Method and system for rapid spectral unmixing using spectrally interpolated background reduction

Abstract

In some instances, a system for rapid spectral unmixing using spectrally interpolated background reduction (SIBR) is provided. The system comprises a device comprising an excitation light source and a detector. The system further comprises a control system configured to: obtain a plurality of excitation spectra for a plurality of fluorophores that are used to stain a sample; determine a plurality of chosen wavelengths and a plurality of chosen light intensities for the plurality of fluorophores; obtain a first detection of the sample based on using a set of first wavelengths from the plurality of chosen wavelengths and a set of first light intensities; obtain a second detection of the sample based on using a set of second wavelengths from the plurality of chosen wavelengths and a set of second light intensities; and output a SIBR quantity.

Description

METHOD AND SYSTEM FOR RAPID SPECTRAL UNMIXING USING SPECTRALLY INTERPOLATED BACKGROUND REDUCTION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 529,866, filed July 31, 2023, which is herein incorporated by reference in its entirety. BACKGROUND

[0002] Existing methods for imaging and unmixing a plurality of fluorescent probes on a single sample typically use two approaches that are not mutually exclusive, and may be used together. For example, the first method may include labelling the sample with a small number (e.g., approximately four) of fluorescent probes, imaging them, removing or inactivating them, and re-staining the sample with a new set of probes. This ‘iterative labeling’ process may be repeated multiple times. It has the advantage that it is very easy to image a small number fluorescent probes with no crosstalk between them. The disadvantages include i) because it is an iterative procedure, it is slow – often taking days and many iterations to image the entire probe set for a sample; ii) the images of the sample taken at different iterations need to be spatially co-registered, which is a computationally intense procedure; and iii) removing or deactivating fluorescent probes is typically performed with harsh solvents that may damage the sample, limiting the number of possible iterations.

[0003] The second method may include labelling the sample with a large number (e.g., greater than six) of fluorescent probes. Images are acquired with a hardware configuration that minimizes, but does not eliminate, crosstalk between images of different fluorophores. These images are then computationally unmixed – e.g., they are separated based on the known level of crosstalk between each fluorophore channel. This approach has the advantage that since a large number of fluorescent probes are being imaged in a single imaging session – fewer staining rounds are necessary. This improves the speed / throughput of the process, reduces the number of computational co-registrations, and reduces the exposure of the sample to harsh solvents. However, unmixing a large number of fluorescent probes is itself a computationally complex process and does not scale well with data size. As the number of fluorophores increases, or as the number of images increases with larger or more numerous samples, the computation required for unmixing becomes prohibitive – often requiring specialized and expensive computers (e.g., graphic processing unit (GPU) clusters and / or supercomputers).SUMMARY

[0004] In an exemplary embodiment, the present application provides a system for rapid spectral unmixing using spectrally interpolated background reduction (SIBR). The system comprises a device and a control system. The device comprises an excitation light source and a detector. The control system is configured to obtain a plurality of excitation spectra for a plurality of fluorophores that are used to stain a sample, determine a plurality of chosen wavelengths and a plurality of chosen light intensities for the plurality of fluorophores based on the plurality of excitation spectra, and obtain, using the detector of the device, a first detection of the sample based on using a set of first wavelengths from the plurality of chosen wavelengths and a set of first light intensities. The set of first wavelengths comprises a wavelength associated with a target fluorophore of the plurality of fluorophores. The control system is further configured to obtain, using the detector of the device, a second detection of the sample based on using a set of second wavelengths from the plurality of chosen wavelengths and a set of second light intensities and output a SIBR quantity of the target fluorophore based on the first detection and the second detection.

[0005] In some examples, the device further comprises a filter. The control system is further configured to provide setup instructions to the filter based on the set of first wavelengths and the set of second wavelengths.

[0006] In some instances, the filter is an acousto-optic tunable filter (AOTF) comprising a plurality of channels. Each of the channels permits light at a certain wavelength and a certain intensity to be emitted from the excitation light source onto the sample. Further, the control system provides the setup instructions by setting up one or more first channels of the plurality of channels based on the set of first wavelengths and the set of first light intensities, and setting up one or more second channels of the plurality of channels based on the set of second wavelengths and the set of second light intensities.

[0007] In some variations, the control system enables the one or more first channels of the AOTF and provides one or more instructions to the excitation light source to emit light. The light passes through the AOTF such that only the set of first wavelengths and the set of first light intensities are emitted onto the sample. The control system obtains, from the detector, the first detection of the sample based on only the set of first wavelengths and the set of first light intensities being emitted onto the sample.

[0008] In some examples, the control system enables the one or more second channels of the AOTF and provides one or more instructions to the excitation light source to emit light.The light passes through the AOTF such that only the set of second wavelengths and the set of second light intensities are emitted onto the sample. The control system obtains, from the detector, the second detection of the sample based on only the set of second wavelengths and the set of second light intensities being emitted onto the sample.

[0009] In some instances, the excitation light source is a tunable light source. The control system provides one or more instructions to the tunable light source to emit light onto the sample at the set of first wavelengths and the set of first light intensities. Based on providing the one or more instructions, the control system obtains, from the detector, the first detection of the sample.

[0010] In some variations, the detector is an image capturing device, the first detection of the sample is a first fluorescence image of the sample based on the set of first wavelengths, and the second detection of the sample is a second fluorescence image of the sample based on the set of second wavelengths.

[0011] In some examples, the control system obtains the plurality of excitation spectra for the plurality of fluorophores by receiving user input indicating the plurality of excitation spectra.

[0012] In some instances, the control system obtains, using the detector of the device, a plurality of first initial detections of a second sample at a plurality of different wavelengths. The second sample is stained with only a first fluorophore of the plurality of fluorophores. The control system further generates a first excitation spectra for the first fluorophore based on the plurality of first initial detections and obtains, using the detector of the device, a plurality of second initial detections of a third sample at the plurality of different wavelengths. The third sample is stained with only a second fluorophore of the plurality of fluorophores. The control system further generates a second excitation spectra for the second fluorophore based on the plurality of second initial detections.

[0013] In some variations, the plurality of chosen wavelengths and the plurality of chosen light intensities for the plurality of fluorophores are determined by receiving first user input indicating the plurality of chosen wavelengths and receiving second user input indicating the plurality of chosen light intensities.

[0014] In some examples, the control system receives user input indicating the target fluorophore of the plurality of fluorophores, selects a plurality of first selected wavelengths for the plurality of fluorophores, determines a plurality of first ratios of light intensities based on the plurality of first selected wavelengths and the plurality of excitation spectra, determines a first SIBR quantity of the target fluorophore based on the plurality of first selected wavelengths,the plurality of excitation spectra, and the plurality of first ratios of light intensities, and based on comparing the first SIBR quantity of the target fluorophore with one or more additional SIBR quantity of the target fluorophore, determines the plurality of chosen wavelengths and the plurality of chosen light intensities. The one or more additional SIBR quantity are determined using a plurality of additionally selected wavelengths that are different from the plurality of first selected wavelengths.

[0015] In some instances, the control system determines a maximum value from the first SIBR quantity and the one or more additional SIBR quantity, and selects the plurality of chosen wavelengths and the plurality of chosen light intensities based on the maximum value.

[0016] In some examples, the plurality of fluorophores comprise three fluorophores, the plurality of chosen wavelengths comprise three chosen wavelengths, the set of first wavelengths comprise the wavelength associated with the target fluorophore from the three fluorophores, and the set of second wavelengths comprise the other two chosen wavelengths associated with the other two fluorophores.

[0017] In some variations, the plurality of chosen light intensities comprise a plurality of positive light intensities and one or more negative light intensities, the one or more negative light intensities indicate a negative value for the light intensity, the one or more negative light intensities is associated with one or more wavelengths from the plurality of chosen wavelengths, the set of first wavelengths comprises the wavelength associated with the target fluorophore and the one or more wavelengths associated with the one or more negative light intensities, and the set of second wavelengths comprise wavelengths from the plurality of chosen wavelengths associated with the plurality of positive light intensities.

[0018] In some instances, the wavelength associated with the target fluorophore of the plurality of fluorophores is used to obtain a total detected signal from the sample, and the total detected signal comprises a signal for the target fluorophore and one or more signals detected for other fluorophores from the plurality of fluorophores.

[0019] In some examples, a method is provided. The method comprises obtaining, by a control system, a plurality of excitation spectra for a plurality of fluorophores that are used to stain a sample, determining, by the control system, a plurality of chosen wavelengths and a plurality of chosen light intensities for the plurality of fluorophores based on the plurality of excitation spectra, and obtaining, by the control system and using a detector of a device, a first detection of the sample based on using a set of first wavelengths from the plurality of chosen wavelengths and a set of first light intensities. The set of first wavelengths comprises a wavelength associated with a target fluorophore of the plurality of fluorophores. The methodfurther comprises obtaining, by the control system and using the detector of the device, a second detection of the sample based on using a set of second wavelengths from the plurality of chosen wavelengths and a set of second light intensities, and outputting, by the control system, a SIBR quantity of the target fluorophore based on the first detection and the second detection.

[0020] In some variations, the device further comprises a filter, and the method further comprises providing setup instructions to the filter based on the set of first wavelengths and the set of second wavelengths.

[0021] In some instances, the filter is an acousto-optic tunable filter (AOTF) comprising a plurality of channels, and each of the channels permits light at a certain wavelength and a certain intensity to be emitted from an excitation light source onto the sample. Further, providing the setup instructions comprises setting up one or more first channels of the plurality of channels based on the set of first wavelengths and the set of first light intensities, and setting up one or more second channels of the plurality of channels based on the set of second wavelengths and the set of second light intensities.

[0022] In some examples, obtaining the first detection of the sample comprises: enabling the one or more first channels of the AOTF; providing one or more instructions to the excitation light source to emit light, and the light passes through the AOTF such that only the set of first wavelengths and the set of first light intensities is emitted onto the sample; and obtaining, from the detector, the first detection of the sample based on only the set of first wavelengths and the set of first light intensities being emitted onto the sample.

[0023] In some variations, a non-transitory computer-readable medium having processor- executable instructions stored thereon is provided. The processor-executable instructions, when executed, facilitate: obtaining a plurality of excitation spectra for a plurality of fluorophores that are used to stain a sample; determining a plurality of chosen wavelengths and a plurality of chosen light intensities for the plurality of fluorophores based on the plurality of excitation spectra; obtaining, and using a detector of a device, a first detection of the sample based on using a set of first wavelengths from the plurality of chosen wavelengths and a set of first light intensities. The set of first wavelengths comprises a wavelength associated with a target fluorophore of the plurality of fluorophores. The processor-executable instructions, when executed, further facilitate: obtaining, using the detector of the device, a second detection of the sample based on using a set of second wavelengths from the plurality of chosen wavelengths and a set of second light intensities; and outputting a SIBR quantity of the target fluorophore based on the first detection and the second detection.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows an exemplary spectrally interpolated background reduction (SIBR) system for rapid spectral unmixing according to one or more examples of the present application.

