Molecular scale resolution optical super-resolution microscopy method

Abstract

The invention relates to the technical field of optical microscope imaging, in particular to a molecular scale resolution optical super-resolution microscopy method. The method comprises the following steps: constructing a single molecule polarization measurement module; performing single-molecule imaging on the sample, dividing single-molecule fluorescence into two beams by using the single-molecule polarization measurement module, and simultaneously imaging in different half regions of the EMCCD; carrying out single molecule identification and center calculation on the collected single molecule image; constructing a conversion matrix, and mapping the positions of the single molecules of two different channels; measuring the fluorescence intensity of the single molecules in the two channels, calculating polarization values, and grouping the positioning of the single molecules according to the difference of the polarization values; calculation of a single molecule positioning distribution center and MIPI positioning precision is carried out on grouped single molecule positioning; and carrying out super-resolution image reconstruction to obtain a molecular scale resolution image. The method utilizes polarization measurement to distinguish adjacent single fluorescent molecules, and is suitable for high-density fluorescence labeled sample imaging.

Description

Technical Field

[0001] The present invention relates to the technical field of optical microscope imaging, in particular to a molecular scale resolution optical super-resolution microscopy method. Background Art

[0002] Single-Molecule Localization Microscopy (SMLM) is a super-resolution imaging technology that breaks through the traditional optical diffraction limit. This technology achieves super-resolution imaging by precisely locating the position of a single fluorescent molecule, achieving a lateral resolution of 20 nanometers and an axial resolution of 60 nanometers. In single-molecule localization microscopy (SMLM), the localization accuracy of a single molecule depends on the total number of photons emitted by a single fluorescent molecule collected in a single image. The actual imaging process often involves multiple imaging of the same fluorescent molecule, resulting in multiple localizations (one localization per imaging). During the reconstruction process of single-molecule localization microscopy, due to the limitation of positioning accuracy, these positioning points often overlap, invisibly increasing the size of the single molecule and thus reducing the resolution of single-molecule localization microscopy.

[0003] In order to solve the above problems, in 2023, Reinhardt1 et al. developed an optical super-resolution microscopy based on single-molecule localization microscopy, called Resolution Enhancement by Sequential Imaging (RESI). RESI can improve the resolution of optical microscopes to the angstrom level. RESI obtains the position of the center by weighted averaging multiple localizations of a single fluorescent molecule and uses it as the center of this single molecule. According to the law of statistics, the standard deviation of multiple localizations (RESI localization accuracy) is equal to the standard deviation of a single localization (SMLM localization accuracy) divided by the square root of the total number of localizations. Therefore, the RESI localization accuracy (σ RESI ) and SMLM positioning accuracy (σ SMLM ) can be described as the relationship between Where K is the total number of position measurements from the same target. If the localization total K is large enough, RESI can achieve spatial resolution of a few nanometers to angstroms. However, RESI is limited in that it is only effective for biological samples with sparse fluorescent labels (the spacing between fluorescent molecules of the same fluorescent label is greater than 20 nanometers, which is the typical resolution of SMLM). It is difficult to apply to samples with high fluorescent density, such as those with immunofluorescence labeling and fluorescent protein fusion expression, thus limiting the widespread use of RESI in biological research.

[0004] CN113933277A discloses a high-density three-dimensional single-molecule localization super-resolution microscopy method. This method uses a first signal channel unit and a second signal channel unit, each containing a first cylindrical lens and a second cylindrical lens oriented orthogonally to project two fluorescent signal beams split by a beam splitter onto different regions of a detector for imaging. However, this method simply splits the luminescence intensity of the fluorescent molecules into two, resulting in a lateral resolution of approximately 20 nanometers for the three-dimensional imaging, similar to the limitations of SMLM.

