Systems and methods for reducing channel crosstalk in fluorescence microscopy.

Abstract

This specification provides methods and systems for crosstalk reduction, such as removing crosstalk interference from a microscope image signal, in order to reconstruct an intended image from an image signal. In one example, the method includes determining a weighted interference signal of a microscope image by determining a weighting constant of the weighted interference signal that provides minimum variance; removing the weighted interference signal from the whole signal; and determining the intended image signal based on the removal of the weighted interference signal from the whole signal.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Patent Application No. 18 / 194,449, filed Mar. 31, 2023, entitled "SYSTEMS AND METHODS FOR FLUORESCENCE MICROSCOPY CHANNEL CROSSTALK MITIGATION". The entire contents of the application listed above are incorporated herein by reference for all purposes.

[0002] Embodiments of the subject matter disclosed herein generally relate to fluorescence microscopy, and more specifically, to reducing channel crosstalk in fluorescence microscopy.

Background Art

[0003] To acquire digital images of cells, biological structures, or other materials, various imaging techniques such as microscopy can be used. Fluorescent dyes in a sample emit light at a lower wavelength than the light used to irradiate them. Dichroic filters are included in such microscopes to block the illumination light and pass the emitted light. Microscopy techniques for illuminating and imaging fluorescent dyes while simultaneously imaging structures are commonly used to study complex biological structures, cells, etc. Due to many factors including potential chemical interactions between the dye and the sample, absorption or irradiation and the corresponding emission wavelengths, and the availability of the dye, bioassays may not be simply adjusted to fit a particular dichroic filter design. As a result, a sub - optimal match between the irradiation wavelength and the emission wavelength can result in an emission spectrum of a particular fluorescent dye that spans two different emission channels.

[0004] Fluorescent dyes that span two different emission channels can cause interference if one of the channels is intended for a different fluorescence color. This interference, or signal degradation, is commonly referred to as inter-channel crosstalk. Inter-channel crosstalk can reduce the image accuracy of a particular channel in an image or signal, and therefore reduce the usefulness of the information obtained from the image or signal.

[0005] Current methods for reducing crosstalk involve image subtraction by binary classification of pixels, achieved by inspecting and comparing pixels to determine which channel a particular pixel belongs to, or by calculating the expected crosstalk using all known parameters. The former approach can determine crosstalk, but interference removal is not achieved. The latter method requires spectral knowledge, including the illumination light, dyes, and characterization of any shifts due to chemical properties, for example, measured by a spectrometer or otherwise known, in order to properly calculate the expected crosstalk. If any of the parameters are unknown, the process becomes impractical. [Overview of the project]

[0006] The inventors have recognized the above-mentioned problems and have devised a method to address them at least partially. In one example, the method may include determining a weighted interference signal of a microscope image, removing the weighted interference signal from the whole signal of the microscope image, and determining the intended image signal from the whole signal. The intended image signal is the image signal for the first channel of the microscope system. The weighted interference signal may be the product of the unweighted interference signal from the second channel of the microscope system and a weighting constant (e.g., a scalar constant). The weighting constant may be determined to provide the minimum variance of the recovered image signal. The whole signal may be known and / or determined by the computing system of the microscope system, and the unweighted interference signal may be known and / or determined by the computing system.

[0007] Thus, by determining weighting constants that provide the minimum variance for the recovered image signal, the intended image signal can be recovered from the overall image signal. The method described herein can be performed by the processor of a computing system without, among other things, knowledge of microscopy system details such as dye spectra, light source details (e.g., illumination scale, etc.), and spectral shifts due to chemical properties, or without requiring the use of a spectrometer to measure spectra.

[0008] The advantages and other features of this specification described above will be readily apparent when the following detailed description is read alone or in conjunction with the accompanying drawings.

