CN-116678863-B - Super-resolution imaging method and device for biological sample

CN116678863BCN 116678863 BCN116678863 BCN 116678863BCN-116678863-B

Abstract

The invention discloses a super-resolution imaging method and device for biological samples, and belongs to the technical field of optical imaging. In the imaging method, activating light and exciting light are emitted to a biological sample, fluorescent light generated by a fluorescent probe is reflected to a detector through an objective lens, and the detector generates a fluorescent image. The visible light generator periodically emits first imaging light and second imaging light, and drift data of the biological sample is predicted according to the generated reference image. And synthesizing the plurality of fluorescent images into a super-resolution image according to the drift data. The invention determines whether to enter a calibration state according to the size of the drift data, and moves the workbench in the calibration state. The intensity of the exciting light is regulated through the light transmittance of the optical filter, the luminous state of the fluorescent probe is controlled, and the photobleaching rate of the fluorescent probe is delayed.

Inventors

ZHANG GUOLONG
YI KE
XU CHENG
LI XIONG
ZHANG HAILIANG
ZHANG YUEJIN

Assignees

华东交通大学

Dates

Publication Date: 20260512
Application Date: 20230616

Claims (7)

1. A method for super-resolution imaging of a biological sample, comprising the steps of: Step 1, injecting a fluorescent probe into a biological sample, fixing the biological sample on a workbench, and sequentially emitting a first imaging light ray and a second imaging light ray to the biological sample by a visible light generator; Step 2, after the first imaging light and the second imaging light pass through the biological sample, generating first contrast data and second contrast data in the detector respectively, and synthesizing a reference image according to the first contrast data and the second contrast data; step 3, entering an imaging state, wherein the first laser emits activating light, the second laser emits exciting light, the activating light and the exciting light are coupled and then emitted to a biological sample through the objective lens, fluorescent light generated by the fluorescent probe is reflected to the detector through the objective lens, and the detector generates a fluorescent image; step 4, entering a detection state, and re-emitting first imaging light rays and second imaging light rays by a visible light generator, and synthesizing a reference image according to first contrast data and second contrast data generated in the detector; calculating the displacement of the reference image when the cross-correlation value of the reference image and the reference image reaches the maximum, and determining drift data of the biological sample according to the displacement; step 6, storing drift data by taking the sequence number of the fluorescent image as an index, if the drift data is larger than a reference value, entering a step 7, otherwise, entering a step 8; Step 7, entering a calibration state, the control unit moves the workbench according to the drift data, the exciting light is coupled with the activating light through an optical filter, the light transmittance k of the optical filter is adjusted according to the time deltat of the calibration state, T 0 is the total time length for acquiring N frames of fluorescent images; And 8, returning to the step 3 if the frame number of the obtained fluorescent images is smaller than the preset value N, otherwise, synthesizing a plurality of fluorescent images into a super-resolution image according to the drifting data.
2. The method of claim 1, wherein in step 2, the reference image I 0 =2(I 1 -I 2 )/(I 1 +I 2 ),I 1 is first contrast data and I 2 is second contrast data.
3. The super-resolution imaging method as claimed in claim 1, wherein in step 5, the cross-correlation value of the reference image and the reference image is calculated Wherein a (x, y) is a pixel value of a coordinate (x, y) on the reference image, B (x-u, y-v) is a pixel value of a coordinate (x, y) on the reference image after the displacement amount (u, v) is moved, μ A is a pixel mean value of the reference image, μ B is a pixel mean value of the reference image, σ A is a pixel variance of the reference image, σ B is a pixel variance of the reference image, and m and n are the numbers of pixels of the horizontal and vertical coordinates of the reference image, respectively.
4. The method of super-resolution imaging of a biological sample according to claim 1, wherein the wavelength of the activating light matches the transition wavelength of the fluorescent probe and the wavelength of the exciting light matches the absorption wavelength of the fluorescent probe.
5. An imaging apparatus for performing the super-resolution imaging method of a biological sample according to claim 1, comprising: a workbench for placing a biological sample; a visible light generator for emitting a first imaging light and a second imaging light to the biological sample; a first laser for emitting an activating light; the second laser is used for emitting exciting light rays, and the exciting light rays are coupled with the activating light rays through a light filter; an objective lens for reflecting the excitation light and the activation light to the biological sample; the detector is used for receiving the first imaging light, the second imaging light and the fluorescent light; the signal analysis unit is used for generating a base image or a reference image and generating drift data according to the base image and the reference image; the control unit is used for moving the workbench according to the drifting data; and the synthesis unit is used for synthesizing the plurality of fluorescent images into a super-resolution image according to the drift data.
6. The imaging apparatus of claim 5, further comprising a first adjustment assembly for adjusting the activation or excitation light, a second adjustment assembly for adjusting the first and second imaging light, and a third adjustment assembly for adjusting the fluorescence light.
7. The imaging apparatus of claim 5, wherein the visible light generator has a plurality of programmable LED light sources.

