Method for automatically positioning Gaussian light sheet center based on confocal scanning light field microscope

Abstract

The invention discloses an automatic Gaussian light sheet center positioning method based on a confocal scanning light field microscope, and relates to the technical field of optical imaging, and the method comprises the steps: setting an initial offset center of a line scanning module based on a target sample, and adjusting the confocal scanning light field microscope; shooting a two-dimensional light field image of the target sample through the adjusted confocal scanning light field microscope, and performing two-dimensional slicing on the two-dimensional light field image according to a preset interval to obtain sub-view-angle images; on the basis of the sub-view-angle images, full-view-field longitudinal energy distribution is obtained, and the full-view-field longitudinal energy distribution is corrected; and calibrating the initial offset center based on the full-view-field longitudinal energy distribution before and after correction. Compared with manual calibration, parameters do not need to be adjusted manually, and automatic Gaussian line light center position positioning and calibration of different systems and different samples are achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical imaging technology, and more particularly to an automatic positioning method of a Gaussian light sheet center based on a confocal scanning light field microscope. BACKGROUND

[0002] The light field microscope uses a microlens array to realize multi-view light field imaging, sacrifices certain spatial resolution to obtain angle information of light, and can obtain three-dimensional tomographic results of a sample through a phase space reconstruction method. The scanning light field microscope adds a two-dimensional galvanometer to the sample for two-dimensional scanning under the premise of ensuring the angle resolution, thereby further improving the lateral resolution of the light field microscope imaging system. The confocal scanning light field microscope introduces a line scanning confocal module on the basis of the scanning light field microscope, realizes lower phototoxicity and faster imaging speed through synchronization with the camera rolling shutter exposure, and significantly improves the signal-to-background ratio of the imaging result.

[0003] Accurate synchronization of line scanning and camera rolling shutter exposure is an important factor for the confocal scanning light field microscope to realize full-field uniform illumination and clear imaging. Different refractive indexes of different samples to be photographed will cause the center position of the line laser to deviate, and then cause the full-field illumination to be non-uniform, thereby affecting the imaging effect. Therefore, for different systems and different samples, the center position of the Gaussian line light needs to be positioned and calibrated.

[0004] In the prior art, the best line light center is obtained through manual traversal and subjective judgment. This method of calibrating the line light center through manual calibration is too dependent on subjective judgment and has poor adaptability to different systems and different samples.

[0005] Therefore, how to realize automatic positioning and calibration of the center position of the Gaussian line light for different systems and different samples, realize accurate synchronization with the camera rolling shutter exposure, and realize full-field uniform illumination is a problem that needs to be solved by those skilled in the art. SUMMARY

[0006] In view of the above problems, the present application provides an automatic positioning method of a Gaussian light sheet center based on a confocal scanning light field microscope to at least solve part of the technical problems mentioned in the background.

[0007] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0008] The present application provides an automatic positioning method of a Gaussian light sheet center based on a confocal scanning light field microscope, comprising the following steps:

[0009] S1, based on a target sample, setting an initial bias center of a line scanning module, and adjusting the confocal scanning light field microscope;

[0010] S2, taking a two-dimensional light field map of the target sample by the adjusted confocal scanning light field microscope, and two-dimensionally slicing the two-dimensional light field map according to a preset interval to obtain a sub-view angle map;

[0011] S3, obtaining a full-view longitudinal energy distribution based on the sub-view angle map, and correcting the full-view longitudinal energy distribution;

[0012] S4, calibrating the initial bias center based on the full-view longitudinal energy distribution before and after correction.

[0013] Further, the S1 specifically comprises:

[0014] Placing the target sample at a shooting position of the confocal scanning light field microscope; setting an initial bias center of the line scanning module as O0;

[0015] Adjusting a focal length of the confocal scanning light field microscope so that a Gaussian line light irradiated on the target sample reaches a target fineness, and the center position of the Gaussian line light is coincided with the focal plane of the objective lens;

[0016] Taking a preview map of the target sample, and dynamically adjusting an exposure time and a light source power according to the intensity of the preview map so that a pixel value dynamic range covers a sensor linear region.

