US-12625357-B2 - Fluorescence microscope system and method

US12625357B2US 12625357 B2US12625357 B2US 12625357B2US-12625357-B2

Abstract

A fluorescence microscope system including an optical detection system configured to capture a raw image of a sample, the raw image including a plurality of pixels, each pixel having a brightness value and a processor, configured to determine one or more invalid pixels in the raw image, assign a predetermined value to each invalid pixel, determine a range of brightness values including the brightness values of a majority of the plurality of pixels excluding the one or more invalid pixels, and generate a processed image of the sample based on the determined range of brightness values.

Inventors

Kai RITSCHEL
Oliver Schlicker

Assignees

LEICA MICROSYSTEMS CMS GMBH

Dates

Publication Date: 20260512
Application Date: 20221213
Priority Date: 20211216

Claims (12)

1 . A fluorescence microscope system comprising: an optical detection system configured to capture a raw image of a sample, the raw image comprising a plurality of pixels, each pixel having a brightness value; and a processor, configured to: determine one or more invalid pixels in the raw image; assign a predetermined value to each invalid pixel; determine a range of brightness values comprising the brightness values of a majority of the plurality of pixels excluding the one or more invalid pixels; and generate a processed image of the sample as a false color image by: assigning a respective color value to each brightness value of the range of brightness values, such that the range of brightness values corresponds to a range of color values that is extended to a full dynamic range of an output unit configured to display the processed image, and assigning a predetermined color value to the predetermined value, wherein the range of color values has a same hue, and the predetermined color value is a complementary color to the hue of the range of the color values.
2 . The fluorescence microscope system according to claim 1 , comprising a memory element, the memory element comprising at least one look-up table; wherein the look-up table correlates brightness values to color values; and wherein the processor is configured to generate the processed image as the false color image based on the look-up table.
3 . The fluorescence microscope system according to claim 1 , wherein the processor is configured to determine whether a pixel of the raw image is saturated, and to determine each saturated pixel as one of the one or more invalid pixels.
4 . The fluorescence microscope system according to claim 1 , wherein the processor is configured to determine whether the brightness value of a pixel of the raw image is a result of a calculation error and/or a result of a detection error, and to determine each such pixel as one of the one or more invalid pixels.
5 . The fluorescence microscope system according to claim 1 , wherein the processor is configured to determine a confidence value for each pixel of the raw image, and to determine each pixel having a confidence value below a predetermined threshold as one of the invalid pixels.
6 . The fluorescence microscope system according to claim 1 , wherein the processor is configured to determine a brightness histogram of the raw image after the processor assigns the predetermined value to each invalid pixel, the brightness histogram comprising the number of pixels for each brightness value; and wherein the processor is configured to determine the range of brightness values based on the brightness histogram.
7 . The fluorescence microscope system according to claim 1 , wherein the predetermined value is a minimum brightness value or a maximum brightness value.
8 . The fluorescence microscope system according to claim 1 , comprising an illumination system configured to emit excitation light for exciting at least one fluorophore located within the sample; wherein the optical detection system is configured to generate the raw image based on fluorescence light emitted by the excited fluorophores; and wherein the brightness values of the raw image correspond to a fluorescence intensity.
9 . The fluorescence microscope system according to claim 1 , wherein the fluorescence microscope system is configured for fluorescence widefield microscopy.
10 . The fluorescence microscope system according to claim 1 , wherein the fluorescence microscope system is configured for confocal laser scanning microscopy.
11 . The fluorescence microscope system according to claim 1 , comprising the output unit configured to display the raw image and/or the processed image.