[0025] FIG. 2 shows an exemplary process for rapid spectral unmixing using SIBR according to one or more examples of the present application.

[0026] FIG. 3 shows a graphical representation of three fluorophores with overlapping excitation spectra according to one or more examples of the present application.

[0027] FIG. 4 shows a graphical representation of “N” fluorophores with overlapping excitation spectra according to one or more examples of the present application.

[0028] FIGs. 5A and 5B show graphical representations of absorption, excitation, and emission spectra for eight fluorophores selected for validation of SIBR according to one or more examples of the present application.

[0029] FIG.6 shows fixed U2OS cells that were stained in an eight well slide.

[0030] FIG. 7 shows a graphical representation of recorded excitation spectra of a target (i.e., Atto Rho6G) and five background fluors that are plotted as a function of AOTF frequency (e.g., a measure of excitation wavelength).

[0031] FIG.8 shows positive images, negative images, and SIBR images of two dyes (Atto Rho6G and DyLight 554)

[0032] FIGs.9-16 show positive images and the SIBR images for multiple dyes and targets. DETAILED DESCRIPTION

[0033] Examples of the presented application will now be described more fully hereinafter with reference to the accompanying FIGs., in which some, but not all, examples of the application are shown. Indeed, the application may be exemplified in different forms and should not be construed as limited to the examples set forth herein; rather, these examples are provided so that the application will satisfy applicable legal requirements. Where possible, any terms expressed in the singular form herein are meant to also include the plural form and vice versa, unless explicitly stated otherwise. Also, as used herein, the term “a” and / or “an” shall mean “one or more” even though the phrase “one or more” is also used herein. Furthermore, when it is said herein that something is “based on” something else, it may be based on one or more other things as well. In other words, unless expressly indicated otherwise, as used herein “based on” means “based at least in part on” or “based at least partially on”.

[0034] Systems, methods, and computer program products are herein disclosed that perform spectrally interpolated background reduction (SIBR). SIBR may rapidly calculate the signal from a single fluorescent probe in a sample labeled with many spectrally distinct fluorescent probes. Iterating this process for all the spectrally distinct fluorophores in a sample provides an ‘unmixed’ set of signals (e.g., a signal from each individual fluorophore, proportional to its concentration, without appreciable crosstalk from other fluorophores).

[0035] For instance, to calculate the fluorescence signal from a single ‘target’ fluorophore in a sample containing many fluorophores, a SIBR system may use the acquisition of two images taken sequentially using excitations with a calculated pattern of wavelength and intensity. The calculated difference between these two images is an image that is proportional to the concentration of the target fluorophore without appreciable crosstalk from the other non- target fluorophores. The simplicity of this approach allows for very high speed (e.g., even real time) spectral unmixing of image data.

[0036] For example, SIBR may be used to unmix fluorescence images of cells / tissue stained with many fluorescent probes. Further, SIBR is a general technique for rapid unmixing of many fluorophores that may be extended to non-imaging based fluorescence techniques (e.g., multiplex flow cytometry, fluorimetry, and / or multiplex Western blot). For example, in flow cytometry, SIBR may use a photomultiplier tube (PMT) to detect the total fluorescence coming from a cell rather than the spatial distribution of the fluorescence as captured by a camera. The first and second detections described in FIG. 2 below may be, in this example, output signals from the PMT, which are then subtracted to obtain the SIBR quantity that measures the total amount of the target fluorescent probe in that cell.

[0037] Rapid spectral unmixing using SIBR is described below. For instance, multiplex fluorescence imaging involves labelling and imaging a single cell / tissue sample with many fluorescent probes (e.g., greater than six probes) with the goal of gathering expansive spatial information from the sample. Labeling a sample with many fluorophores, however, presents a challenge for quantitatively measuring the concentration and distribution of each individual fluorophore. The excitation and emission spectra of fluorophores are typically broad (e.g., approximately 100 nanometers (nm)), which leads to significant spectral overlap between them as the number of fluorophores on the sample increases. The imaging channels for individual fluorophores, which may be defined by an excitation wavelength and an emission band-pass filter, receive crosstalk from other fluorophores. If the spectra are known, or if the crosstalk can be directly measured, then systems may computationally correct it, yielding images of the individual fluorophores without crosstalk. This process is known as ‘unmixing’ or ‘spectralunmixing’, and it is an essential part of multiplex imaging as it converts convoluted image data into interpretable information.

[0038] By using SIBR, a SIBR system (e.g., the SIBR system shown in FIG.1) may rapidly calculate the signal from a single fluorescent probe in a sample labeled with many spectrally distinct fluorescent probes. Iterating this process for all the spectrally distinct fluorophores in a sample provides an ‘unmixed’ set of signals (e.g., a signal from each individual fluorophore, proportional to its concentration, without appreciable crosstalk from other spectrally distinct fluorophores). To calculate the fluorescence signal from a single ‘target’ fluorophore in a sample containing many fluorophores, the SIBR system obtains (e.g., acquires) two images taken sequentially using excitations with a calculated pattern of wavelength and intensity. The calculated difference between these two images is an image that is proportional to the concentration of the target fluorophore without appreciable crosstalk from the other non-target fluorophores. The simplicity of this approach allows for very high speed (even real time) spectral unmixing of image data.

[0039] As described in certain examples below, SIBR is used in the context of fluorescence imaging and fluorescence microscopes. For instance, the measurements of fluorescence are images, e.g., there is a spatially varying distribution of the fluorescence. However, in other examples, the SIBR technique might not be limited to imaging. For instance, the SIBR technique may be applied to any context in which multiple fluorophores are being excited and the signal from each fluorophore needs to be isolated without crosstalk from the other fluorophores. Examples include a mixture of multiple fluorophores in solution being measured by a fluorometer, cells stained with multiple fluorophores flowing through a fluorescence activated cell sorter (FACS), and so on.

[0040] Among other advantages and uses, the SIBR system and method may be used for multiple commercial classes of products such as multiplex fluorescence imaging and / or high content imaging. These related and overlapping fields may use fluorescence imaging to extract as much information as possible from cell / tissue samples, and may be used extensively in academia, healthcare, and the biotech / pharmaceutical industry. Advantages of using the SIBR system and method may include, but are not limited to, increasing the speed / throughput of many existing platforms for multiplex and high content imaging. The SIBR system may further provide a particular advantage in situations where the amount of image data generated is so large that image computation becomes a limiting step in the data pipeline such as multiplex high throughput imaging (e.g., automated imaging in multi-well plates – common in drug discovery) and / or multiplex cleared tissue imaging. Non-imaging based implementations ofthe SIBR system and method may be useful in multiplex flow cytometry / fluorescence activated cell sorting (FACS), or multiplex fluorescence Western blotting. For example, these may benefit from the ability to rapidly unmix fluorescence signals as it is a very high-speed technique.

[0041] FIG. 1 shows an exemplary spectrally interpolated background reduction (SIBR) system for rapid spectral unmixing according to one or more examples of the present application. The SIBR system 100 includes a control system 102, a device 108, and a sample 116. The sample 116 may be dyed or stained with fluorescent dyes and / or fluorophores. In some examples, the sample 116 may also express fluorescent proteins, or exhibit native, endogenous fluoresce (e.g., autofluorescence or intrinsic fluorescence). For example, the sample 116 may be a cell, tissue, or micro-organism, and a plurality of fluorophores may be applied to the sample 116. Each fluorophore may be associated with an absorption spectrum, an emission spectrum, and / or an excitation spectrum (e.g., change in fluorescence intensity as a function of wavelength of excitation light). For instance, the fluorophore may absorb light at certain wavelengths (e.g., based on an absorption spectrum) and emit light at other wavelengths (e.g., based on an emission spectrum). For example, one particular fluorophore may absorb blue light and emit green light, another fluorophore may absorb yellow light and emit orange light. As such, each fluorophore may be associated with a different absorption spectrum, excitation spectrum, and / or emission spectrum. By using multiple fluorophores, certain aspects of the sample 116 may be observed. For instance, one particular fluorophore may be used to show one particular component or molecule such as actin and another fluorophore may be used to show another component or molecule. The sample 116 may be dyed / stained with multiple different fluorophores (e.g., an “N” number of fluorophores such as three or more fluorophores) that have different excitation spectra.

[0042] The control system 102 includes a processor 104 and memory 106. The control system 102 controls the operation of the SIBR system 100 (e.g., the device 108) and is configured to perform rapid spectral unmixing based on SIBR. The processor 104 may be any type of device, controller, apparatus, circuitry, and / or logic used to control the operation of the device 108. The processor 104 may be contained within a single device, or there may be multiple processors in a plurality of devices connected together to provide any of the functionality described herein.

[0043] The memory 106 may be a non-transitory computer readable medium that includes processing instructions and / or may be used to store information. The processor 104 may execute the processing instructions stored in the memory 106 to control the operation the device108. The memory 106 may be any type of memory including a computer-readable carrier such as solid-state memory, magnetic disk, and optical disk comprising the processing instructions and data structures that would cause a processor 104 to carry out any of the functions described herein.

[0044] The device 108 is any type of device that is associated with a fluorescence-based platform. For instance, the device 108 may be a fluorescence microscope such as a light sheet microscope. In other instances, the device 108 may be another type of fluorescence microscope (e.g., widefield fluorescence microscope, structured illumination microscope, and / or confocal microscope) and / or another type of microscope altogether. In some examples, the device 108 may be a non-imaging-based fluorescence measurement device such as fluorimeters, fluorescence activated cell sorters (FACS) or flow cytometers, Western blots, and so on.

[0045] The device 108 includes an excitation light source 110 that is configured to excite the sample 116 and a detector 114 that is configured to determine an intensity of emission from the sample 116. The filter 112 is shown in dotted lines to reflect that it is optional. For example, the excitation light source 110 is configured to provide light (e.g., emit or shine light) onto the sample 116. As mentioned previously, the sample 116 may be stained with a plurality of fluorophores. Based on providing light onto the sample 116, the sample 116 re-emits light (e.g., light of another wavelength). The detector 114 detects the re-emitted light from the sample 116. For instance, the detector 114 may detect an intensity (e.g., quantity) of light that is re-emitted from the sample 116.

