pyramid. Each focus point in between the center focus point 1602 and another of the base focus points may be used as a basis to approximate focal distances based on the pyramid shape. Although the focus map described herein includes five focus points, any number of focus points may be present in a particular focus map. In such examples, other shapes may be used to approximate focal distances.

[00125] In some embodiments, autofocusing may be performed using an electronic focusing system in which attributes associated with capturing an image of the sample are used to autofocus images of the sample. For example, attributes such as edge sharpness, contrast, brightness, and/or other image sensor parameters (e.g., as measured by the image capture device) may be used as a basis in which to autofocus images captured by the image capture device.

[00126] In some embodiments, autofocusing techniques may be improved by the use of metal slide surfaces. For example, a slide may be composed of metal and/or have a polished metal surface on a slide side facing the emission beam path (e.g., emission beam path 108 shown in FIG. 1). The use of a metal slide (and/or metal polished slide surface) may enhance a fluorescence measurement obtained from a sample by improving contrast of the image of the sample. The improved contrast may be a direct result of the use of metal slides, which can enable brightfield microscopy when using fluorescence microscopes even when such microscopes are typically unequipped to perform brightfield microscopy. That is metal (e.g., steel) slides are typically not used in conventional brightfield microscopy techniques because brightfield microscopy generally uses transmission of light through a clear slide and through a sample to visualize the sample. However, a technical effect of using a metal slide (e.g., polished stainless steel slide) with the brightfield microscopy embodiments described herein includes an improved sample carrier system and an improved image focus of images captured by an image capture device of the microscope. For example, a slide (or slide surface or coating) composed of steel improves autofocusing performance of image capture over that of image capture using glass slides.

[00127] FIG. 17 illustrates an example of using an offset value for focusing on an image plane. As shown, the sample 1604 is receiving an incoming beam 1702 and reflecting an outgoing beam 1704. A reflective plane and an imaging plane are shown and are separated by an offset value 1706.

[00128] Typical biological applications prepare cells that are immobilized onto a glass slide with a glass coverslip mounted on top of the cells and/or any mounting medium. Further, to optimize image quality in such a configuration, both the mounting medium and the glass coverslip may have an identical refractive index. Therefore, an applied laser beam will be reflected from the top of the coverslip or the bottom of the slide, as shown in FIG. 17 depicting an air-glass interface and the use of an offset value 1706 for focusing on an image plane.

[00129] Biological laser-based autofocus systems can compensate for the air-glass interface by allowing an offset value 1706 to focus on the imaging plane. That is, the offset value 1706 may be used to focus on the sample, rather than the top reflective plane (e.g., the coverslip) or the bottom reflective plane (e.g., the bottom of slide). Conventional use of glass slides and coverslips with such offset values can introduce errors in capture and/or image focus if the horizontal coverslip position is moved during use and/or if a particular glass slide is marred. In addition, another drawback of using an offset value with conventional glass slides and coverslips is that a portion of the laser beam light is not reflected but is instead lost due to the refractive nature of two glass layers. Replacing a conventional glass slide with a metal slide ensures that the reflecting surface is at the sample surface (i.e., in the imaging plane), to provide an improved reflection of the laser beam over reflections provided from conventional glass slides.

[00130] As used in the description and claims, the singular form “a”, “an” and “the” include both singular and plural references unless the context clearly dictates otherwise. For example, the term “image capture device” may include, and is contemplated to include, a plurality of image capture devices. At times, the claims and disclosure may include terms such as “a plurality,” “one or more,” or “at least one;” however, the absence of such terms is not intended to mean, and should not be interpreted to mean, that a plurality is not conceived. [00131] The term “about” or “approximately,” when used before a numerical designation or range (e.g., to define a length or pressure), indicates approximations which may vary by ( + ) or ( - ) 5%, 1% or 0.1%. All numerical ranges provided herein are inclusive of the stated start and end numbers. The term “substantially” indicates mostly (i.e., greater than 50%) or essentially all of a device, substance, or composition.

[00132] As used herein, the term “comprising” or “comprises” is intended to mean that the devices, systems, and methods include the recited elements, and may additionally include any other elements. “Consisting essentially of’ shall mean that the devices, systems, and methods include the recited elements and exclude other elements of essential significance to the combination for the stated purpose. Thus, a system or method consisting essentially of the elements as defined herein would not exclude other materials, features, or steps that do not materially affect the basic and novel characteristic(s) of the claimed disclosure. “Consisting of’ shall mean that the devices, systems, and methods include the recited elements and exclude anything more than a trivial or inconsequential element or step. Embodiments defined by each of these transitional terms are within the scope of this disclosure. [00133] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. Other embodiments may be utilized and derived therefrom, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is in fact disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