[0009] It should be understood that the above summary is provided in a simplified form to introduce a selection of concepts that will be further explained in the detailed description. It is not intended to identify any important or essential features of the claimed subject matter, whose scope is uniquely defined by the claims that follow the detailed description. Furthermore, the claimed subject matter is not limited to any implementation that solves any defects described above or in any part of this disclosure. [Brief explanation of the drawing]

[0010] [Figure 1] A diagram of the microscope system is shown. [Figure 2] A high-level flowchart illustrating an exemplary method for recovering the image of the first channel is shown. [Figure 3] A flowchart illustrating an exemplary method for determining the recovered image signal from an image signal with crosstalk interference is shown. [Figure 4] An example of an image with crosstalk interference is shown. [Figure 5] The interference image of the image in Figure 4 is shown. [Figure 6] An example of a recovered image is shown. [Modes for carrying out the invention]

[0011] This specification relates to the image quality obtained through a fluorescence microscope assembly. Images obtained through fluorescence microscopy are subject to crosstalk interference, thereby degrading the signal from the first channel by interference signals from the second channel due to a fluorescent dye spanning the first and second channels. Methods for crosstalk reduction are described herein, in which weighted interference signals are removed from the image signal having crosstalk interference in order to restore the image of the first channel. Crosstalk reduction may be used by a controller (e.g., a computing system) communicating with a fluorescence microscope system, such as the microscope system 100 shown in Figure 1. The controller may use one or more methods, such as those shown in Figures 2-3, to remove crosstalk interference from the image and restore the image signal of the first channel. Figure 4 shows an example of an image with crosstalk interference. Figure 5 shows an example of an interfered image, and Figure 6 shows an example of a restored image.

[0012] Referring here to Figure 1, a schematic diagram of the microscope system 100 (hereinafter referred to as system 100) is shown. In one example, system 100 may be configured as a fluorescence microscope system. The imager 190 of system 100 may include a light source 102 that provides incident light to components arranged in the path of the incident light, as indicated by the arrow 104. The light source 102 may be a mercury lamp, a xenon arc lamp, a laser, or one or more light-emitting diodes (LEDs). In some examples, system 100 may be included in a multi-detector microscope system.

[0013] The incident light may be directed towards a filter cube 106 (also called a filter block, for example). The filter cube 106 may contain components that filter the incident light so that a target wavelength is transmitted to a target to be analyzed, for example, one or more samples supported on a sample holder 108. In one example, the sample holder 108 may be a microplate. In the example in Figure 1, three filtering components, including an excitation filter 110, a dichroic filter 112, and an emission filter 114, are arranged within the filter cube 106. The incident light may first pass through the excitation filter 110, which filters the light so that a selected wavelength, for example, a target wavelength, continues to pass through the excitation filter 110 while blocking light of other wavelengths. The target wavelength may be a wavelength that excites electrons in a particular fluorophore or fluorescent dye, resulting in the emission of photons when the excited electrons relax to the ground state.

[0014] Excitation light, for example, light filtered by the excitation filter 110, then strikes a dichroic filter 112 (or dichroic beam splitter), as indicated by arrow 116. The dichroic filter 112 may be a mirror, for example, arranged at a 45-degree angle to the optical path of system 100, or angled at a 45-degree angle to the incident light path, as indicated by arrow 104. The surface of the dichroic filter 112 may include a coating that reflects the excitation light, for example, light filtered by the excitation filter 110, but allows fluorescence emitted from the sample in the sample holder 108 to pass through it. The reflected excitation light passes through the objective lens 118, as indicated by arrow 116, and illuminates the sample holder 108. If the sample positioned in the sample holder 108 emits fluorescence, light is emitted, generating emitted light, for example, as indicated by arrow 120, which is collected by the objective lens 118. The emitted light passes through a dichroic filter 112 and then to an emission filter 114, which blocks undesirable excitation wavelengths from passing through. The filtered emitted light is received by a detector 122. In one example, the detector 122 may be a camera, such as a charge-coupled device (CCD) camera. In other examples, the detector 122 may be another type of camera, such as a CMOS camera or a photomultiplier tube.

[0015] The detector 122 can convert emitted light into electronic data. For example, if the detector 122 is a CMOS camera, the detector 122 may include a photosensor configured as a transistor on an integrated circuit. The photons of the emitted light may generate charges that are incident on the photosensor and converted into electronic data representing the photon pattern of the emitted light captured within the camera's field of view (FOV). The electronic data may be stored in the camera's memory, such as random access memory, and may be retrieved by the computing system 124.