[0046] In some instances, the excitation light source 110 is a white light (broad spectrum) source and / or a tunable light source. The white light source may be a light source (e.g., a laser or other entity) that emits white light. When the excitation light source 110 is a white light source, the filter 112 is present. For instance, the filter 112 is an excitation filter that selects the excitation wavelength and / or intensity that is provided to the sample 116. For example, the filter 112 receives the light from the excitation light source 110 (e.g., a white light source) and filters the light such that only selected wavelength(s) of light at certain intensities is passed to the sample 116. In some examples, the filter 112 is an acousto-optic tunable filter (AOTF) that includes a plurality of channels (e.g., eight channels). Each of the channels may be tuned (e.g., tunable) such that a certain wavelength and / or intensity of light are allowed to be passed through. For instance, a first channel may allow light at 530 nm at an intensity of 100 milli- Watts (mW). The second channel may allow light at 500 nm at an intensity of 70 mW. The control system 102 may provide instructions to set the plurality of channels of the AOTF. Additionally, and / or alternatively, the filter 112 may be an excitation filter. An excitation filteris an optical-glass filter that passes through a specific band of wavelengths of light from a light source (e.g., the excitation light source 110).

[0047] In some instances, when the excitation light source 110 is a laser or other coherent light source, the device 108 may include a mechanism, component, and / or other aspect that reduces the spatio-temporal coherence of the excitation light such as a rapidly spinning optical diffuser. For example, as mentioned above, in some examples, the device 108 may be a widefield fluorescence microscope (e.g., a modified widefield fluorescence microscope), and the widefield fluorescence microscope may include a mechanism (e.g., a rapidly spinning optical diffuser) for reducing the spatio-temporal coherence of the excitation light.

[0048] In some variations, the excitation light source 110 may be a tunable light source (e.g., a tunable laser). The tunable light source may be configured to provide light at different wavelengths and / or intensities. For instance, the control system 102 may control the tunable light source to output light to the sample 116 at different wavelengths and / or intensities. In some examples, the excitation light source 110 is a tunable light source, and the device 108 may further include a secondary filter device that is configured to provide light at different intensities. For example, the tunable light source may be tuned to provide a user specified wavelength and the secondary filter device (e.g., a secondary intensity filter device such as a Pockels Cell and / or other type of electro-optic device) may control the intensity of light that passes through it.

[0049] The detector 114 may be configured to detect intensities (e.g., fluorescence intensities) that are emitted from the sample 116 after light is shone onto the sample 116. For instance, in some variations, the detector 114 is an image capturing device (e.g., camera). The detector 114 is configured to obtain an image of the sample 116 after the excitation light source 110 and / or the filter 112 provides light onto the sample 116. The image of the sample 116 may indicate the spatial distribution of the fluorescence intensity (e.g., brightness or luminance) emitted by the sample 116. For instance, the image of the sample 116 may include pixels (e.g., 2000 x 2000 pixels) and each pixel may have an associated intensity value that is based on the light emitted by the sample at that spatial location. In other words, the fluorophores may be stained onto certain aspects of the sample 116 such as the proteins, nucleic acids, or other portions. Based on shining light from the excitation light source 110, the fluorophores may emit light at certain wavelengths. The detector 114 detects this light. In some instances, the excitation and emission spectra of different fluorophores may have significant spectral overlap, which impedes being able to distinguish particular fluorophores (e.g., a target fluorophore). Assuch, the control system 102 may perform process 200 shown in FIG. 2 to perform rapid spectral unmixing of the fluorophores.

[0050] In some instances, the detector 114 is a non-imaging device such as a photomultiplier tube. The detector 114 may still obtain / detect the intensities that are emitted from the sample 116.

[0051] It will be appreciated that the exemplary system depicted in FIG. 1 is merely an example, and that the principles discussed herein may also be applicable to other situations and system configurations.

[0052] FIG. 2 shows an exemplary process 200 for rapid spectral unmixing using SIBR according to one or more examples of the present application. The exemplary SIBR system 100 from FIG.1 may perform the process 200. However, it will be recognized that any of the following blocks may be performed in any suitable order and that the process 200 may be performed in any suitable environment by any suitable device. The descriptions, illustrations, and processes of FIG.2 are merely exemplary and the process 200 may use other descriptions, illustrations, and processes.

[0053] In operation, at block 202, the control system 102 obtains a plurality of excitation spectra for a plurality of fluorophores that are used to stain a sample (e.g., sample 116). For instance, as mentioned previously, the sample 116 may be stained with a plurality of fluorophores that have different excitation spectra. The excitation spectrum indicates changes in fluorescence intensity as a function of wavelength of excitation light. FIG. 3 shows a graphical representation 300 of three fluorophores with overlapping excitation spectra according to one or more examples of the present application. For instance, referring to FIG. 3, three excitation spectra for fluorophore zero (e0) 302, fluorophore first (e1) 304, and fluorophore second (e2) 306 are shown. The x-axis is the wavelength 316 in nm and the y-axis 314 shows the normalized excitation (e.g., a normalized version of the excitation intensity). Each fluorophore has a different curve. For instance, for fluorophore zero (spectrum e0) 302, the peak of the curve (e.g., the maximum normalized excitation shown on the y-axis) is around a wavelength of 535 nm. For the fluorophore first (spectrum e1) 304, the peak of the curve is around a wavelength of 515 nm. Therefore, the control system 102 may obtain excitation spectra for a plurality of fluorophores (e.g., the excitation spectra 302, 304, and 306 for the three fluorophores shown in FIG.3).

[0054] In some instances, the control system 102 obtains excitation spectra for the fluorophores based on user input. For instance, the control system 102 may include a user input device. Using the user input device, a user may provide the excitation spectra to thecontrol system 102. Additionally, and / or alternatively, the control system 102 may import the excitation spectra (e.g., the curves for the different fluorophores that are used to stain the sample 116) from another source.

[0055] In some examples, the control system 102 may perform a process to obtain the excitation spectra for the fluorophores. For instance, a user may stain a sample with a single fluorophore. Then, the control system 102 may provide light (e.g., instruct the excitation light source 110 to provide light) onto the stained sample with the single fluorophore. The control system 102 may provide the light at different wavelengths, and may use the detector to obtain a quantity (e.g., an image) at each of the different wavelengths. For instance, the control system 102 may provide instructions to output excitation light at a first wavelength, 500 nm, and obtain a first quantity (e.g., fluorescent intensity) at this wavelength. Then, the control system 102 may provide instructions to output light at second wavelength, 505 nm, obtain a second quantity at this wavelength, and so on. After obtaining the plurality of quantities at the plurality of wavelengths, the control system 102 may generate the excitation spectra for the fluorophore. The control system 102 may use the same process to obtain the excitation spectra for the other fluorophores that stained the sample 116.

[0056] At block 204, the control system 102 determines a plurality of chosen wavelengths and a plurality of chosen light intensities for the plurality of fluorophores based on the plurality of excitation spectra. For instance, based on the excitation spectra that are obtained in block 202, the control system 102 determines multiple wavelengths such as the wavelengths 308-312 (e.g., λ0to λ2). For example, as shown in FIG. 3, the three wavelengths are associated with the excitation spectra 302-306 for the three fluorophores. For instance, the control system 102 determines the plurality of wavelengths (e.g., wavelengths 308-312 such as at 527 nm, 511 nm, and 545 nm) and excitation intensities based on the excitation spectra of the fluorophores (e.g., the excitation spectra 302-306) so as to maximize the SIBR quantity (e.g., the SIBR quantity that is described in block 210 below) of the target fluorophore (e.g., the fluorophore e0that has the excitation spectra 302) and minimize the SIBR quantity of the non-target fluorophores (e1 that has spectra 304 and e2 that has spectrum 306).

[0057] In some variations, the control system 102 may receive user input indicating the plurality of chosen wavelengths. For instance, the control system 102 may include a user input device. Using the user input device, a user may provide the plurality of wavelengths (e.g., the wavelengths 308-310) to the control system 102. For example, the control system 102 may receive user input indicating the wavelengths 308-312. Using the user input wavelengths, the control system 102 may then calculate the excitation intensities at these wavelengths tomaximize the SIBR quantity of the target fluorophore and minimize the SIBR quantity for non- target fluorophores. For instance, the control system 102 may receive user input indicating the plurality of wavelengths (e.g., wavelength 310, wavelength 308, and wavelength 312), and the control system 102 may calculate the excitation intensities at those wavelengths (e.g. a light intensity of 1 mW for wavelength 310, a light intensity of 1.5 mW for wavelength 308, and a light intensity of 0.7 mW for wavelength 312).

[0058] In some examples, the control system 102 may use an algorithm (e.g., a script) and / or one or more mathematical equations to determine the plurality of chosen wavelengths and the plurality of chosen intensities based on the excitation spectra. For example, the control system 102 may use one or more of the below equations to determine the intensities for the wavelengths and the optimal wavelengths to use for blocks 206 and 208:

[0059] For example, the control system 102 may use equations (Eq.) A-D (shown as Equations 9-10 and 14-15 below) to determine the intensities for the wavelengths and the optimal wavelengths. As mentioned previously, the optimal wavelengths (e.g., selected wavelengths) may be the wavelengths that maximize the SIBR quantity' of the target fluorophore while reducing the SIBR quantity for all other non-target (background) fluorophores.

[0060] For instance, in operation, the control system 102 receives user input indicating a. target fluorophore to be examined (e.g., a fluorophore that is used to stain a particular molecule of interest such as actin). For example, referring to FIG. 3, the control system 102 receives user input indicating that the target fluorophore is the fluorophore with the excitation spectra of e0302. The control system 102 may select wavelengths (e.g., the wavelengths 308-312) to determine the wavelengths that maximize the SIBR quantity of the target fluorophore based on the equations above and / or to minimize the SIBR quantity of non-target fluorophores. For instance, initially, the control system 102 may select three wavelengths 308-312, and use theexcitation spectra 302-306 to determine values (e.g., normalized excitation intensity values) for the wavelengths. For example, referring to Eq. A, based on the selected wavelengths, thecontrol system 102 may determine values of the excitation spectra at the wavelengths, such as e1(λ0) that represents the normalized excitation of the excitation spectra 304 at λ0308. e2(λ0) that represents the normalized excitation of the excitation spectra 306 at λ0308, and so on. By using Eq. A, the control system 102 may utilize a set of simultaneous equations for i1and i2, which represent ratios of the intensities between the plurality of wavelengths. For instance, i1represents the ratio of I1(excitation intensity at λ1310) and I0(excitation intensity at λ0308), and i2represents the ratio of I2(excitation intensity at λ2312) and I0(excitation intensity at λ0308). The control system 102 may then solve the simultaneous equations to determine i1and i2for the selected three wavelengths.