WHAT IS CLAIMED IS:

1. A method of performing rapid channel -by-channel fluorescence microscopy with a fluorescence microscope having a plurality of image capture devices comprising: exciting at least n+1 emission wavelengths from a sample, wherein n is equal to a number of the plurality of image capture devices; capturing a first set of images of the sample in a first scan of the sample wherein each image of the first set of images is captured simultaneously and separately on a distinct image capture device of the plurality of image capture devices; capturing a second set of images of the sample in a second scan of the sample wherein each image of the second set of images is captured simultaneously and separately on a distinct image capture device of the plurality of image capture devices; calculating an alignment shift between at least the first image and second image; applying the alignment shift to at least one image of the first set of images or to at least one image of the second set of images; and combining the at least one image of the first set with the at least one image of the second set to generate an aligned composite image.

2. The method of claim 1, wherein at least a first image of the first set and at least a second image of the second set capture an identical selection of one or more of the at least n+1 emission wavelengths from the sample;

3. The method of claim 1, wherein the first set of images further comprises at least one image capturing a different selection of one or more of the at least n+1 emission wavelengths than all the images of the second set.

4. The method of claim 1, wherein the second set of images further comprises at least one image capturing a different selection of one or more of the at least n+1 emission wavelengths than all the images of the first set.

5. The method of claim 1, wherein n is 2.

6. The method of claim 1, wherein the first set of images comprises two images.

7. The method of claim 1, wherein the second set of images comprises two images.

8. The method of claim 1, wherein n is 3.

9. The method of claim 1, wherein the first set of images comprises three images.

10. The method of claim 1, wherein the second set of images comprises three images.

11. The method of claim 1, wherein n is greater than 3.

12. The method of claim 1, wherein the first set of images comprises more than three images.

13. The method of claim 1, wherein the second set of images comprises more than three images.

14. The method of claim 1, wherein combining comprises forming a Z-stack of the at least one image of the first set and the at least one image of the second set.

15. The method of claim 1, wherein the first image and the second image are reference images.

16. The method of claim 1, wherein each image capture device of the plurality of image capture devices are independently selected from the group consisting of: a camera, a portion of a camera’s CCD array, and a portion of a camera’s sCMOS chip.

17. The method of claim 1, wherein no pair of images within the first set of images and no pair of images within the second set of images capture a selection of emission wavelengths with a crosstalk intensity greater than 5%.

18. A fluorescence microscope comprising: at least one light source; an excitation beam path that passes light from the at least one light source through a sample stage, wherein at least one of the excitation beam path and the sample stage is configured to move an area of the sample stage through the excitation beam path as a scan; an emission beam path that passes emitted light of a plurality of predetermined emission wavelengths towards at least a first and second image capture device, wherein the emission beam path comprises at least one beam splitter to separate a first and second subdivision of the plurality of emission wavelengths and wherein the at least one beam splitter directs the first subdivision towards the first image capture device and the second subdivision towards the second image capture device; and a processor and a memory storing machine-readable instructions that, when executable by the processor, cause the processor to perform a method comprising: capturing a first set of images of a sample in a first scan wherein each image of the first set of images is captured simultaneously and separately on one of the at least first or second image capture devices; capturing a second set of images of the sample in a second scan wherein each image of the second set of images is captured simultaneously and separately on one of the at least first or second image capture devices; wherein at least a first image of the first set and at least a second image of the second set capture an identical selection of one or more emission wavelengths of a total of at least n+1 emission wavelengths, wherein n is equal to a total number of image capture devices; calculating an alignment shift between at least the first and second images that capture an identical selection of one or more of the at least n+1 emission wavelengths; applying the alignment shift to at least one of the first set of images or the second set of images combining at least one image of the first set with at least one image of the second set to create an aligned composite image.

19. The device of claim 18, wherein the first set of images further comprises at least one image capturing a different selection of one or more of the at least n+1 emission wavelengths than all the images of the second set.

20. The device of claim 18, wherein the second set of images further comprises at least one image capturing a different selection of one or more of the at least n+1 emission wavelengths than all the images of the first set.

21. The device of claim 18, wherein at least one of the excitation beam path and the emission beam path comprises a filter turret configured to automatically add, remove, or replace a first filter with a second filter during a period of time between the first scan and the second scan.

22. The device of claim 21, wherein the filter turret is positioned between the beam splitter and the first or second image capture device.

23. The device of claim 21, wherein the filter turret is positioned between the sample stage and the light source.

24. The device of claim 18, wherein the emission beam path further comprises a beam splitter turret configured to automatically add, remove, or replace a first beam splitter with a second beam splitter during a period of time between the first scan and the second scan.

25. The device of claim 18, wherein the at least one beam splitter of the emission path is a dichroic filter or a dichroic mirror.