[0016] The computing system 124 may be a computing device or other computer. The computing system 124 may include a processor 126 and memory 128. The processor 126 may have one or more computing components that can be used to execute machine-readable instructions. For example, the processor 126 may include a central processing unit (CPU) or, for example, a graphics processing unit (GPU). The processor 126 may be located within the computing system 124 or may be coupled to the computing system 124 in a communicative manner via a suitable remote connection.

[0017] Memory 128 may comprise one or more types of computer-readable media, including volatile and / or non-volatile memory. Volatile memory may include, for example, random-access memory (RAM), and non-volatile memory may include read-only memory (ROM). Memory 128 may include one or more hard disk drives (HDDs), solid-state drives (SSDs), flash memory, etc. Memory 128 is available for storing machine-readable instructions that can be executed by processor 126. Memory 128 is further configured to store images 130, which may include digital images captured or created using various techniques, including digital imaging, digital illustration, etc. Images 130 may further include one or more reference images and / or one or more acquired images.

[0018] At least a portion of image 130 can be acquired via system 100. Memory 128 further includes an image processing module 132 containing machine-readable instructions that can be executed by processor 126 to remove crosstalk interference from image 130. Thus, the image processing module 132 contains machine-readable instructions for manipulating the digital image (e.g., image 130), such as instructions for implementing crosstalk mitigation methods. For example, the machine-readable instructions stored in the image processing module 132 may correspond to one or more methods, examples of which are provided with respect to Figures 2 and 3.

[0019] System 100 further includes a user interface 140 which may include one or more peripherals and / or input devices, including but not limited to a keyboard, mouse, touchpad, or substantially any other input device technology that is communicatively coupled to the computing system 124. The user interface 140 may enable the user to interact with the computing system 124 for purposes such as selecting one or more images to evaluate, or selecting one or more parameters of the imager 190.

[0020] The system 100 further includes a display device 142 which may be configured to display the results of crosstalk removal, the image itself, and possible parameter options and selections related to image acquisition, such as one or more dye wavelengths, channels, and emission spectra. The user may select or otherwise input parameters via the user interface 140 based on the options displayed via the display device 142.

[0021] The computing system 124 may be communicatively coupled to components of system 100. For example, the computing system 124 may be configured to command the activation / deactivation of the light source 102 when prompted based on user input. In another example, the computing system 124 may command the adjustment of the position of the sample holder 108 and focus the excitation light onto different areas of the sample holder. The computing system 124 may command the operation of a motor 160 coupled to the sample holder 108 to vary the position of the sample holder 108 relative to the objective lens 118 and the excitation light, and provide commands on how the sample holder position should be corrected. In some examples, a position sensor 162 may monitor the actual position of the sample holder 108 and may be communicatively coupled to the computing system 124 to relay the sample holder position to the computing system 124.

[0022] Computing system 124 may also be communicatively coupled to detector 122. Thus, the electronic data collected by detector 122 can be retrieved by computing system 124 for further processing and display at an interface such as a computer monitor. It will be appreciated that computing system 124 may be further coupled to other sensors and actuators of system 100. In one example, communication between computing system 124 and the sensors and actuators of system 100 can be enabled by various electrical cables, such as hardwiring. In other examples, computing system 124 may communicate with the sensors and actuators via wireless protocols such as, for example, Wi-Fi®, Bluetooth®, Long Term Evolution (LTE®).

[0023] It will be understood that the system 100 depicted in FIG. 1 is a non-limiting example of a fluorescence microscopy system. Other examples may include variations in the quantities of individual components, such as the number of dichroic, excitation, and emission filters, the configuration of the light source, the relative positioning of the components, and the like. In one example, a fluorescence microscopy system, such as system 100 of FIG. 1, can be used for high-throughput screening of biological samples.

[0024] Referring now to FIG. 2, a high-level flowchart showing an exemplary method 200 for recovering an image of a first channel is shown. Method 200, and other methods included herein, can be executed by a processor of a computing system, such as processor 126 of computing system 124 of FIG. 1, according to instructions stored in a non-transitory memory of the computing system (e.g., within image processing module 132 of memory 128 of FIG. 1).

[0025] In 202, method 200 includes obtaining an image acquired by a microscope system. The microscope system may be system 100 in Figure 1, or it may be configured as a fluorescence microscope system. The image acquired by the microscope system may be the original image containing multiple channel signals. For example, the first channel may have a first signal, and the second channel may have a second signal. The second signal may be an interference signal. Both the first and second signals may be included in the image, and as a result, the image may have crosstalk interference. In some examples, the image may be obtained from the memory of a computing system. In other examples, the image may be obtained from external memory (e.g., an external drive such as a flash drive or optical storage device).