[0061] After determining the ratio of intensities i1and i2, the control system 102 may use Eq. B to determine the SIBR quantity of the target fluorophore (e.g., F0- F1- F2). For example, F0represents the total signal detected from the sample when excited with wavelength λ0308, F1represents the total signal detected from the sample when excited with wavelength 310, and F2represents the total signal detected from the sample when excited with wavelength λ2312. C0represents the concentration of the target fluorophore. In some variations, the control system 102 might not explicitly calculate the target fluorophore concentration C0, and instead calculate a quantity (e.g., F0- F1- F2from Eq. B) that is directly proportional to C0.

[0062] In other words, in an image, it may be enough in process 200 for the control system 102 to know (e.g., obtain) that the value (e.g., the intensity) of any and every pixel is directly proportional to the concentration of the fluorophore at that pixel location even though the constant of proportionality is not known. The constant of proportionality between the SIBR quantity ( F0- F1- F2) and the concentration of the target fluorophore (C0) is the same for every pixel in an image, hence if the concentration of the fluorophore at pixel 1 is twice that at pixel 2, then the calculated SIBR quantity at pixel 1 will be twice that at pixel 2. The relative values of the SIBR quantity vary proportionally to the concentration of the target fluorophore from pixel to pixel - this is true regardless of the specific value of the constant of proportionality. The absolute values of the SIBR quantity at each pixel do however depend on the specific value of the constant of proportionality - which depends on the selected wavelengths (λ0- λ1- λ2), the excitation spectrum e0(λ), and the calculated excitation intensities (i1, i2) (Eq. B). The control system 102 varies the wavelengths (λ0- λ1- λ2) in simulation, calculates the excitation intensities (i1, i2) necessary to zero out all but the target fluorophore concentration (C0) contribution to the SIBR quantity for every choice of wavelengths (λ0- λ1- λ2), and calculatesthe constant of proportionality in Eq. B for every choice of wavelengths (λ0, λ1, λ2). The control system 102 then finds (e.g., determines) those values of wavelengths (λ0, λ1, λ2) that maximize the constant of proportionality, and therefore maximizes the absolute values of the SIBR quantity at each pixel. For any choice of wavelengths (λ0, λ1, λ2), by using the values of excitation intensities (i1, i2) calculated using Eq. A, it may be guaranteed that the SIBR quantity will be pixel-by-pixel proportional to the concentration of the target fluorophore (C0). By optimizing the choice of the wavelengths (λ0, λ1, λ2), the control system 102 ensures that this constant of proportionality is maximal, and therefore the absolute values of the SIBR quantity are maximized. In some instances, the maximum value of the constant of proportionality maybe limited not by its theoretical value, but by the configuration of the device 108, e.g., certain excitation wavelengths might not be adequately filtered out by the emission filter prior to reaching the detector 114. These excitation wavelengths might not be used and therefore may constrain the possible values of (λ0, λ1, λ2) that may be used to maximize the value of the constant of proportionality.

[0063] In some instances, as described above, using process 200 (e.g.. the SIBR algorithm), the control system 102 maximizes the SIBR quantity (e.g., F0- F1- F2) of the target fluorophore while try ing to minimize the SIBR quantity for non-target fluorophores. The SIBR quantity is proportional to the concentration of the target fluorophore, which is shown in Eq. B, and the control system 102 maximizes the constant of proportionality to increase the SIBR signal.

[0064] The control system 102 may select new wavelengths and repeat the process to determine a new SIBR quantity for the target fluorophore. For instance, the control system 102 may continuously select new wavelengths, and determine the ratio of excitation intensities, the new SIBR quantity of the target fluorophore, and / or the concentration of the target fluorophore. Afterwards, the control system 102 may determine the maximum SIBR quantity for the target fluorophore from the set of selected wavelengths (e.g., the highest value of the SIBR quantity). The control system 102 may determine the plurality of chosen wavelengths as the wavelengths associated with the maximum SIBR quantity, and may determine the plurality of chosen excitation light intensities as the light intensity ratios associated with the maximum SIBR quantity. For instance, if the wavelengths 510 nm, 530 nm, and 550 nm shown in FIG. 3 produce the highest value SIBR quantity from Eq. B, then the control system 102 determines the chosen wavelengths as these wavelengths and the chosen light intensities as the light intensity ratios associated with these wavelengths. In other words, the control system102 compares SIBR quantity of the target fluorophores (e.g., compares the first SIBR quantity of the target fluorophore with one or more additional SIBR quantities of the target fluorophore), and determines the chosen wavelengths and the chosen excitation light intensities based on a maximum value of the SIBR quantities. The control system 102 selects the chosen wavelengths and light intensities based on the maximum value.

[0065] The control system 102 may use Eq. A and Eq. B for samples 116 with three fluorophores. In some variations, the sample 116 may include more than three fluorophores. In such variations, the control system may use Eq. C and Eq. D for samples 116. Eq. C and Eq. D are substantially similar to Eq. A and Eq. B, except they are for an “N” number (e.g., where “N” is greater than three) of fluorophores such as four, eight, and / or more fluorophores for the sample. For instance, similar to Eq. A, the control system 102 may receive user input indicating a target fluorophore, and select a plurality of wavelengths. The control system 102 may use Eq. C to determine intensity ratios for the selected wavelengths based on solving a set of simultaneous equations. Then, the control system 102 may use Eq. D to determine the SIBR quantity of the target fluorophore (e.g., F0— (F1+ F2+ ... + FN_1)).

[0066] In some instances, the control system 102 may determine that one or more of the excitation intensity ratios is negative. The control system 102 may group the negative intensity ratios together and group the positive intensity ratios together. Therefore, unlike Eq. B, where F1 and F2 are subtracted from F0, for negative intensity ratios, the control system 102 may change the sign by using an addition sign rather than a subtraction sign (e.g.,where Fnrepresents the negative intensity ratios (in< 0) and Fmrepresents thepositive intensity ratios (im> 0)). By switching the sign using an addition sign, the negative intensity ratios are still subtracted from F0(e.g., the total signal from sample upon excitation with wavelength λ0308).

[0067] The control system 102 may continuously select new wavelengths and determine the SIBR quantity for the target fluorophore as well as the excitation intensity ratios. After, similar to the above, the control system 102 may determine the maximum SIBR quantity from the set of selected wavelengths (e.g., the highest value of the SIBR quantity). The control system 102 may determine the plurality of chosen wavelengths as the wavelengths associated with the maximum SIBR quantity for the target fluorophore, and may determine the plurality of chosen excitation light intensities as the light intensity ratios associated with the maximum SIBR quantity for the target fluorophore.

[0068] At block 206, the control system 102 obtains a first detection (e.g. , a positive image) of the sample 116 based on using a set of first wavelengths from the plurality of chosen wavelengths and a set of first light intensities. The set of first wavelengths includes a wavelength associated with a target fluorophore. For example, the wavelength associated with the target fluorophore (e.g., the wavelength λ0308) may be used by the control system 102 to determine F0, which as mentioned above, represents the total detected signal from the sample when excited with wavelength λ0308. As shown below in Eqs. 2 and 11 (e.g., Eq. 11 shows Fm, which is F0when m is equal to 0), F0indicates the total detected signal from the sample including the signal detected for the target fluorophore (e.g., associated with C0) as well as signals detected from the other fluorophores. Thus, because F0indicates the total signal detected from the sample, including from the target fluorophore. then the wavelength used to determine F0is associated with the target fluorophore.

[0069] In other words, in some variations, the wavelength associated with the target fluorophore may be the wavelength λ0308. For instance, for every round of calculating the SIBR image / quantity for a specific target fluorophore, all the selected excitation wavelengths in that round may be said to be “associated” with that target, as the values of those wavelengths are set specifically and solely in order to cancel out the contributions of all other fluorophores to the SIBR image / quantity - leaving only signal from the target, or at least to maximize the target signal and minimize the non-target signal in the SIBR quantity. For every new round with a different target, all the wavelengths are set to whatever values that may leave only that target signal in the SIBR image / quantity. The wavelength λ0308 may be considered more “specific” in its association with the target (e.g., Fluor 0) as it is typically, though not always, set to a value that excites more fluorescence from Fluor 0 than any of the other selected wavelengths. The wavelength λ0308 may excite other fluorophores besides Fluor 0, but it typically excites Fluor 0 to a greater extent than does λ1, λ2, and so on. This is usually, though not strictly always, the case.

[0070] To put it another way, in some examples, the SIBR process may involve imaging many (e.g.. eight) fluorescent probes on a sample. In this example, for every round of imaging there would be one target fluor and seven non-target or background fluors; but in some instances, there be only three or four excitation wavelengths used. All of these 3three or four wavelengths may be accurately described to be associated with all the fluors in that round as the values of these wavelengths are selected taking into consideration the excitation spectra of all the fluorophores - with the goal of canceling out the contribution of all the non-target fluorsto the SIBR quantity, leaving only a contribution from the target fluor. The wavelength λ0308 may have a special or more specific association with the target (Fluor 0) as it typically, though not always, is selected such that it excites the target fluor more than any other selected wavelength. The wavelength λ0308 excites other fluorophores besides Fluor 0, but, it may typically excite Fluor 0 to a greater extent than does λ1, λ2, and so on.

[0071] At block 208. the control system 102 obtains a second detection of the sample 116 based on using a set of second wavelengths and a set of second light intensities.

[0072] For example, referring to FIG. 1, the control system 102 may provide instructions to the device 108 to perform setup of the device 108 based on the set of first and second wavelengths. In some instances, the control system 102 may determine a first set of wavelengths from the plurality of chosen wavelengths as the wavelength for the target fluorophore (e.g., λ0308 such as 530 nm). For example, in the three fluorophore example and using Eq. A and Eq. B, the control system 102 may determine the wavelength λ0308, and the first set of wavelengths may include only this wavelength. The control system 102 may determine the second set of wavelength as the other wavelengths determined using Eq. A and Eq. B such as the wavelengths 310 and λ2312.

[0073] The control system 102 may further determine the set of first and second excitation light intensities. The set of first light intensities correspond to the set of first wavelengths (e.g., the light intensity for λ0308). and may be predefined and / or user defined. For instance, the control system 102 may obtain user input indicating the light intensity for the wavelength λ0. Based on the light intensity of the wavelength λ0, the control system 102 may determine the set of second light intensities. For example, as mentioned previously, the control system 102 may use Eq. A and Eq. B to determine the ratio of intensities (e.g.. i1and i2). Using the ratio of intensities and the light intensity of the wavelength λ0, the control system 102 may determine the set of second light intensities (e.g., determine I1and I2based on i1and i2and I0).