26. The device of claim 18, wherein n is 2.

27. The device of claim 18, wherein the first set of images comprises two images.

28. The device of claim 18, wherein the second set of images comprises two images.

29. The device of claim 18, wherein n is 3.

30. The device of claim 18, wherein the first set of images comprises three images.

31. The device of claim 18, wherein the second set of images comprises three images.

32. The device of claim 18, wherein n is greater than 3.

33. The device of claim 18, wherein the first set of images comprises more than three images.

34. The device of claim 18, wherein the second set of images comprises more than three images.

35. The device of claim 18, wherein combining comprises forming a Z-stack of the at least one image of the first set and the at least one image of the second set.

36. The device of claim 18, wherein the first image and the second image are reference images.

37. The device of claim 18, wherein each image capture device of the plurality of image capture devices are independently selected from the group consisting of: a camera, a portion of a camera’s CCD array, and a portion of a camera’s sCMOS chip.

38. The device of claim 18, wherein no pair of images within the first set of images and no pair of images within the second set of images capture a selection of emission wavelengths with a crosstalk intensity greater than 5%.

39. The device of claim 18, wherein light of the excitation beam path is reflected by one of a plurality of dichroic mirrors onto the sample stage, and wherein emission wavelengths from the sample are transmitted through the dichroic mirror; and wherein the plurality of dichroic mirrors can be exchanged in an operable position.

40. The device of claim 39, wherein at least one dichroic mirror of the plurality of dichroic mirrors that reflects light of the excitation beam path reflects the excitation wavelengths and transmits the emission wavelengths of at least one of stains selected from the group consisting of PE, PE-Cy7, PerCP, DRAQ5, BV 421, BV 480, AF 488, AF 594, and AF 800.

41. A fluorescence microscope comprising: at least one light source; an excitation beam path that passes light from the at least one light source through a sample stage, wherein at least one of the excitation beam path and the sample stage is configured to move an area of the sample stage through the excitation beam path as a scan; an emission beam path that passes emitted light of a plurality of predetermined emission wavelengths towards at least a first and second image capture device, wherein the emission beam path comprises at least one beam splitter to separate a first and second subdivision of the plurality of emission wavelengths and wherein the at least one beam splitter transmits the first subdivision towards the first image capture device and reflects the second subdivision towards the second image capture device; wherein light from the excitation beam path is reflected by a first, second, or third dichroic mirror onto the sample stage, and wherein emission wavelengths from the sample are transmitted through the first, second or third dichroic mirror; wherein the first dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 488, BV 421, BV 480, DRAQ5, and PerCP or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the second dichroic mirror reflects the excitation wavelengths of the fluorescent stains of PE and DRAQ5 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the third dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 594 and DRAQ5 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the first, second, and third dichroic mirror can be exchanged in an operable position; wherein the at least one beam splitter is a 660 nm long pass dichroic mirror; wherein a first filter wheel is filter is positioned between the first image capture device and the beam splitter wherein the first filter wheel comprises filters of 810/90 and 697/60; and wherein a second filter wheel is positioned between the second image capture device and the beam splitter wherein the second filter wheel comprises filters of 431/28, 480/30, 537/29, 572/23, and 630/28.

42. A fluorescence microscope comprising: at least one light source; an excitation beam path that passes light from the at least one light source through a sample stage, wherein at least one of the excitation beam path and the sample stage is configured to move an area of the sample stage through the excitation beam path as a scan; an emission beam path that passes emitted light of a plurality of predetermined emission wavelengths towards at least a first and second image capture device, wherein the emission beam path comprises at least one beam splitter to separate a first and second subdivision of the plurality of emission wavelengths and wherein the at least one beam splitter transmits the first subdivision towards the first image capture device and reflects the second subdivision towards the second image capture device; wherein light from the excitation beam path is reflected by a first, second, third, or fourth dichroic mirror onto the sample stage, and wherein emission wavelengths from the sample are transmitted through the first, second, third, or fourth dichroic mirror; wherein the first dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 488, BV 421, BV 480, DRAQ5, and PerCP or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the second dichroic mirror reflects the excitation wavelengths of the fluorescent stains of PE-Cy7 and BV 480 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the third dichroic mirror reflects the excitation wavelengths of the fluorescent stains of PE and DRAQ5 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the fourth dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 594 and DRAQ5 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the first, second, third, and fourth dichroic mirror can be exchanged in an operable position; wherein the at least one beam splitter is a 647 nm short pass dichroic mirror; wherein a first filter wheel is positioned between the first image capture device and the beam splitter, wherein the first filter wheel comprises filters of 431/28, 480/30, 537/29, 572/23, and 630/28; and wherein a second filter wheel is positioned between the second image capture device and the beam splitter wherein the second filter wheel comprises filters of 810/90 and 697/60.