[0026] In 204, method 200 includes determining a recovered image by removing crosstalk interference from the image. Removing crosstalk interference may include determining a weighted interference signal that minimizes variance, as will be described in more detail with respect to Figure 3. The weighted interference signal may be a second signal weighted by a constant. The weighted interference signal may be used to determine a first signal, which is the signal of the recovered image. The computing system described herein may include executable instructions for generating and / or constructing the recovered image from the first signal.

[0027] In 206, method 200 includes outputting the recovered image onto a display device. The display device may be the display device 142 in Figure 1. The display device may communicate with a computing device to display an image generated by the computing device. The display device may display the recovered image for viewing by a user. In this way, the user can visualize the first signal from the first channel without a second interfering signal, such as that contained in the original image.

[0028] In 208, method 200 includes storing the recovered image in memory. The memory may be the memory 128 of the computing system 124 in Figure 1. Storing the recovered image may allow the recovered image to be used for downstream applications. For example, the method may be repeated if two or more adjacent channels have crosstalk. As an example, as described above, the second channel may crosstalk with the first channel, and the first channel may crosstalk with the third channel. In such an example, the interference from the second channel to the first channel may be removed as described above, and the first image of the first channel may be recovered. The recovered first image of the first channel may be removed from the third channel in a similar manner.

[0029] In any iteration, an interfering but non-crosstalk-interfering adjacent channel (e.g., the second channel described above) can be removed from the adjacent channel (e.g., the first channel described above). The recovery of an image (e.g., the first image) allows that image to be removed from another adjacent image with which it is interfering. In this way, the method allows the recovery of multiple images from multiple channels.

[0030] Referring here to Figure 3, a flowchart illustrating an exemplary method 300 for determining an image signal recovered from an image signal with crosstalk interference is shown. As described above, method 300 can be executed by the processor of a computing system according to instructions stored in non-temporary memory. In some examples, method 300 can be implemented as part of method 200 in Figure 2 (for example, in 204).

[0031] In 302, method 300 includes obtaining a whole image signal. The whole image signal may be an image signal with crosstalk interference, and for example, the whole image may include a first channel signal and a weighted second channel signal (for example, the whole image signal may be the sum of the first channel signal and the weighted second channel signal). The whole image signal may be known and / or determined by a computing system following the acquisition of a whole image by a microscope system (e.g., system 100 in Figure 1). The whole image signal may be the output for the first channel, and the second channel signal may interfere due to crosstalk, thereby creating an image signal with crosstalk interference. The whole image signal may be a real number or a complex number. The value of the whole image signal is given by equation (1).

number

[0032] In 304, method 300 includes determining an unweighted interference signal. As described, the overall image signal value may be known based on image acquisition. Since the second channel signal does not contain interference from adjacent channels, the second channel signal value may be known in a similar manner. The second channel signal value may be an unweighted interference signal. Therefore, determining an unweighted interference signal may include obtaining the second channel signal.

[0033] In method 306, method 300 includes determining a weighting constant. The weighted interference signal may be the product of an unweighted interference signal (e.g., a second channel signal value) and a scalar constant c (e.g., a weighting constant). To determine the weighted interference signal, a scalar constant c that gives the minimum variance may be determined. To determine the variance when the first and second signals are nearly uncorrelated, equation (1) can be squared to give equation (2).

number

[0034] The determination of the scalar constant c that provides the minimum variance is described by equations (3) and / or (4).

number

[0035] In 308, method 300 includes calculating a weighted interference signal based on a determined weighting constant. The value of the scalar constant c that provides the minimum variance may be determined by an approximation of a gradient descent algorithm, thereby monitoring the change in variance stepwise with respect to the change in the scalar constant so that the resulting variance continues to decrease. The determination of the scalar constant c in this manner is given by equation (5).

number

[0036] Instead of determining the minimum variance, in some examples the minimum energy of the recovered image signal can be determined. With respect to equations (4) and (5), as explained with reference to equation (2), when the first and second signals are nearly uncorrelated, the product 2*s1*s2*c or the product 2*s1*s2*c2 (both derived by factorizing the squares) is nearly zero. In this way, equation (1) can be used with either equation (5) or equation (4) to find the minimum energy or variance over c, respectively, and give rise to equations (6) and (7).

number

[0037] In 310, method 300 includes removing the weighted interference signal determined in 308 from the overall image signal. Using equation (1), the first image signal can be solved by removing (e.g., subtracting) the weighted interference signal (e.g., the product of the second image signal and a scalar constant) from the overall image signal.