[0074] In some examples, the set of first wavelengths from the plurality of chosen wavelengths may include multiple wavelengths. For instance, the first set of wavelengths may include the wavelength for the target fluorophore (e.g., λ0308 such as 530 nm) and one or more additional wavelengths. For example, as mentioned above with Eq. C and Eq. D, and in the context of an “N” number of fluorophores, certain light intensities determined from Eq. C may be negative light intensities. As such, the control system 102 may determine the first set of wavelengths as wavelengths for the target fluorophore and additional wavelengths that have negative excitation light intensities. The control system 102 may further determine the set ofsecond wavelengths as the remaining wavelengths and / or the wavelengths that have positive light intensities (e.g., excitation light intensities). In addition, the control system 102 may determine the set of first and second light intensities associated with the set of first and second wavelengths (e.g., based on the light intensity ratios determined using Eq. C and the light intensity for the wavelength of the target fluorophore, I0).

[0075] After determining the set of first wavelengths and the set of second wavelengths, the control system 102 may provide instructions to the device 108 to setup the device 108. For instance, in some examples, the device 108 includes a filter 112 such as an AOTF. The AOTF includes a plurality of channels (e.g., three channels). Each of the channels may be configured with a particular wavelength (e.g., from the set of first and second wavelengths) and a particular light intensity. For example, for a first channel, the AOTF may allow light from the excitation light source 110 at a first wavelength (e.g., λ0308 such as 530 nm) and at a particular intensity (e.g., 10 mW). For a second channel, the AOTF may allow light from the excitation light source 110 at a second wavelength (e.g. λ1310 such as 510 nm) and at a particular intensity(e.g., 7 mW), and so on. In other words, the control system 102 may provide instructions to set the channels of the AOTF based on the set of first and second wavelengths. For example, the control system 102 may set one or more channels of the AOTF (e.g., a first channel) based on the set of first wavelengths and the first set of light intensities (e.g., light intensity associated with the set of first wavelengths). The control system 102 may set one or more other channels of the AOTF (e.g.. a second and third channel) based on the set of second wavelengths and the second set of light intensities (e.g., light intensity associated with the set of second wavelengths). As mentioned previously, the first set of wavelengths may include only one wavelength (e.g., λ0308 for the target fluorophore), and the control system 102 may set one channel for this wavelength and associated intensity. In some instances, the first set of wavelengths may include multiple wavelengths, and the control system 102 may set multiple channels for these wavelengths and associated intensities. In some examples, as some of the light intensities are negative, the control system 102 may use an absolute value for the light intensities that are negative when setting up the channels.

[0076] Afterwards, at block 206, the control system 102 obtains a first detection (e.g., an image such as a fluorescent image) based on using the set of first wavelengths and set of first light intensities. For example, after setting the channels of the AOTF, the control system 102 may provide an instruction to the AOTF to enable the channels associated with the set of first wavelengths (e.g., the first channel). Then, the control system 102 may provide an instructionto the excitation light source 110 (e.g. , a white light source) to emit white light. The white light passes through the filter 112 (e.g.. the AOTF with the enabled channel(s)), and then onto the sample 116. For instance, the enabled channels permit light of a certain wavelength and intensity to pass through. In the context of the first channel being enabled (e.g., the three fluorophore case), the light of the λ0308 such as 530 nm passes through at an intensity of I0such as 10 mW. In some variations, the set of first wavelengths may include multiple wavelengths, and the filter 112 may allow light to pass through of multiple wavelengths at multiple intensities.

[0077] After the light passes through the filter 112 and hits the sample 116, the fluorophores of the sample 116 may re-emit light. The detector 114 detects the re-emitted light. For instance, the first detection may be an image (e.g., a fluorescent image). The detector 114 may be an image capturing device such as a camera that obtains the fluorescent image and provides the fluorescent image to the control system 102. For example, the fluorescent image may be an image of the sample 116 such as a pixelated image (e.g., a 2048 x 2048 pixel image), and each pixel may have an associated intensity value that represents the fluorescence intensity emitted by the sample 116 at the location in space that is imaged onto that pixel.

[0078] In some instances, the detector 114 may be a non-imaging detector. After the light passes through the filter 112 and hits the sample 116, the detector 114 may detect the re-mitted light such as a quantity of light (e.g., an intensity of fluorescent light) that is re-emitted from the sample 116 by the fluorophores. In other words, the first detection may be an image such as a fluorescent image and / or a quantity or intensity of fluorescent light that is not in image form.

[0079] At block 208, similar to block 206, the control system 102 obtains a second detection (e.g.. a negative image) of the sample. For instance, the control system 102 may provide an instruction to the AOTF to enable the channels associated with the set of second wavelengths (e.g., the second and third channels). Then, the control system 102 may provide an instruction to the excitation light source 110 to emit light. The light passes through the filter 112 with the enabled channel(s), and then onto the sample 116. The detector 114 obtains the second detection (e.g.. a fluorescent image and / or a quantity or intensity of fluorescent light that is not in image form) and provides the second detection to the control system 102.

[0080] At block 210, the control system 102 outputs a SIBR quantity (e.g., a SIBR quantity of a target fluorophore) based on the first and second detections. For instance, the first and second detections may be a first and a second fluorescent image. The control system 102 may perform a subtraction of the first and second fluorescent image to obtain the SIBR quantity(e.g., a SIBR image). For example, for each pixel, the control system 102 may subtract the brightness or fluorescent intensity value of the first fluorescent image (e.g., based on the set of first wavelengths and light intensities) with the brightness or fluorescent intensity value of the second fluorescent image (e.g., the set of second wavelengths and set of second light intensities). The control system 102 may generate the SIBR image based on the subtractions. As such, for the three fluorophore case (e.g., using Eq. A and B). the control system 102 may subtract the first fluorescent image associated with the target fluorophore from the second fluorescent image associated with the other fluorophores (e.g., fluorophores associated with 310 and λ2312). For the “N” fluorophore case (e.g., using Eq. C and D), the control system 102 may subtract the second fluorescent image associated with the other non-target fluorophores (e.g.. the fluorophores with positive light intensities) from the first fluorescent image associated with the target fluorophore and fluorophores with negative light intensities. The control system 102 may then output the SIBR quantity (e.g., display or cause display of the generated SIBR image on a display device, and / or save the image to memory).

[0081] In some instances, the first and second detections may be a quantity or value. The control system 102 may subtract the second detection from the first detection to determine the SIBR quantity.

[0082] In some variations, the excitation light source 110 is a tunable light source. The control system 102 may provide instructions to the tunable light source to emit light at different wavelengths and different intensities. As such, for blocks 206 and 208. the control system 102 may provide instructions to the tunable light source to emit light at each of the first set of wavelengths and first set of intensities and at each of the second set of wavelengths and second set of intensities. At block 210, the control system 102 may determine and output a SIBR quantity. For instance, the control system 102 may use Eq. D to determine the SIBR quantity.

[0083] In some examples, the device 108 may include an emission filter. The emission filter may be between the sample 116 and the detector 114. For instance, the emission filter may filter light that is emitted from the sample 116 prior to the detector 114 obtaining the light.

[0084] In some variations, the process 200 uses the excitation light intensities for the blocks 202-208. In other variations, the process 200 may use other types of light intensities such as fluorescence light intensities and / or other light intensities.

[0085] Examples of the SIBR system 100 and process 200 will be discussed in more detail below. For instance, using the SIBR system 100 for three fluorophores is described below, and the SIBR system 100 and process 200 may be used to unmix (e.g., computationally render free of crosstalk from the other fluorophores) the fluorescence signal from single fluorophores. Thethree fluorophores (e.g., fluors) with substantially overlapping excitation spectra are shown in FIG. 3. For instance, as mentioned above, fluor zero 302 with excitation spectrum eo is the target, fluor first 304 and fluor second 306 with excitation spectra e1and. e2are ‘background’.

[0086] The ‘target’ fluorophore (Fluor 0) is the fluorophore whose fluorescence is to be selectively computed, the other two (Fluor 1 and Fluor 2) are considered ‘background’ fluorophores whose fluorescence is to be selectively reduced or ideally eliminated entirely. The excitation spectra of these fluors overlap enough that it is not possible to excite the target fluor without also exciting the background fluors. The concentrations of Fluor 0, 1, 2 are C0, C1, C2respectively. The normalized excitation spectra are e 0(λ), e 1(λ), e 2(λ), which is defined as the product of the extinction coefficient (ε(λ)) and tire quantum yield (0), normalized to a peak value of 1 .

[0087] This is shown by the following equation:

[0088] If the sample is excited at wavelength λ0near the excitation peak of Fluor 0, with intensity I0(FIG. 3), the total emitted fluorescence from all 3 fluors is:

[0089] Similarly, if the sample is excited at wavelengths λ1and λ2to the blue (i.e., lower wavelength) and red (i.e., higher wavelength) sides of the Fluor 0 peak, with intensities I1and I2respectively, the total emitted fluorescence due to each excitation is:

[0090] The SIBR quantity (e.g., the SIBR image) can now be calculated based on F0- F1- F2. For example, from Eq. 2-4:

[0091] In Eq. 5, the terms are grouped by the concentrations Cn. In this quantity (e.g., F0- F1- F2), it is wanted for the contributions from the two background fluors, Fluor 1 and. Fluor 2, to vanish leaving only a non-zero contribution from the target Fluor 0. In other words, it is wanted for the C1and C2terms to cancel out leaving only the C0term. For the C1and. C2terms to cancel out, the below occurs:

[0092] In the above and as mentioned earlier, i1= I1 / I0and i2= I2 / I0. Eq. 6 can be solved for 11 and 12 below:

[0093] As long as det(e) ≠ 0 (e.g., the determinant of e is not equal to 0), for these values of i1and. 12, the C1and C2terms in (F0- F1- F2) → 0, leaving only the C0term, which is shown below:

[0094] This demonstrates that for a set of three spectrally overlapping fluorophores, the system 100 may choose a set of excitation wavelengths and calculate the necessary excitation intensities such that the calculated quantity: (F0- F1- F2) contains only fluorescence signal from the target Fluor 0 with no contribution from the two background fluors. Thus, the quantity (e.g., F0-- F1-- F2) is linear in concentration C0and therefore faithfully maps the spatial distribution of the target Fluor 0. As such, the control system 102 may use Eq. 9 and 10, which are shown above as Eq. A and B, for process 200.

[0095] In some instances, to implement the calculation of the SIBR image, the system 100 may utilize a white light source such as a super continuum white light laser that provides a broad spectrum of excitation light, and an acousto-optic tunable filter (AOTF) to select a specified wavelength with a specified power from the broad spectrum light source. The AOTF may have a minimum number of channels (e.g., four channels).