[0017] Further, in the step S2, the preset interval is an image pixel corresponding to a microlens.

[0018] Further, the step S3 specifically comprises:

[0019] S31, performing column projection on the sub-view angle map to obtain a full-view longitudinal energy distribution L0, which is expressed as:

[0020]

[0021] Wherein, C0 represents a sub-view angle map; x represents a pixel horizontal coordinate of the sub-view angle map; y represents a pixel vertical coordinate of the sub-view angle map; W represents a width of the sub-view angle map; H represents a height of the sub-view angle map;

[0022] S32, considering a full-view longitudinal energy distribution baseline, calculating a local background mean value of the full-view longitudinal energy distribution L0 by using a sliding window as a corrected full-view longitudinal energy distribution ; which is expressed as:

[0023]

[0024] Wherein, w represents a window width; k represents a middle parameter;

[0025] S33, calculating a local background mean value A baseline flatness test is performed.

[0026] Further, the step S33 specifically comprises:

[0027] Obtaining the corrected full-field longitudinal energy distribution The standard deviation in the non-peak region, if the standard deviation is less than a preset value, indicates that the correction is complete; otherwise, the window width is increased to re-execute steps S32-S33.

[0028] Further, the step S4 specifically comprises:

[0029] S41, the maximum value of the position corresponding to the full-field longitudinal energy distribution L0 as the initial Gaussian light center position;

[0030] S42, taking the initial Gaussian light center position as the midpoint, based on the corrected full-field longitudinal energy distribution , the central region of the full-field longitudinal energy distribution L0 is intercepted to obtain the central energy distribution L1; represented as:

[0031]

[0032] Wherein, Y represents the initial Gaussian light center position; H represents the height of the sub-view angle diagram;

[0033] S43, one-dimensional Gaussian function fitting is performed on the central energy distribution L1 to obtain the first energy distribution peak coordinate Y0;

[0034] S44, the first energy distribution peak coordinate Y0 is judged:

[0035] If , it indicates that the line scanning module is centered, and at this time, there is no need to calibrate the initial offset center of the line scanning module.

[0036] Further, the step S44 further comprises:

[0037] If , the initial offset center of the line scanning module is calibrated, specifically comprising:

[0038] Setting the new offset center of the line scanning module as O1, obtaining the second energy distribution peak coordinate Y1 of the line scanning module at the new offset center O1;

[0039] According to the first energy distribution peak coordinate Y0 and the second energy distribution peak coordinate Y1, the coefficient K of the offset center with respect to the pixel size is calculated, and the initial offset center O0 is calibrated according to the coefficient K; represented as:

[0040]

[0041] Wherein, O represents the initial bias center after calibration.

[0042] Further, the center energy distribution L1 is approximated to a one-dimensional Gaussian function, in the form as follows:

[0043]

[0044] Wherein, B represents the center point of the Gaussian function; C 2 represents the data distribution variance; y2 represents the pixel coordinate of the center energy distribution.

[0045] Through the above technical solution, compared with the prior art, the application provides an automatic positioning Gaussian light sheet center method based on a confocal scanning light field microscope, which has the following beneficial effects.

[0046] The application adjusts the confocal scanning light field microscope before shooting, which is conducive to adjusting the signal interval distribution and improving the accuracy of subsequent step fitting,

[0047] The application corrects the longitudinal energy distribution of the full field of view, which is conducive to distinguishing the signal and background components and avoiding uncontrollable errors caused by noise.

[0048] Compared with manual calibration, the application does not need to manually adjust parameters, and realizes automatic positioning and calibration of the Gaussian line light center position of different systems and different samples.

[0049] The technical solutions of the application will be further described in detail below with the help of the drawings and examples. DETAILED DESCRIPTION

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the application, and those skilled in the art can obtain other drawings according to the provided drawings without creative labor.