[0038] In 312, method 300 determines whether the difference between the whole image signal and the weighted interference signal has minimum variance. The difference between the whole image signal and the weighted interference signal may be the reconstructed image signal. Gradient descent approximations may allow for determining a value of a scalar constant that does not remove too much signal from the whole image signal, and therefore the weighted interference signal. For example, if the value of the scalar constant is too large, the reconstructed image signal may be negative, while if the value of the scalar constant is too small, not enough signal may be removed from the whole image signal, resulting in a reconstructed image that still has crosstalk interference. Minimum variance aims to determine a value of a scalar constant that can minimize these too small and too large values ​​and removes enough interference signal from the whole image signal to properly reconstruct the intended image signal. If the reconstructed image signal has minimum variance (yes), method 300 proceeds to 314. If the recovered image signal does not have minimum variance, for example, if the weighting constant is too large or too small as described above, method 300 returns to 306 to determine a weighting constant that provides minimum variance. This process, returning to 306 after proceeding to 308 and 310, can describe the gradient descent approximation described above, where values ​​are sequentially tried until a value that provides minimum variance is reached, at which point method 300 may proceed to 314.

[0039] In 314, method 300 includes determining a recovered (e.g., restored) image signal. The recovered image signal may be a first image signal s1 included in the overall image signal, including crosstalk interference. Removing the weighted interference signal determines the difference between the overall image signal and the weighted interference signal. The difference may be the recovered image signal. The recovered image signal may be the intended signal from a first channel without crosstalk. As described with reference to method 200 in Figure 2, the recovered image signal may then be displayed on a display device or otherwise output.

[0040] In this way, by determining the weighted interference signal, and by determining the weighting constant that provides the minimum variance, the first image signal can be reconstructed from the overall image signal including the crosstalk interference. The method described herein can be carried out without knowing the spectrum of the fluorescent dye used, the specifications of the light source (e.g., LED) used during imaging, or any other optical specifications of the microscope system.

[0041] In addition, the methods described herein can be applied to scenarios involving varying amounts of interference, from mild to severe. For example, interference resulting in a signal-to-noise ratio in the range of -20 dB may be considered severe. The methods considered herein can restore the image signal when the interference results in a signal-to-noise ratio in the range of -20 dB.

[0042] As described above, in some examples, the overall image signal may be a first image signal containing a second image signal of the first channel interfering with a third image signal of the second channel that causes a first crosstalk interference. The third image signal may further interfere with a fourth image signal of the third channel, causing a second crosstalk interference. The fifth image signal may contain the third and fourth image signals. Following method 300 described above, the third image signal may be reconstructed from the first image signal by removing a first weighted interference with the second image signal. Then, using the reconstructed third image signal, the fourth image signal may be reconstructed from the fifth image signal by removing a second weighted interference with the third image signal. In this way, method 300 can be applied to multiple channels of crosstalk interference, as long as at least one of the channel signals is not interfered with by an adjacent channel. Thus, each of the multiple channels may be recovered and separated from adjacent channels without knowledge of imaging details (e.g., dye spectrum, LED specifications, etc.).

[0043] Referencing Figures 4 to 6, illustrative images are shown. The first image 400 is depicted in Figure 4, the second image 500 is depicted in Figure 5, and the third image 600 is depicted in Figure 6. The first image 400 in Figure 4 may be an example of an overall image (e.g., a microscopic image) including inter-channel crosstalk interference. The second image 500 in Figure 5 may be an example of an image of an unweighted interference signal (e.g., an interference image). The third image 600 in Figure 6 may be an example of a reconstructed image output with crosstalk interference removed.