[0096] As such, the system 100 (e.g., the control system 102) may use and / or perform the procedure below:

[0097] First, the control system 102 may record, the excitation spectra of the individual fluorophores used on multiply stained sample. As with all unnnxing techniques - the excitation or emission spectra may be known before unmixing. In SIBR, the excitation spectrum might need, to be precisely known.

[0098] Second, the control system 102 may select λ0, λ1, λ2in simulation in order to maximize the pre-factor of C0(e.g., the constant of proportionality) in Eq. 10: [ ε0(λ0) - ε0(λ1)i1- ε0(λ2)i2]. Since i1and i2are calculated using Eq. 8, the C1and C2terms m (F0- F1- F2) are guaranteed to be 0. For instance, as described above, because the SIBR quantity (F0- F1- F2)and the C0are related (e.g., (F0- F1- F2) = pre-factor x C0), then by maximizing the pre-factor, the SIBR quantity is also maximized. As such, by determining F0, F1, and F2, the control system102 may calculate the SIBR quantity. During the simulation phase (e.g., block 204 above), before the data, acquisition begins, the control system 102 may determine (e.g., calculate) the pre-factor based on the excitation spectra. The wavelengths may be selected and used to calculate intensities so as to maximize the pre-factor. By doing this, when F0, F1, and F2at the chosen wavelengths and calculated intensities are recorded, the control system 102 knows that the SIBR quantity that is calculated (e.g., F0- F1- F2) is at its maximum value.

[0099] Third, the control system 102 may set the AOTF Channels to: Channel 1: wavelength = λ0, power = I0; AOTF Channel 2: wavelength = λ1, power = i1-I0; and AOTF Channel 3: wavelength = λ2, power = i2.I0. I0may be chosen by the user as whatever intensity produces a convenient level of fluorescence (e.g., the control sy stem 102 may obtain user input indicating I0).

[0100] Four, the control system 102 may enable AOTF Channel 1, and excite the sample and acquire an image of the fluorescence. This image is F0but as a function of space; F0(x,y).

[0101] Five, the control system 102 may enable AOTF Channel 2 and Channel 3 simultaneously, and excite the sample and. acquire an image of the fluorescence. This image is F1+ F2- also as a function of space F1(x,y ) + F2.(x,y).

[0100] Six, the control system 102 may calculate the SIBR image F0- (F1+ F2).

[0101] The SIBR image is the unmixed image of Fluor 0. In some instances, further processing might not be used. In some examples, only two acquisitions and one subtraction may be used by the control system 102 to obtain a. de-multipl exed image of the target fluor. This process may be repeated for the other fluorophores - setting each in turn as the Target’ and calculating the unmixed SIBR image for those.

[0102] The “N” fluorophore case is described below. For instance, the three fluorophore treatment may be extended in a very straightforward way to the case of “N” fiuorophores. Following the three fluor case, as before, the control system 102 determines a single ‘target’ fluorophore: Fluor 0, and N-1 background fluorophores: Fluor 1 , 2, 3... (N-l). These have concentrations C0, C1, C2. . . CN-1, and normalized excitation spectra e0(λ), e1(λ), . . . eN-1(λ)(FIG. 4). For instance, FIG. 4 shows a graphical representation 400 of “N” fluorophores with overlapping excitation spectra according to one or more examples of the present application. As shown, “N” is eight in FIG. 4 (e.g., eight representative overlapping excitation spectra are shown). Fluor 0 402 is the target fluor, and all others are background fluors. The dotted box 404 indicates the typical range of excitation wavelengths used to obtain a SIBR image of thetarget. The dotted box 406 indicates the band-pass of a typical emission filter used while imaging the target.

[0103] If the sample is excited at wavelength with intensity Im, the total emitted fluorescence from all “N” fluors may be:

[0104] The SIBR image is defined as:

[0105] For N-1 background fluors, “N” excitation wavelengths may be used for F0-- (F1+ F2+.. . FN-1) to have only the C0term, e.g., for other Cn>o terms to vanish. The intensity of these excitation wavelengths may be calculated as before:

[0106] In some examples, when solving this system of equations, not all in(e.g., light intensities) may be calculated as positive. The interpretation of a negative in is that the image Fnresulting from excitation at wavelength λnwith intensity |in| (e.g., the absolute value of in) may be on the other side of the subtraction in the SIBR image calculation FO --- (F1 + F2 +. . . FN- 1). Taking the possibility of negative values of intensity into account, the SIBR image expression may be rewritten as:

[0107] As such, the system 100 (e.g., the control system 102) may use and / or perform the procedure below:

[0108] First, the control system 102 may record excitation spectra of all “N” fluorophores.

[0109] Second, the control system 102 may select λ0, λ1... λN-1in simulation in order to maximize the C0pre-factor in Eqn 12: [ e0(λ0) - e0(λ1)i1. . . - e0(λN-1)iN-1]. Calculating in using Eq. 14 ensures the C1. . . CN-1terms in F0- (F1+ F2+. . . FN-1) → 0.

[0110] Third, the control system 102 may set the AOTF channels to be: AOTF Channel 1: wavelength = λ0, power = I0; AOTF Channel 2: wavelength = λ1, power = |i1|.I0;. . . AOTF Channel N: wavelength = λN-1, power = |iN-1|.I0. In some instances, AOTFs may not have more than eight channels - the below indicates that even for N>8 fluorophores, the system 100 might not typically use more than three or four channels.

[0118] Fourth, the control system 102 may enable AOTF Channel 1 and all other channels with in<0, and excite the sample and acquire an image of the fluorescence. This image, called the ‘positive image,’ is:[01.19] Five, the control system 102 may enable all AOTF channels with in<0, and excite the sample and acquire an image of the fluorescence. This image, called the ‘negative image’, is:

[0120] Six, the control system 102 may subtract these two images to obtain the SIBR image:

[0121] In some instances, even though the spectral and computational complexity of the “N” fluor case is significantly greater than the three fluor case, the system 100 may only use two image acquisitions and one subtraction to obtain an unmixed image of the target. The scaling of the acquisition / computation with the number of fluorophores makes SIBR a particularly attractive approach for unmixing as the number of fluorophores increases.

[0122] An implementation of SIBR is described below. For instance, the realistic value of “N” is described. For example, the “N” fluorophore treatment above outlines how to unmix the image of a single fluorophore from a set of “N” fluorophores - e.g., how to zero out the contributions of N-l background fluorophores. In operation, in a ty pical set of fluorescent probes used for multiplex labeling of a. sample (FIG. 4), the choices of excitation wavelengths used to unmix Fluor 0 in the SIBR protocol (box 404; FIG. 4) might not excite all N-1 background fluorophores. In some examples, it may be determined that only the two fluors (e.g., e3 and e2 in FIG. 2) immediately to the blue (i.e., shorter wavelength) of the ‘target’ (eo in FIG. 4) may be excited, in addition to most of the fluors to the red (i.e., longer wavelength) of the target. Furthermore, a typical emission filter used to image Fluor 0 (box 406; FIG. 4) passes fluorescence from the three fluorophores immediately to the red. of the target and no redder, in addition to all tire bluer fluors. Hence, for typical choices of excitation wavelength and emission filter, any given target fluor may experiences potential crosstalk from five otherbackground fiuorophores. As such, the control system 102 may therefore use N=6 in the treatment for “N” fiuorophores.

[0126] The required number of excitation wavelengths is described below. In the three fluor case (one target, two background), the control system 102 may select λ0, λ1, λ2 in order to maxi mize the magnitude of the CO term. The constraint:

[0127] ensures that for any for any choice of λ0, λ1, λ2, the excitation intensities i1and i2. may be determined such that the C1and C2terms in F0- (F1+ F2) vanish. The particular choices of λ0, λ1, λ2are therefore degrees of freedom. If there are more background fluors beyond F1and F2, λ0, λ1, λ2may be varied to minimize the contributions to F0--- (F1+ F2) from other background fluors, being assured that i1and i2may be set to fully eliminate the contributions of F1and F2.

[0128] As discussed above, even for a large total number of fluorophores, any given target fluor may experience crosstalk from five background fluors. Using three excitation wavelengths, it may be guaranteed to be able to eliminate the contribution from two of those background fluors for any choice of λ0, λ1, λ2. By judiciously choosing λ0, λ1, λ2however, the contribution from the remaining three background fluors may be reduced by greater than 95% (often greater than 99%) while sacrificing an acceptable amount of the target signal.

[0129] Hence, in many cases, three excitation wavelengths are adequate to perform SIBR unmixing of a target from a large total set of fluorophores. In other words, regardless of the number of fluorophores in the sample 116, in some variations, the control system 102 may use three excitation wavelengths and their corresponding intensities to perform process 200.

[0130] The simulation software for SIBR unmixing is described below. The process 200 may be performed by an EXCEL based program to simulate SIBR unmixing for N=3 and N=4 models. This allows the user to load excitation spectra and vary the excitation wavelengths, the program may automatically calculate the necessary excitation intensities to eliminate all but the target fluorescence, and calculate the magnitude of the residual target fluorescence, as well as the magnitude of residual signal from all the background fluors. Additionally, and / or alternatively, the process 200 may be performed using another version of this program such as in PYTHON / MATLAB.

[0131] The experimental validation is described below. For instance, sample preparation is described. Eight commercially available fluorescent dyes were selected after simulating SIBR unmixing on their known (publicly available) excitation spectra to ensure each could beunmixed from the set. The dyes were Atto 465, CF488, CF514, Atto Rhodamine 6G, DyLight 554, Atto Rhodamine 12, Atto 590, and DyLight 633. This is shown in FIGs. 5A and 5B. Forinstance, FIGs. 5A and 5B show graphical representations 500 and 550 of absorption, excitation, and emission spectra for the eight fluorophores selected for validation of SIBR according to one or more examples of the present application. These are eight conventional fluorophores with emission peaks ranging from 506 to 650 nm. This corresponds to a 20.6 nm inter-peak interval - comparable to or smaller (better) than other state-of-the-art attempts at multiplex imaging.

[0133] Each fluor shown in FIGs. 5A and 5B was obtained in its N-Hydroxysuccinimide (NHS)-ester form, dissolved (0.1% dimethylsulfoxide (DMSO) in phosphate-buffered saline (PBS)) and applied directly to Paraformaldehyde (PF A) fixed monolayers of human bone osteosarcoma epithelial (U2OS) cells in an eight well slide. This is shown in FIG. 6. For instance, FIG. 6 shows two representations 600 of fixed U2OS cells that were stained in an eight well slide. The top portion of FIG. 6 shows the eight well slide 602, and the bottom portion 604 of FIG. 6 shows a representation 604 of the eight well side with the U2OS cells. Each well was stained with a single fluorophore. The wells walls were then removed to give a single slide with different regions (bottom) of cells labeled with different fluorophores. The NHS ester form of a dye binds to any free amine in the cell - leading to a whole cell labeling pattern with particular enrichment in the nucleus. Each well of the 8 well slide was stained with a different dye. After, staining the well walls were removed leaving a slide with distinct regions of cells stained with a single dye. This sample was used to test the validity of SIBR. Applying the SIBR method (e.g.. process 200) to image a particular target fluor, e.g., CF514 yielded clear images of cells in the well region where cells were stained with CF514, and no signal from the cells in the other well regions.