[0005] The present invention develops a RESI-based optical super-resolution microscopy technique, named Molecular Imaging by Polarization Imaging (MIPI). Similar to RESI, MIPI uses single-molecule localization averaging to improve resolution. However, MIPI distinguishes adjacent fluorescent molecules with the same fluorescent label (within 20 nanometers) by measuring the polarization of individual fluorescent molecules. This overcomes the limitations of RESI and is a molecular-scale resolution optical super-resolution microscopy technique with a wider range of applications. Summary of the Invention

[0006] To address the shortcoming of resolution-enhanced sequential imaging (RESI) microscopy, which is only applicable to sparsely fluorescently labeled biological samples, the present invention proposes a new type of molecular-scale resolution optical super-resolution microscopy: molecular-scale imaging through polarization imaging (MIPI). By measuring the differences in the polarization of individual fluorescent molecules, the adjacent different fluorescent molecules can be distinguished. This microscopy overcomes the shortcomings of RESI, making MIPI a molecular-scale resolution optical super-resolution microscopy with a wider range of application scenarios, especially for high-density fluorescent-labeled samples.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A molecular-scale resolution optical super-resolution microscopy method comprising:

[0009] Step 1: Build a single-molecule polarization measurement module;

[0010] Step 2: Perform single-molecule imaging on the sample. Use the single-molecule polarization measurement module to split the single-molecule fluorescence into two beams and simultaneously image them on different halves of the EMCCD.

[0011] Step 3: Perform single molecule identification and center calculation on the collected single molecule images;

[0012] Step 4: Construct a conversion matrix to map the positions of single molecules in two different channels;

[0013] Step 5: Measure the single-molecule fluorescence intensity in the two channels, calculate the polarization value, and group the single-molecule localization based on the difference in polarization values;

[0014] Step 6: Calculate the single-molecule localization distribution center and MIPI localization accuracy for the grouped single-molecule localization;

[0015] Step 7: Perform super-resolution image reconstruction to obtain molecular-scale resolution images.

[0016] Furthermore, the single-molecule polarization module includes a polarization beam splitting unit and two detection channels, wherein the polarization beam splitting unit splits the single-molecule fluorescence into two beams of light with mutually perpendicular polarizations according to the polarization of the single molecule, and the two detection channels respectively introduce the two beams of light into different half-areas of the EMCCD.

[0017] According to a specific embodiment, the polarization beam splitting unit is a 50:50 polarization beam splitting cube.

[0018] In step 2, the method for performing single-molecule imaging on the sample is: performing single-molecule imaging on the sample using single-molecule localization microscopy to obtain single-molecule fluorescence.

[0019] According to a specific embodiment, in step 3, the method of performing single molecule identification and center calculation on the collected single molecule image includes:

[0020] An algorithm written in MATLAB was used to locate single molecules in the image. The method involves four steps: 1) obtaining the average intensity of all pixels in a single frame; 2) searching for pixels in the image with fluorescence intensities greater than 1.2 to 1.3 times the average intensity of the single frame; 3) centering these pixels, selecting 5x5 pixels adjacent to each pixel to obtain the fluorescence intensity within the 5x5 region; 4) calculating the average fluorescence intensity within the 5x5 region, and selecting regions with an average fluorescence intensity greater than 1.1 times the average fluorescence intensity of the single frame for the next step of calculating the center of the single molecule.

[0021] Calculation of single molecule centers:

[0022] For each located single molecule, a two-dimensional Gaussian function is used to fit its average fluorescence intensity distribution. The center of the fitted two-dimensional Gaussian function is used as the center of the single molecule. The two-dimensional Gaussian function f(x, y) used is as follows:

[0023]

[0024] Where A represents the peak height, (x0, y0) is the coordinate of the center of the Gaussian distribution, σ is the standard deviation along the X-axis and Y-axis, and (x, y) is the pixel position coordinate.

