JP-2026514420-A - 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. [Selection Diagram] Figure 1

Inventors

• ファーガソン ケビン

Assignees

• アラセリ バイオサイエンシズ インク．

Dates

Publication Date

20260511

Application Date

20240320

Priority Date

20230331

Claims (15)

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 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 the 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 contains a fluorescent dye that emits a spectrum of wavelengths, and the spectrum of wavelengths is contained within two or more channels.
12. 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, according to claim 11.
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.

Description

Cross-reference of related applications This application claims priority to U.S. Patent Application No. 18/194,449, entitled “SYSTEMS AND METHODS FOR FLUORESCENCE MICROSCOPY CHANNEL CROSSTALK MITIGATION,” filed on 31 March 2023. The entirety of the above-listed applications is incorporated herein by reference for all purposes. The embodiments of the subject matter disclosed herein generally relate to fluorescence microscopy, and more specifically, to channel crosstalk reduction in fluorescence microscopy. Various imaging techniques, such as microscopy, can be used to obtain digital images of cells, biological structures, or other materials. Fluorescent dyes in a sample emit light at wavelengths lower than the light used to illuminate them. Dichroic filters are included in such microscopes to block illumination light and allow synchroic light to pass through. Microscopy techniques for illuminating and imaging fluorescent dyes and simultaneously imaging structures are routinely used to study complex biological structures, cells, and the like. Due to many factors, including potential chemical interactions between the dye and the sample, the wavelengths of absorption or irradiation and corresponding emission, and the availability of the dye, bioassays may not be simply tailored to fit a particular dichroic filter design. As a result, imperfect matches between irradiation and emission wavelengths can lead to emission spectra of specific fluorescent dyes spanning two different emission channels. 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 degrade 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. Current methods for crosstalk reduction 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. The inventors of this invention recognize 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 overall signal of the microscope image, and determining the intended image signal from the overall 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 overall 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. 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 executed by a computing system processor without, among other things, knowledge of microscopy system details such as dye spectra, light source details (e.g., illumination scale), and spectral shifts due to chemical properties, or without requiring the use of a spectrometer to measure spectra. The advantages and other features of this specification, as described above, will be readily apparent upon reading the following detailed description alone or in conjunction with the accompanying drawings. 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 significant 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 resolves any defects described above or in any part of this disclosure. A diagram of the microscope system is shown.A high-level flowchart illustrating an exemplary method for recovering the image of the first channel is shown.A flowchart i