[0134] The preparation and calibration is described below. For instance, imaging was earned out on a light sheet microscope (e.g., the CT-DISPIM microscope from APPLIED SCIENTIFIC INSTRUMENTATION). Other microscopes (as mentioned above) may also be used for imaging. A white light laser (e.g., Super K Compact laser from NKT PHOTONICS) was used as the broad spectrum light source, and an 8 channel AOTF (AA OPTOELECTRONICS) was used to pass user selected wavelengths at user specified intensities into the microscope to excite the sample.

[0135] While the fluorophores used here have known, published spectra, excitation spectra may change somewhat depending on factors such as protein binding - therefore, as described above, the excitation spectra may be measured in the same environment / context in which SIBR imaging may be performed. The excitation spectrum of each fluor was measured by acquiring a series of images of a field of cells stained only with that fluor using a range of excitationwavelengths set by the AOTF. The integrated fluorescence of the field as a function of excitation wavelength is then calculated as the excitation spectrum. In some instances, for SIBR, it might not be necessary to correct for wavelength dependent transmission of the optical path or wavelength dependent variation in power output by the source - as long as these do not change between acquiring the excitation spectrum and performing the SIBR imaging procedure.

[0136] Once all the excitation spectra are recorded from cells, for each fluor, the control system 102 may determine the λ0, λ1, λ2, i1, i2that may maximize the SIBR image of the target while minimizing the SIBR image intensity from the five background fluors (two immediately bluer fluors, three immediately redder fluors). By way of example, FIG. 7 shows this calculation for Atto Rhodamine 6G as the target. For instance, FIG. 7 shows a graphical representation 700 of the recorded excitation (exc) spectra of the target - in this case, Atto Rho6G and five background fluors are plotted as a function of AOTF frequency (which determines the wavelength of light passed by the AOTF) in megahertz (MHz). For instance, excitation wavelengths (λ0, λ1, λ2) are selected by the user to minimize the background fluor contribution to the SIBR image. The excitation intensities 11 and 12 are calculated.

[0137] While spectra are usually plotted as a function of wavelength, it may be more convenient during actual calculations to consider spectra as a function of the AOTF frequency. The SIBR calculations are agnostic to the choice of wavelength or AOTF frequency as the measure of the color of light - however, it can be kept in mind that wavelength and AOTF frequency are inversely related, so a higher AOTF frequency corresponds to a lower / bluer wavelength of light passed by the AOTF.

[0138] The SIBR imaging is described below-. For instance, three excitation lines per target fluor are used. Each fluor on the slide was selected as the ‘target’ in turn. For the selected target, channel 1 of the AOTF w as set to the wavelength λ0with an intensity I0determined in simulation for that target fluor. The choice of I0is one of convenience that provides adequate signal and does not affect the SIBR calculation. Channel 2 of the AOTF was set to λ1with an intensity I1= i1.I0and Channel 3 was set to λ2 with an intensity I2= i2.I0; i1and i2are calculated in simulation using Eq. 9.

[0139] AOTF Channel 1 may be enabled, and a stage scanned image may be acquired on the microscope. This gives the image Fo that may be the minuend of the SIBR Image calculation - also called the ‘positive image’. Then AOTF Channels 2 and 3 may be enabled simultaneously, and a stage scanned image may be acquired of the same field of cells. This gives the image (F1+ F2) that may be the subtrahend of the SIBR Image calculation - alsocalled the ‘negative image’. The difference, F0- (F1+ F2), may be calculated giving the SIBR image of the target fluor.

[0140] Continuing with the example outline above in FIG. 7 with Atto Rho6G set as the target, λ0, λ1, λ2, i1, i2are chosen / calculated and programmed into the AOTF. The positive and negative images are acquired and the SIBR image is calculated as the difference of the two images (e.g., a first detection / positive image and a second detection / negative image), which is shown in FIG. 8. FIG. 8 shows a set of images 800. For instance. FIG. 8 shows that the target is Atto Rhodamine 6G, and positive and negative images 802, 804 (e.g., the first detection and second detection described in blocks 206 and 208 above) are acquired and the SIBR image 806 is calculated based on process 200.

[0141] Using the (λ0, λ1, λ2, i1, i2) set of values specific for an Atto Rho6G target, the SIBR image 806 of cells stained with Atto Rho6G (e.g., the left side in FIG. 8) is just a reduced intensity version of the positive image of the cells. The positive image 802 may be considered the ground tmth of the target fluorescence as it is excited under “ordinary” conditions, e.g., a single wavelength near its excitation peak. The SIBR image 806 therefore faithfully reproduces the spatial distribution of the target fluorophore. The same (λ0, λ1, λ2, i1, i2) set of values for an Atto Rho6G target, when used to image a background fluorophore such as Dy Light 554 (e.g., the right side in FIG. 8) gives a SIBR image that is a near homogenous field of zero values, which is shown by the positive image 808. negative image 810, and SIBR image 812 for a sample stained with Dy Light 554. This demonstrates how SIBR imaging can eliminate the fluorescence from background fluorophores, while only somewhat attenuating the fluorescence from the target fluorophore.

[0142] Each fluorophore may be set in turn to be the target - each time setting the AOTF to the (λ0, λ1, λ2, i1, i2) values specifically calculated for that target (e.g., “standard” excitation (exc) and / or emission (em) for the target). The SIBR image for each target is attenuated in comparison to the positive image, while SIBR images of the background fluors are largely completely extinguished. These results are presented below as maximum z projections of the image stacks and are shown in FIGs. 9-16. For instance, FIGs. 9-16 show images 900-1600. For each target, only the background fluors that exhibit appreciable detected fluorescence under the excitation / emission conditions for that target are presented. The top row in each figure shows the positive images ( F0) - e.g., images taken under what would be considered regular fluorescence imaging conditions for the target. The bottom row show s the SIBR images. The contrast of the images in the top rows have been enhanced for visual clarity, however thevertical pairs of images are displayed on the same intensity scale to accurately show the loss in signal in the SIBR image.

[0143] FIGs. 9-16 show the SIBR image for additional targets using the exemplary process of FIG. 2. For instance, FIG. 9 shows the target 902 as Atto 465. The fluors that appreciably crosstalk with the target are displayed here - all other background fluors gave negligible signal under these excitation / emission filter conditions. For instance, the top row of images 904 are for the target 902 (i.e., Atto 465). CF 488, CF 514. and Atto Rho6G. The bottom row of images 906 are the SIBR images. As shown, the SIBR images 906 for the target 902 are visible, and the rest are at a near homogenous field of zero values. Further, contrast in the top row 904 is enhanced for visual clarity, vertical pairs are displayed on the same intensity scale to accurately represent the signal loss. Atto 465 signal is attenuated by 32% while the signal from the other background fluors is extinguished almost entirely (>98% reduction).

[0144] FIG. 10 shows the target 1002 as CF 488. For instance, the top row of images 1004 are positive images for Atto 465, the target 1002 (CF 488), CF 514, Atto Rho6G, and DL554. The bottom row of images 1006 are SIBR images. As shown, the CF488 signal is attenuated by 56% while the signal from the other background fluors is extinguished almost entirely (>99% reduction).

[0145] FIG. 11 shows the target 1102 as CF 514. For instance, the top row of images 1104 show positive images for CF 488, the target 1102 (CF 514), Atto Rho6G, DL 554, Atto Rho12, and Atto 590. The bottom row of images 1106 show SIBR images. As shown, the CF514 signal is attenuated by 34% while the signal from the other background fluors is extinguished almost entirely (>99% reduction).

[0146] FIG. 12 shows the target 1202 as Atto Rho6G. For instance, the top row of images 1204 show positive images for CF 488, CF 514, the target 1202 (Atto Rho6G), DL 554. Atto Rho12, and Atto 590. The bottom row of images 1206 show SIBR images. As shown, the Atto Rho6G signal is attenuated by 52% while the signal from the other background fluors is extinguished almost entirely (>99% reduction).

[0147] FIG. 13 shows the target 1302 as DyLight (DL) 554. For instance, the top row of images 1304 show positive images for CF 514. Atto Rho6G, the target 1302 (DL 554). Atto Rho12, Atto 590, and DL 633. The bottom row ofimages 1306 show SIBR images. As shown, the DyLight 554 signal is attenuated by 39% while the signal from the other background fluors is extinguished almost entirely (>99% reduction).

[0148] FIG. 14 shows the target 1402 as Atto Rho12. For instance, the top row of images 1404 show positive images for Atto Rho6G, DL 554, the target 1402 (Atto Rho12), Atto 590,and DL 633. The bottom row of images 1406 show SIBR images. As shown, the Atto Rho12 signal is attenuated by 60% while the signal from the other background fluors is extinguished almost entirely (>99% reduction).

[0149] FIG. 15 shows the target 1502 as Atto 590. For instance, the top row of images 1504 show positive images for DL 554, Atto Rho12, the target 1502 (Atto 590), and DL 633. The bottom row of images 1506 show SIBR images. As shown, the Atto 590 signal is attenuated by 39% while the signal from the other background fluors is extinguished almost entirely (>99% reduction).

[0150] FIG. 16 shows the target 1602 as DyLight 633. For instance, the top row of images 1604 show positive images for DL 554, Atto Rho12, Atto 590, and the target 1602 (DL 633). The bottom row of images 1606 show SIBR images. The DyLight 554 SIBR image shows imperfect extinction because its low signal in the DyLight 633 emission channel makes it difficult to accurately measure the DyLight 554 spectrum. The intensity' of the DL 554 SIBR image is approximately 30 times lower than the intensity of the DL 633 SIBR image. All other background fluors are extinguished almost entirely (>99% reduction).

[0151] The above is demonstrated using only three excitation lines per target. In a set of eight fluorophores with highly overlapping excitation spectra, the control system 102 may unmix all eight using SIBR.

