Method and system for reconstructing microscopic fluorescence image of thick sample

Abstract

The invention provides a method and system for reconstructing a microscopic fluorescence image of a thick sample, which collects field of view images of different focal planes at equal intervals with a step length smaller than the depth of field of the microscope to obtain a data cube; the field of view images are divided into For different grids, use the brightness value of the brightest point in the grid and the variance of the brightness value of each pixel in the grid to construct a sharpness evaluation function, and establish a grid confidence map; after correcting the first focus position of each grid , the focus position of each pixel is accurate, and the edge of the grid is smoothed in the position space and the field of view image to obtain a preliminary reconstructed image; the pixels in the preliminary reconstructed image are clustered with fluorescence features, and the fluorescence features The number of pixels occupied determines the size of the segmentation grid, and each fluorescent feature is reconstructed to obtain the final reconstructed image. It solves the problem of different focal planes at different positions in the same field of view of thick samples, and improves the quality of the reconstructed fluorescence image and the overall clarity.

Description

technical field [0001] The invention relates to the field of microscopy and image processing, in particular to a method and system for reconstructing microscopic fluorescence images of thick samples. Background technique [0002] Microscopic fluorescence imaging is an important means of quantitative analysis in the field of life sciences. Limited by the extremely small depth of field of high-magnification objective lenses, fluorescence microscopic imaging of thick samples cannot achieve clear collection of fluorescence features in the field of view, especially in focal planes at different positions in the same field of view of thick samples. At the same time, fluorescence image acquisition is more difficult and the overall clarity is not enough. [0003] The prior art related to this application is the patent document CN1071111118A, which discloses an epi-illumination Fourier overlapping correlation imaging system and method for high-resolution imaging of thick samples, incl...

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Problems solved by technology

[0002] Microscopic fluorescence imaging is an important means of quantitative analysis in the field of life sciences. Limited by the extremely small depth of field of high-magnification objective lenses, fluorescence microscopic imaging of thick samples cannot achieve clear collection of fluorescence features in the field of view, especially in focal planes at different positions in the same field of view of thick samples. At the same time, fluorescence image acquisition is more difficult, and the overall clarity is not enough

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