[0051] Figure 1 The automatic positioning Gaussian light sheet center method based on a confocal scanning light field microscope provided by the embodiment of the application is shown in the flowchart.

[0052] Figure 2 The line light position before and after calibration and the comparison of the collected images provided by the embodiment of the application are shown in the schematic diagram. DETAILED DESCRIPTION

[0053] With reference to the drawings and embodiments of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0054] The present application aims to realize accurate synchronization of line scanning and camera rolling shutter exposure by calibrating the offset center of the line scanning module of the confocal scanning light field microscope. To this end, the present application proposes an automatic positioning Gaussian light sheet center method based on the confocal scanning light field microscope. The position of the Gaussian line light is determined through column projection and Gaussian fitting to realize calibration of the offset center of the line scanning module. Referring to Figure 1 As shown, it comprises:

[0055] S1, based on the target sample, setting the initial offset center of the line scanning module, and adjusting the confocal scanning light field microscope;

[0056] S2, taking a two-dimensional light field image of the target sample through the adjusted confocal scanning light field microscope, and two-dimensionally slicing the two-dimensional light field image according to a preset interval to obtain a sub-view angle image;

[0057] S3, based on the sub-view angle image, obtaining a full field of view longitudinal energy distribution, and correcting the full field of view longitudinal energy distribution;

[0058] S4, based on the full field of view longitudinal energy distribution before and after correction, calibrating the initial offset center.

[0059] Next, each of the above steps will be described in detail.

[0060] In the above step S1, based on the target sample, the initial offset center of the line scanning module is set, and the confocal scanning light field microscope is adjusted; specifically including:

[0061] S11, placing the target sample at the shooting position of the confocal scanning light field microscope; setting the initial offset center of the line scanning module as O0;

[0062] S12, adjusting the focal length of the confocal scanning light field microscope so that the Gaussian line light irradiated on the target sample reaches the target fineness, and the center position of the Gaussian line light coincides with the focal plane of the objective lens;

[0063] S13, taking a preview image of the target sample, and dynamically adjusting the exposure time and light source power according to the intensity of the preview image, so that the pixel value dynamic range covers the sensor linear region, avoiding overexposure or too dark.

[0064] In the step S2, a two-dimensional light field image of the target sample is captured by the adjusted confocal scanning light field microscope, and two-dimensional slices of the two-dimensional light field image are obtained according to a preset interval.

[0065] The preset interval is an image pixel corresponding to each microlens. Assuming that each microlens corresponds to N image pixels, the two-dimensional slices of the two-dimensional light field image are obtained at an interval of N.

[0066] In the step S3, the longitudinal energy distribution of the full field of view is obtained based on the sub-view angle image, and the longitudinal energy distribution of the full field of view is corrected. Specifically, the step S3 includes:

[0067] In the step S3, the longitudinal energy distribution of the full field of view is obtained based on the sub-view angle image, and the longitudinal energy distribution of the full field of view is corrected. Specifically, the step S3 includes:

[0068]

[0069] In the step S3, the longitudinal energy distribution of the full field of view is obtained based on the sub-view angle image, and the longitudinal energy distribution of the full field of view is corrected. Specifically, the step S3 includes:

[0070] In the step S3, the longitudinal energy distribution of the full field of view is obtained based on the sub-view angle image, and the longitudinal energy distribution of the full field of view is corrected. Specifically, the step S3 includes:

[0071]

[0072] In the step S3, the longitudinal energy distribution of the full field of view is obtained based on the sub-view angle image, and the longitudinal energy distribution of the full field of view is corrected. Specifically, the step S3 includes:

[0073] In the step S3, the longitudinal energy distribution of the full field of view is obtained based on the sub-view angle image, and the longitudinal energy distribution of the full field of view is corrected. Specifically, the step S3 includes: In the step S3, the longitudinal energy distribution of the full field of view is obtained based on the sub-view angle image, and the longitudinal energy distribution of the full field of view is corrected. Specifically, the step S3 includes:

[0074] In the step S3, the longitudinal energy distribution of the full field of view is obtained based on the sub-view angle image, and the longitudinal energy distribution of the full field of view is corrected. Specifically, the step S3 includes: In the step S3, the longitudinal energy distribution of the full field of view is obtained based on the sub-view angle image, and the longitudinal energy distribution of the full field of view is corrected. Specifically, the step S3 includes: In the step S3, the longitudinal energy distribution of the full field of view is obtained based on the sub-view angle image, and the longitudinal energy distribution of the full field of view is corrected. Specifically, the step S3 includes:

[0075] In the step S4, the initial bias center is calibrated based on the longitudinal energy distribution of the full field of view before and after correction. Specifically, the step S4 includes:

[0076] In the step S4, the initial bias center is calibrated based on the longitudinal energy distribution of the full field of view before and after correction. Specifically, the step S4 includes:

[0077] ​

[0078] S42, taking the initial Gaussian line light center position Y estimated roughly as the midpoint, based on the corrected full field of view longitudinal energy distribution , intercepting the center region of the full field of view longitudinal energy distribution L0 to obtain the center energy distribution L1; represented as:

[0079]

[0080] Wherein Y represents the initial Gaussian line light center position; H represents the height of the sub-view angle diagram;

[0081] S43, wherein the center energy distribution L1 is approximated to a one-dimensional Gaussian function, in the form as follows:

[0082]

[0083] Wherein B represents the center point of the Gaussian function; C 2 represents the data distribution variance; y2 represents the pixel coordinate of the center energy distribution; the one-dimensional Gaussian function fitting is performed on the center energy distribution L1 to obtain the first energy distribution peak coordinate Y0;

[0084] S44, the first energy distribution peak coordinate Y0 is judged:

[0085] If , it indicates that the line scanning module is centered, and at this time, there is no need to calibrate the initial offset center of the line scanning module;

[0086] If , the initial offset center of the line scanning module is calibrated, specifically including:

[0087] (1) setting the new offset center of the line scanning module as O1, repeating the above steps to obtain the second energy distribution peak coordinate Y1 of the line scanning module at the new offset center O1;

[0088] (2) according to the first energy distribution peak coordinate Y0 and the second energy distribution peak coordinate Y1, the coefficient K of the offset center about the pixel size is calculated, and the initial offset center O0 is calibrated according to the coefficient K; represented as:

[0089]

[0090] Wherein O represents the calibrated initial offset center.

[0091] Figure 2The calibration before and after the line light position and the contrast schematic diagram of the collected image are provided. Wherein, the upper left diagram is the line light position under the condition that the line scanning center is correctly calibrated, and the lower left diagram is the collected image under the condition that the line scanning and the camera rolling shutter are matched; the upper right diagram is the line light position under the condition that the line scanning center is not correctly calibrated, and the lower right diagram is the collected image under the condition that the line scanning and the camera rolling shutter are not matched; it should be noted that, in the lower right diagram, the line light center is not calibrated, so that the line light scanning and the camera rolling shutter exposure are not matched, and thus there is no signal.

[0092] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the difference from other embodiments. The same or similar parts between the various embodiments can be referred to each other. For the device disclosed by the embodiments, since it corresponds to the method disclosed by the embodiments, the description is relatively simple, and the related parts can be referred to the method part.

[0093] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to the embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for automatically locating the center of a Gaussian light sheet based on a confocal scanning light field microscope, characterized in that: The steps include: S1. Based on the target sample, set the initial offset center of the line scan module and adjust the confocal scanning light field microscope; S2. Using the adjusted confocal scanning light field microscope, capture a two-dimensional light field image of the target sample, and perform two-dimensional slicing on the two-dimensional light field image at preset intervals to obtain sub-viewing angle images; S3. Obtaining a full-field longitudinal energy distribution based on the sub-viewing angle image, and correcting the full-field longitudinal energy distribution; S4. Calibrate the initial offset center based on the full-field longitudinal energy distribution before and after correction.