[0152] The advantages of SIBR over existing unmixing approaches are described below. For instance, the scaling of computation is described. The computations used to unmix “N” fluorescent labels using existing approaches scale as N2. This is because each fluorophore potentially receives crosstalk from N-1 fluorophores. If Cmis the concentration of Fluor m and Fmis the fluorescence image with crosstalk acquired with excitation / emission optimized for Fluor m, then the unmixed, images (Cm) are calculated by a system of equations of the form:

[0153] The number of computations required here scales as N2. Hence, while conventional approaches to unmixing require only “N” acquisitions, the computations required scale as N2In comparison, SIBR may use two acquisitions per target fluor and one subtraction, meaning for “N” fluorophores, SIBR may use “2N”acquisitions and “N”computations. Furthermore,that computation is a simple subtraction, which may be performed extremely quickly. This presents a. significant computational advantage for large sets of fluorophores.

[0157] The larger acceptable emission filter bandwidth is described. In the SIBR calculation, since each non-target fluor cancels itself out - larger bandwidth emission filters may be used that allow more crosstalk. By way of comparison - the multiplex acquisition performed in PICASSO used 10-15 nm bandwidth filters for regular dyes. SIBR may use 30- 40 nm bandwidth filters that are conventional in fluorescence imaging. This ameliorates some of the loss of signal due to the subtraction used to calculate the SIBR image.

[0158] The channel co-registration for unmixing is automatic. Conventional multiplex imaging requires perfect spatial co-registration of multiple colors to accurately unmix. This may be difficult in some samples, e.g., large cleared tissue samples where only one color is acquired in a long stage scan, follow ed by a reset of the stage and the next scan with the next color. Multiple colors cannot typically be acquired within the same scan as the emission filters cannot be changed fast enough. Sub-micron level co-registration after centimeter scale motion typically may use software correction in post-processing. This becomes prohibitively difficult for the terabyte scale image volumes common for large samples. SIBR may acquire positive and negative images in immediate succession since, for a given target, the emission filter does not change, only the excitation wavelengths change - which may be done very quickly (millisecond (ms) time scale) with an AOTF. The two images used for unmixing the target are already co-registered and require no further computationally expensive co-registration.

[0159] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0160] The use of the terms “a” and “an:and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "‘such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0161] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMS:

1. A system for rapid spectral unmixing using spectrally interpolated background reduction (SIBR), comprising: a device comprising: an excitation light source; and a detector; and a control system configured to: obtain a plurality of excitation spectra for a plurality of fluorophores that are used to stain a sample; determine a plurality of chosen wavelengths and a plurality of chosen light intensities for the plurality of fluorophores based on the plurality of excitation spectra; obtain, using the detector of the device, a first detection of the sample based on using a set of first wavelengths from the plurality of chosen wavelengths and a set of first light intensities, wherein the set of first wavelengths comprises a wavelength associated with a target fluorophore of the plurality of fluorophores; obtain, using the detector of the device, a second detection of the sample based on using a set of second wavelengths from the plurality of chosen wavelengths and a set of second light intensities; and output a SIBR quantity of the target fluorophore based on the first detection and the second detection.

2. The system of claim 1, wherein the device further comprises a filter, and wherein the control system is further configured to: provide setup instructions to the filter based on the set of first wavelengths and the set of second wavelengths.

3. The system of claim 2, wherein the filter is an acousto-optic tunable filter (AOTF) comprising a plurality of channels, wherein each of the channels permits light at a certain wavelength and a certain intensity to be emitted from the excitation light source onto the sample, and wherein providing the setup instructions comprises: setting up one or more first channels of the plurality of channels based on the set of first wavelengths and the set of first light intensities; andsetting up one or more second channels of the plurality of channels based on the set of second wavelengths and the set of second light intensities.

4. The system of claim 3, wherein obtaining the first detection of the sample comprises: enabling the one or more first channels of the AOTF ; providing one or more instructions to the excitation light source to emit light, wherein the light passes through the AOTF such that only the set of first wavelengths and the set of first light intensities are emitted onto the sample; and obtaining, from the detector, the first detection of the sample based on only the set of first wavelengths and the set of first light intensities being emitted onto the sample.

5. The system of claim 3, wherein obtaining the second detection of the sample comprises: enabling the one or more second channels of the AOTF; providing one or more instructions to the excitation light source to emit light, wherein the light passes through the AOTF such that only the set of second wavelengths and the set of second light intensities are emitted onto the sample; and obtaining, from the detector, the second detection of the sample based on only the set of second wavelengths and the set of second light intensities being emitted onto the sample.

6. The system of claim 1, wherein the excitation light source is a tunable light source, and wherein obtaining the first detection of the sample comprises: providing one or more instructions to the tunable light source to emit light onto the sample at the set of first wavelengths and the set of first light intensities; and based on providing the one or more instructions, obtaining, from the detector, the first detection of the sample.

7. The system of any of claims 1-6, wherein the detector is an image capturing device, wherein the first detection of the sample is a first fluorescent image of the sample based on the set of first wavelengths, and wherein the second detection of the sample is a second fluorescent image of the sample based on the set of second wavelengths.

8. The system of any of claims 1-7, wherein obtaining the plurality of excitation spectra for the plurality of fluorophores comprises: receiving user input indicating the plurality of excitation spectra.

9. The system of any of claims 1-7, wherein obtaining the plurality of excitation spectra for the plurality of fluorophores comprises: obtaining, using the detector of the device, a plurality of first initial detections of a second sample at a plurality of different wavelengths, wherein the second sample is stained with only a first fluorophore of the plurality of fluorophores; generating a first excitation spectra for the first fluorophore based on the plurality of first initial detections; obtaining, using the detector of the device, a plurality of second initial detections of a third sample at the plurality of different wavelengths, wherein the third sample is stained with only a second fluorophore of the plurality of fluorophores; and generating a second excitation spectra for the second fluorophore based on the plurality of second initial detections.

10. The system of any of claims 1-9, wherein determining the plurality of chosen wavelengths and the plurality of chosen light intensities for the plurality of fluorophores comprises: receiving first user input indicating the plurality of chosen wavelengths; and receiving second user input indicating the plurality of chosen light intensities.

11. The system of any of claims 1-9, wherein determining the plurality of chosen wavelengths and the plurality of chosen light intensities for the plurality of fluorophores comprises: receiving user input indicating the target fluorophore of the plurality of fluorophores; selecting a plurality of first selected wavelengths for the plurality of fluorophores: determining a plurality of first ratios of light intensities based on the plurality of first selected wavelengths and the plurality of excitation spectra; determining a first SIBR quantity of the target fluorophore based on the plurality of first selected wavelengths, the plurality of excitation spectra, and the plurality of first ratios of light intensities; based on comparing the first SIBR quantity of the target fluorophore with one or more additional SIBR quantity of the target fluorophore, determining the plurality of chosen wavelengths and the plurality of chosen light intensities, wherein the one or more additionalSIBR quantity are determined using a plurality of additionally selected wavelengths that are different from the plurality of first selected wavelengths.

12. The system of claim 11, wherein determining the plurality of chosen wavelengths comprises: determining a maximum value from the first SIBR quantity and the one or more additional SIBR quantity; and selecting the plurality- of chosen wavelengths and the plurality of chosen light intensities based on the maximum value.

13. The system of any of claims 1-12, wherein the plurality of fluorophores comprise three fluorophores, wherein the plurality of chosen wavelengths comprise three chosen wavelengths, wherein the set of first wavelengths comprise the wavelength associated with the target fluorophore from the three fluorophores, and wherein the set of second wavelengths comprise the other two chosen wavelengths associated with the other two fluorophores.

14. The system of any of claims 1-12, wherein the plurality of chosen light intensities comprise a plurality- of positive light intensities and one or more negative light intensities, wherein the one or more negative light intensities indicate a negative value for the light intensity, wherein the one or more negative light intensities is associated with one or more wavelengths from the plurality of chosen wavelengths, wherein the set of first wavelengths comprises the wavelength associated with the target fluorophore and the one or more wavelengths associated with the one or more negative light intensities, and wherein the set of second wavelengths comprise wavelengths from the plurality of chosen wavelengths associated with the plurality of positive light intensities.

15. The system of any of claims 1-14, wherein the wavelength associated with the target fluorophore of the plurality of fluorophores is used to obtain a total detected signal from the sample, wherein the total detected signal comprises a signal for the target fluorophore and one or more signals detected for other fluorophores from the plurality of fluorophores.

16. A method, comprising: obtaining, by a control system, a plurality of excitation spectra for a plurality of fluorophores that are used to stain a sample;determining, by the control system, a plurality of chosen wavelengths and a plurality of chosen light intensities for the plurality of fluorophores based on the plurality of excitation spectra; obtaining, by the control system and using a detector of a device, a first detection of the sample based on using a set of first wavelengths from the plurality of chosen wavelengths and a set of first light intensities, wherein the set of first wavelengths comprises a wavelength associated with a target fluorophore of the plurality of fluorophores; obtaining, by the control system and using the detector of the device, a second detection of the sample based on using a set of second wavelengths from the plurality of chosen wavelengths and a set of second light intensities; and outputting, by the control system, a SIBR quantity of the target fluorophore based on the first detection and the second detection.

17. The method of claim 16, wherein the device further comprises a filter, and wherein the method further comprises; providing setup instructions to the filter based on the set of first wavelengths and the set of second wavelengths.

18. The method of claim 17, wherein the filter is an acousto-optic tunable filter (AOTF) comprising a plurality of channels, wherein each of the channels permits light at a certain wavelength and a certain intensity to be emitted from an excitation light source onto the sample, and wherein providing the setup instructions comprises: setting up one or more first channels of the plurality of channels based on the set of first wavelengths and the set of first light intensities; and setting up one or more second channels of the plurality of channels based on the set of second wavelengths and the set of second light intensities.

19. The method of claim 18, wherein obtaining the first detection of the sample comprises; enabling the one or more first channels of the AOTF ; providing one or more instructions to the excitation light source to emit light, wherein the light passes through the AOTF such that only the set of first wavelengths and the set of first light intensities is emitted onto the sample; and obtaining, from the detector, the first detection of the sample based on only the set of first wavelengths and the set of first light intensities being emitted onto the sample.

20. A non-transitory computer-readable medium having processor-executable instructions stored thereon, wherein the processor-executable instmctions, when executed, facilitate: obtaining a plurality of excitation spectra for a plurality of fluorophores that are used to stain a sample; determining a plurality of chosen wavelengths and a plurality of chosen light intensities for the plurality of fluorophores based on the plurality of excitation spectra; obtaining, using a detector of a device, a first detection of the sample based on using a set of first wavelengths from the plurality of chosen wavelengths and a set of first light intensities, wherein the set of first wavelengths comprises a wavelength associated with a target fluorophore of the plurality of fluorophores; obtaining, using the detector of the device, a second detection of the sample based on using a set of second w avelengths from the plurality of chosen wavelengths and a set of second light intensities; and outputting a SIBR quantity of the target fluorophore based on the first detection and the second detection.

Images