12 . A method for generating a processed image of a sample with a fluorescence microscope system, the method comprising: capturing a raw image of the sample with an optical detection system of the microscope system, the raw image comprising a plurality of pixels, each pixel having a brightness value; determining one or more invalid pixels in the raw image; assigning a predetermined value to each invalid pixel; determining a range of brightness values comprising the brightness values of a majority of the plurality of pixels excluding the one or more invalid pixels; and generating a processed image of the sample as a false color image by: assigning a respective color value to each brightness value of the range of brightness values, such that the range of brightness values corresponds to a range of color values that is extended to a full dynamic range of an output unit configured to display the processed image, and assigning a predetermined color value to the predetermined value, wherein the range of color values has a same hue, and the predetermined color value is a complementary color to the hue of the range of the color values.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit to European Patent Application No. EP 21215122.9, filed on Dec. 16, 2021, which is hereby incorporated by reference herein. FIELD The invention relates to a fluorescence microscope system and a method for generating a processed image of a sample with a fluorescence microscope system. BACKGROUND A fluorescence microscopic image comprises a plurality of pixels that each correspond to a data point of a fluorescence microscopic measurement. In most cases, each pixel has a brightness value that corresponds to a detected fluorescence intensity. In other cases, the fluorescence microscopic image is the result of a mathematical reconstruction, for example by means of a spectral unmixing algorithm. In these cases, each pixel may also be assigned a confidence value that represents a certainty with which the algorithm has determined the brightness value of the respective pixel. There are various reasons why a pixel of the fluorescence microscopic image could be considered invalid. For example, the pixel could be saturated, for example due to the detected fluorescence intensity exceeding the dynamic range of the detector. It is also possible that an error occurred during the detector readout resulting in one or more pixels having a brightness value that does not correspond to the fluorescence intensity. Further, a pixel might be considered invalid if an error occurred during the mathematical reconstruction of the fluorescence intensity resulting in a low confidence value associated with the respective pixel. The invalid pixel are artifacts in the fluorescence microscopic image that have to be taken into account when displaying or further processing the image. In fluorescence microscopy, it is common to work with weak signals that utilize only a small range of the dynamic range of the detector. In order to display this weak signal well, a range of brightness values including the signal, i.e. that comprises information about the sample, is selected and extended to the full dynamic range of a display unit. However, since the invalid pixels comprise no information about the sample, the invalid pixels interfere with the determination of the range of brightness values. SUMMARY In an embodiment, the present disclosure provides a fluorescence microscope system comprising an optical detection system configured to capture a raw image of a sample, the raw image comprising a plurality of pixels, each pixel having a brightness value and a processor, configured to determine one or more invalid pixels in the raw image, assign a predetermined value to each invalid pixel, determine a range of brightness values comprising the brightness values of a majority of the plurality of pixels excluding the one or more invalid pixels, and generate a processed image of the sample based on the determined range of brightness values. BRIEF DESCRIPTION OF THE DRAWINGS Subject matter of the present disclosure will be described in even greater detail below based on the exemplary figures. All features described and/or illustrated herein can be used alone or combined in different combinations. The features and advantages of various embodiments will become apparent by reading the following detailed description with reference to the attached drawings, which illustrate the following: FIG. 1 illustrates a schematic view of a fluorescence microscope system according to an embodiment; FIG. 2 illustrates a flowchart of the method for generating a processed image of the sample with the fluorescence microscope system according to FIG. 1; FIG. 3a illustrates a schematic view of a raw image; FIG. 3b illustrates a schematic view of a processed image; and FIG. 4 illustrates a brightness histogram of a raw image. DETAILED DESCRIPTION In an embodiment, the present invention provides a fluorescence microscope system and a method for generating a processed image of a sample with a fluorescence microscope system that can extract relevant information about the sample from a raw image of the sample in a reliable and robust manner. In an embodiment, a fluorescence microscope system comprises an optical detection system configured to capture a raw image of the sample. The raw image comprises a plurality of pixels, each pixel having a brightness value. The fluorescence microscope system further comprises a processor. The processor is configured to determine invalid pixel in the raw image, to assign a predetermined value to each invalid pixel, to determine a range of brightness values comprising the brightness values of a majority of the plurality of pixel excluding the invalid pixel, and to generate a processed image of the sample based on the determined range of brightness values. The predetermined value may be a numerical brightness value, NaN or another numerical or non-numerical data type. By assigning the invalid pixel the predetermined value, the invalid pixel is marked. Since the predetermined value