Description

Super-resolution imaging method and device for biological sample Technical Field The invention relates to the technical field of optical imaging, in particular to a super-resolution imaging method and device for biological samples. Background The super-resolution fluorescence imaging technology enables a specific area of a sample to be lightened to form a fluorescence image by exciting a fluorescence probe (fluorescence molecule) in the sample, and a plurality of fluorescence images are synthesized into a super-resolution image, so that the limit of diffraction limit on resolution can be broken through. The super-resolution fluorescence imaging time is long, only one fluorescence image is generated at a time, and in the imaging time of tens of minutes, the light spot deformation and the accumulation of sample drift can cause the gradual distortion of the fluorescence image. CN201811589999.2 discloses a super-resolution imaging device, after the quenching light generated by the first laser and the excitation light generated by the second laser are injected into the multimode optical fiber, the light spot at the exit end of the multimode optical fiber is imaged onto the camera of the correction system, the modulation signal on the spatial light modulator is continuously transformed, and the light spot intensity information collected by the camera is used as the data base for the correction of the multimode optical fiber. In the technical scheme, an excitation light path is avoided to be used as an error correction standard, and can be used as a reference. But the technical solution can only be used for correcting errors caused by the deformation of the light spot. CN202010723855.2 discloses a three-channel fluorescence positioning super-resolution biological microscopic system. In the imaging process of the microscopic system, three channels of lasers are simultaneously irradiated on a sample, three fluorescent molecules for marking the sample are simultaneously luminous and flash, a detector acquires an original image with a flash signal, and the center positions of flash points of all the original images are overlapped together to obtain an ultrahigh resolution image of the three channels. According to the technical scheme, through registering each fluorescent image, the problem of drifting of the sample in a three-dimensional space is solved. Image registration may eliminate minor sample drift, but as drift accumulation increases, the similarity of fluorescent image center positions decreases, making image registration difficult to reduce distortion. Disclosure of Invention In order to solve the defects in the prior art, the invention provides a super-resolution imaging method and an imaging device for a biological sample, so as to solve the problem of image distortion caused by biological sample drift in the fluorescence imaging process in the prior art. After the calibration state is increased, the intensity of the exciting light rays is adjusted through the light transmittance of the optical filter. The technical scheme of the invention is realized as follows: A super-resolution imaging method of a biological sample, comprising the steps of: Step 1, injecting a fluorescent probe into a biological sample, fixing the biological sample on a workbench, and sequentially emitting a first imaging light ray and a second imaging light ray to the biological sample by a visible light generator; Step 2, after the first imaging light and the second imaging light pass through the biological sample, generating first contrast data and second contrast data in the detector respectively, and synthesizing a reference image according to the first contrast data and the second contrast data; step 3, entering an imaging state, wherein the first laser emits activating light, the second laser emits exciting light, the activating light and the exciting light are coupled and then emitted to a biological sample through the objective lens, fluorescent light generated by the fluorescent probe is reflected to the detector through the objective lens, and the detector generates a fluorescent image; step 4, entering a detection state, and re-emitting first imaging light rays and second imaging light rays by a visible light generator, and synthesizing a reference image according to first contrast data and second contrast data generated in the detector; calculating the displacement of the reference image when the cross-correlation value of the reference image and the reference image reaches the maximum, and determining drift data of the biological sample according to the displacement; step 6, storing drift data by taking the sequence number of the fluorescent image as an index, if the drift data is larger than a reference value, entering a step 7, otherwise, entering a step 8; Step 7, entering a calibration state, the control unit moves the workbench according to the drift data, the exciting light is coupled with the activating light throu