[0044] As described above, a sample imaged by a microscope assembly (e.g., imager 190 of system 100 in Figure 1) may contain a fluorescent dye that emits a spectrum of wavelengths. The spectrum of wavelengths may span or be contained within two or more channels. One of the two or more channels may be the channel intended for the fluorescent dye, while the other channels may be intended for other fluorescent dyes. However, because the fluorescent dye emits wavelengths contained within two or more channels, interference occurs with the signal of another dye.

[0045] For example, the first image 400 may be a microscopic image obtained from the spectrum of wavelengths contained in two or more channels. As a result, the first image 400 may contain crosstalk interference. The third image 600 may be from the first channel intended for a fluorescent dye, such as a red channel. The second image 500 may be from the second channel intended for another fluorescent color, such as a far-red channel. The signal in the second image 500 may interfere with the signal in the third image 600, resulting in the first image 400 containing interference due to crosstalk from the far-red channel to the red channel.

[0046] The third image 600 can be reconstructed from the first image 400 by the method provided above. The weighted interference signal of the second image 500 can be determined based on a calculated weighting constant (e.g., the scalar constant c described with reference to Figure 3) and the signal of the second image 500. The weighted interference signal may be a measure of the degree of interference in which the signal of the second image 500 produces the first image 400 in the third image 600.

[0047] The technical effect of the systems and methods provided herein is that image degradation due to interchannel crosstalk in fluorescence microscopy can be mitigated by removing interference signals from the intended signal. Removal of interference signals can enable the generation of more accurate images with reduced noise and interference. In this way, the sample can be imaged by fluorescence microscopy in a more accurate manner.

[0048] The Disclosure also supports a method comprising: determining a weighted interference signal of a microscope image; removing the weighted interference signal from the whole signal of the microscope image; and determining the reconstructed image signal based on the removal of the weighted interference signal from the whole signal. In a first example of the method, the method further comprises determining a weighting constant for the weighted interference signal that provides the minimum variance of the reconstructed image signal. In a second example of the method, optionally including the first example, the method further comprises storing the reconstructed image signal in memory and outputting an image corresponding to the reconstructed image signal to a display device. In a third example of the method, optionally including one or both of the first and second examples, the weighted interference signal is the product of an unweighted interference signal and a weighting constant. In a fourth example of the method, optionally including one or more of the first through third examples, determining the weighting constant involves performing an approximation of a gradient descent algorithm. In a fifth example of the method, optionally including one or more of the first through fourth examples, the reconstructed image signal is a first channel signal. In the sixth example of the method, one or more of the first through fifth examples are optionally included, and the unweighted interference signal is the second channel signal. In the seventh example of the method, one or more of the first through sixth examples are optionally included, and the whole signal of the microscope image includes a weighted interference signal combined with the reconstructed image signal.

[0049] This disclosure also provides support for a microscope system comprising an imager configured to acquire a microscopic image of a sample, and a computing device including a processor communicatively coupled to the imager, the computing device being configured to execute instructions stored in non-temporary memory, which, when executed, cause the processor to obtain a microscopic image acquired by the imager, determine the overall image signal of the microscopic image, determine a weighted interference signal of the overall image signal, remove the weighted interference signal from the overall image signal to restore the intended image signal, and output the intended image signal for display on a display device. In a first example of the system, the imager is configured for a fluorescence microscope. In a second example of the system, optionally including the first example, the sample comprises a fluorescent dye emitting a spectrum of wavelengths, the spectrum of wavelengths comprising two or more channels. In a third example of the system, optionally including one or both of the first and second examples, the non-temporary memory comprises further instructions, which, when executed by the processor, cause the processor to determine an unweighted interference signal and a scalar constant, the scalar constant providing the minimum variance of the intended image signal. In the fourth example of this system, one or more of the first through third examples are optionally included, and the weighted interference signal is the product of the unweighted interference signal and a scalar constant. In the fifth example of this system, one or more of the first through fourth examples are optionally included, and the overall image signal is the sum of the weighted interference signal and the intended image signal. In the sixth example of this system, one or more of the first through fifth examples are optionally included, and the intended image signal is from the first channel of two or more channels, and the unweighted interference signal is from the second channel of two or more channels.