43. A fluorescence microscope comprising: at least one light source; an excitation beam path that passes light from the at least one light source through a sample stage, wherein at least one of the excitation beam path and the sample stage is configured to move an area of the sample stage through the excitation beam path as a scan; an emission beam path that passes emitted light of a plurality of predetermined emission wavelengths towards at least a first, second, and third image capture device, wherein the emission beam path comprises at least a first and second beam splitter; wherein the first beam splitter separates a first and second subdivision of the plurality of emission wavelengths, and wherein the first beam splitter transmits the first subdivision towards the first image capture device and reflects the second subdivision towards the second image capture device; wherein the second beam splitter separates a third subdivision of the plurality of emission wavelengths from the first and second subdivision of plurality of emission wavelengths, and wherein the second beam splitter transmits the first and second subdivision towards the first beam splitter and reflects the third subdivision towards the third image capture device; wherein light from the excitation beam path is reflected by a first, second, or third dichroic mirror onto the sample stage, and wherein emission wavelengths from the sample are transmitted through the first, second, or third dichroic mirror; wherein the first dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 594, BV 480, and DRAQ5 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the second dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 488, DRAQ5, and BV 421 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the third dichroic mirror reflects the excitation wavelengths of the fluorescent stains of PE, PerCP, and BV 421 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the first, second, and third dichroic mirror can be exchanged in an operable position; wherein the first beam splitter is a 455 nm long pass dichroic mirror; wherein the second beam splitter is a 647 nm short pass dichroic mirror; wherein a first filter wheel is positioned between the first image capture device and the first beam splitter, wherein the first filter wheel comprises filters 537/29, 572/23, and 630/28; wherein a second filter wheel is positioned between the second image capture device and the first beam splitter; and wherein a third filter wheel is positioned between the third image capture device and the second splitter wherein the third filter wheel comprises filters of 810/90 and 697/60.

44. A fluorescence microscope comprising: at least one light source; an excitation beam path that passes light from the at least one light source through a sample stage, wherein at least one of the excitation beam path and the sample stage is configured to move an area of the sample stage through the excitation beam path as a scan; an emission beam path that passes emitted light of a plurality of predetermined emission wavelengths towards at least a first, second, and third image capture device, wherein the emission beam path comprises at least a first and second beam splitter; wherein the first beam splitter separates a first and second subdivision of the plurality of emission wavelengths, and wherein the first beam splitter transmits the first subdivision towards the first image capture device and reflects the second subdivision towards the second image capture device; wherein the second beam splitter separates a third subdivision of the plurality of emission wavelengths from the first and second subdivision of plurality of emission wavelengths, and wherein the second beam splitter transmits the first and second subdivision towards the first beam splitter and reflects the third subdivision towards the third image capture device; wherein light from the excitation beam path is reflected by a first, second, third, or fourth dichroic mirror onto the sample stage, and wherein emission wavelengths from the sample are transmitted through the first, second, third, or fourth dichroic mirror; wherein the first dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 594, BV 480, and DRAQ5 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the second dichroic mirror reflects the excitation wavelengths of the fluorescent stains of AF 488, DRAQ5, and BV 421 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the third dichroic mirror reflects the excitation wavelengths of the fluorescent stains of PE, PerCP, and BV 421 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the fourth dichroic mirror reflects the excitation wavelengths of the fluorescent stains of PE-Cy7 and BV 480 or one or more equivalent fluorophores thereof and transmits their emission wavelengths; wherein the first, second, third, and fourth dichroic mirror can be exchanged in an operable position; wherein the first beam splitter is a 455 nm long pass dichroic mirror; wherein the second beam splitter is a 647 nm short pass dichroic mirror; wherein a first filter wheel is positioned between the first image capture device and the first beam splitter, wherein the first filter wheel comprises filters 537/29, 572/23, and 630/28; wherein a second filter wheel is positioned between the second image capture device and the first beam splitter; and wherein a third filter wheel is positioned between the third image capture device and the second splitter wherein the third filter wheel comprises filters of 810/90 and 697/60.

45. A method for calibrating a microscope that comprises at least two image capture devices, the method comprising: providing a calibration diagram to a microscope comprising at least two image capture devices; capturing a unique image of the calibration diagram with the at least two image capture devices simultaneously; locating on each unique image two endpoints of each terminal spline; calculating the midpoint of each terminal spline to generate at least one calibration coordinate for each unique image; calculating at least one transformation to equate the at least one calibration coordinates of the unique images; and and calibrating the at least two image capture devices with the at least one transformation.

46. The method of claim 45, where the at least one transformation can include one or more of a translation, a rotation, or a scale.