2. The method for automatically locating the center of a Gaussian light sheet based on a confocal scanning light field microscope according to claim 1, characterized in that: Said S1 specifically includes: Place the target sample at the shooting position of the confocal scanning light field microscope; set the initial offset center of the line scanning module to O0; Adjusting the focal length of the confocal scanning light field microscope so that the Gaussian line light irradiated on the target sample reaches the target fineness and the center position of the Gaussian line light coincides with the focal plane of the objective lens; A preview image is taken of the target sample, and the exposure time and light source power are dynamically adjusted according to the intensity of the preview image so that the dynamic range of the pixel value covers the linear region of the sensor.

3. The method for automatically locating the center of a Gaussian light sheet based on a confocal scanning light field microscope according to claim 1, characterized in that: In step S2, the preset interval is the image pixel corresponding to the microlens.

4. The method for automatically locating the center of a Gaussian light sheet based on a confocal scanning light field microscope according to claim 1, characterized in that: The step S3 specifically includes: S31, performing column projection on the sub-viewing angle image to obtain the full-field longitudinal energy distribution L0, which is expressed as: ； Wherein, C0 represents the sub-view image; x represents the pixel horizontal coordinate of the sub-view image; y represents the pixel vertical coordinate of the sub-view image; W represents the width of the sub-view image; H represents the height of the sub-view image; S32, considering the full field of view longitudinal energy distribution baseline, using a sliding window to calculate the local background mean of the full field of view longitudinal energy distribution L0 as the corrected full field of view longitudinal energy distribution ; expressed as: ； Among them, w represents the window width; k represents the intermediate parameter; S33, local background mean Perform a baseline flatness test.

5. The method for automatically locating the center of a Gaussian light sheet based on a confocal scanning light field microscope according to claim 4, characterized in that: The step S33 specifically includes: Obtain the corrected full-field longitudinal energy distribution In the non-peak area, if the standard deviation is less than a preset value, it means that the correction is completed; otherwise, the window width is increased and steps S32-S33 are executed again.

6. The method for automatically locating the center of a Gaussian light sheet based on a confocal scanning light field microscope according to claim 1, characterized in that: The step S4 specifically includes: S41, the maximum value of the position corresponding to the full field of view longitudinal energy distribution L0 is used as the center position of the initial Gaussian line light; S42, with the center of the initial Gaussian line light as the midpoint, based on the corrected full field longitudinal energy distribution , intercept the central area of ​​the full field longitudinal energy distribution L0 to obtain the central energy distribution L1; expressed as: ； Where Y represents the center position of the initial Gaussian line light; H represents the height of the sub-view image; S43, performing one-dimensional Gaussian function fitting on the central energy distribution L1 to obtain the first energy distribution peak coordinate Y0; S44, judging the first energy distribution peak coordinate Y0: like , it means that the line scan module is centered. In this case, there is no need to calibrate the initial offset center of the line scan module.

7. The method for automatically locating the center of a Gaussian light sheet based on a confocal scanning light field microscope according to claim 6, characterized in that: The step S44 further includes: like , then calibrate the initial offset center of the line scan module, specifically including: Setting a new offset center of the line scan module to O1, and obtaining a second energy distribution peak coordinate Y1 when the line scan module is at the new offset center O1; According to the first energy distribution peak coordinate Y0 and the second energy distribution peak coordinate Y1, the coefficient K of the offset center with respect to the pixel size is calculated, and the initial offset center O0 is calibrated according to the coefficient K; it is expressed as: ； Where O represents the initial bias center after calibration.

8. The method for automatically locating the center of a Gaussian light sheet based on a confocal scanning light field microscope according to claim 6, characterized in that: The central energy distribution L1 is expressed as: ； Among them, B represents the center point of the Gaussian function; C 2 represents the data distribution variance; y2 represents the pixel coordinates of the central energy distribution.

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