[0050] This disclosure also provides support for a method comprising: obtaining a first image signal of a microscope image acquired by a microscope assembly; obtaining a second image signal of a first channel; and determining a third image signal of the second channel, wherein the first image signal includes a first crosstalk interference of the first channel to the second channel, and the first crosstalk interference is a first weighted interference of the second image signal. In a first example of the method, determining the third image signal comprises determining a first weighted interference of the second image signal and removing the first weighted interference from the first image signal. In a second example of the method, optionally including the first example, the method further comprises determining a fourth image signal of the third channel, wherein the fifth image signal includes a second crosstalk interference of the second channel to the third channel. In a third example of the method, optionally including one or both of the first and second examples, determining the fourth image signal of the third channel comprises removing a second weighted interference of the third image signal from the fifth image signal. In the fourth example of the method, the method optionally includes one or more of the first to third examples, or each of them, and the microscope assembly is configured as a fluorescence microscope assembly.

[0051] When used herein, an element or step described in the singular and preceded by the words "a" or "an" should be understood not to exclude multiple such elements or steps unless explicitly stated to exclude them. Furthermore, a reference to "one embodiment" of the invention is not intended to be interpreted as excluding the existence of additional embodiments that also incorporate the described features. Moreover, unless explicitly stated to the contrary, an embodiment that "comprises," "includes," or "having" one or more elements having a particular characteristic may include additional such elements that do not possess that characteristic. The terms "including" and "in which" are used as plain language equivalents of the terms "comprising" and "wherein," respectively. Furthermore, terms such as "first," "second," and "third" are used merely as labels and are not intended to impose numerical requirements or a specific positional order on their subjects.

[0052] The specification of this document uses examples to disclose the invention, including in best mode, and to enable a person skilled in the art to practice the invention, including by fabricating and using any device or system, and by performing any incorporated method. The patentable scope of the invention is defined by the claims and may include other examples that a person skilled in the art may conceive. Such other examples are intended to be within the scope of the claims if they have structural elements that are not different from the language of the claims, or if they include equivalent structural elements that have no substantive differences from the language of the claims.

Claims

1. Determining the weighted interference signal of a microscope image, Removing the weighted interference signal from the overall signal of the microscope image, A method comprising determining a reconstructed image signal based on the removal of the weighted interference signal from the overall signal.

2. The method according to claim 1, further comprising determining weighting constants for the weighted interference signal that provide the minimum variance of the restored image signal.

3. The method according to claim 1, further comprising storing the restored image signal in a memory and outputting an image corresponding to the restored image signal to a display device.

4. The method according to claim 2, wherein the weighted interference signal is the product of an unweighted interference signal and the weighting constant.

5. The method according to claim 2, wherein determining the weighting constants includes performing an approximation of a gradient descent algorithm.

6. The method according to claim 1, wherein the restored image signal is a first channel signal.

7. The method according to claim 4, wherein the unweighted interference signal is a second channel signal.

8. The method according to claim 1, wherein the overall signal of the microscope image includes the weighted interference signal combined with the reconstructed image signal.

9. An imager configured to acquire a microscopic image of a sample, The computing device includes a processor communicatively coupled to the imager, wherein the computing device is configured to execute instructions stored in non-temporary memory, and when an instruction is executed, the processor, The imager obtains a microscope image, The overall image signal of the aforementioned microscope image is determined, The weighted interference signal of the overall image signal is determined, The weighted interference signal is removed from the overall image signal to restore the intended image signal. A microscope system that outputs the intended image signal for display on a display device.

10. The microscope system according to claim 9, wherein the imager is configured for fluorescence microscopy.

11. The microscope system according to claim 9, wherein the sample comprises a fluorescent dye that emits a spectrum of wavelengths, and the spectrum of wavelengths is contained within two or more channels.

12. The microscope system according to claim 11, wherein the non-temporary memory includes further instructions, which, when executed by the processor, cause the processor to determine an unweighted interference signal and a scalar constant, the scalar constant providing the minimum variance of the intended image signal.

13. The microscope system according to claim 12, wherein the weighted interference signal is the product of the unweighted interference signal and the scalar constant.

14. The microscope system according to claim 9, wherein the overall image signal is the sum of the weighted interference signal and the intended image signal.

15. The microscope system according to claim 12, wherein the intended image signal is from the first channel of the two or more channels, and the unweighted interference signal is from the second channel of the two or more channels.

Images