[0025] According to a specific embodiment, in step 4, the method of constructing a conversion matrix to map the positions of single molecules in two different channels includes:

[0026] The single molecule center is determined by two-dimensional Gaussian fitting, and the coordinates of n single molecule centers are as follows:

[0027] X=(x1,x2,…,xn)X′=(x1′,x2′,…,xn′)

[0028] Y=(y1,y2,…,yn)Y′=(y1′,y2′,…,yn′)

[0029] Where x1, x2, …, xn and y1, y2, …, yn are the center coordinates of a single molecule in one detection channel, x1′, x2′, …, xn′ and y1′, y2′, …, yn′ are the center coordinates of the single molecule in another detection channel, and the relationship between (X, Y) and (X′, Y′) is as follows:

[0030]

[0031] Among them, M represents the rotation matrix, f represents the scaling factor, and B represents the translation vector. M, f, and B can be determined by fitting using the MATLAB nonlinear least squares fitting function lsqcurvefit.

[0032] The constructed conversion matrix is used to map the single molecule positions of two different channels (x i ,y i )value.

[0033] According to a specific embodiment, in step five, the single-molecule fluorescence intensity in two channels is measured, the polarization value is calculated, and the single-molecule localization is grouped according to the difference in the polarization value.

[0034] The polarization value is calculated according to the following formula:

[0035]

[0036] Where P represents the polarization value, I p and I s Represent the single-molecule fluorescence intensity in the two detection channels, respectively, and g represents the system correction factor, which is used to correct the difference in photon collection efficiency between the two detection channels;

[0037] Grouping of single-molecule localization:

[0038] The polarization of each single-molecule localization in each frame was calculated, and single-molecule localizations with polarization fluctuations within 10%, position deviations less than 20 nm, and appearing in consecutive frames were defined as coming from the same fluorescent molecule, and these single-molecule localizations were grouped into the same group.

[0039] According to a specific embodiment, in step 6, the calculation of the single molecule localization distribution center is:

[0040] The single-molecule localization distribution obtained by multiple independent measurements of the target position is Gaussian. The two-dimensional Gaussian function described in formula (1) is used to fit the localization distribution from the same fluorescent molecule to obtain the center and standard deviation σ of the localization distribution. SMLM , where the nonlinear least squares fitting function lsqcurvefit of MATLAB is used for fitting;

[0041] Calculation of MIPI positioning accuracy:

[0042] MIPI positioning accuracy σ MIPI Use the following formula to calculate:

[0043]

[0044] According to one embodiment, in step seven, the method of performing super-resolution image reconstruction to obtain a molecular-scale resolution image includes:

[0045] For each fluorescent molecule, 200 data points are used to generate a σ with the center of the fluorescent molecule as the center and a standard deviation of σ. MUPI Molecular-scale resolution image of a two-dimensional Gaussian distribution.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] The present invention relates to the construction of a single-molecule polarization measurement module, single-molecule polarization calculation, and the establishment of a method for grouping single-molecule locations based on polarization. The present invention performs beam splitting based on the polarization of a single molecule. The ratio of the fluorescence intensities within the two channels after beam splitting reflects the polarization state of the fluorescent molecule. The present invention utilizes polarization measurement to distinguish adjacent single fluorescent molecules. This method can achieve a molecular-scale resolution of 2-4 nanometers. The optical super-resolution microscopy proposed in the present invention can achieve molecular-scale resolution imaging of high-density fluorescent-labeled biological samples, greatly expanding the application scenarios of molecular-scale resolution optical super-resolution microscopy and providing a more powerful imaging tool for research in the fields of biology and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a flow chart of MIPI proposed by the present invention;

[0049] Figure 2 Schematic diagram of the single-molecule polarization measurement module proposed in the present invention;

[0050] Figure 3This is the super-resolution imaging effect of the MIPI simulation proposed in the present invention. DETAILED DESCRIPTION

[0051] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0052] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they have no substantial technical significance. Any modification of the structure, change in the proportional relationship or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the efficacy and purpose that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0053] The following is a further detailed description with reference to the accompanying drawings.

[0054] A molecular-scale resolution optical super-resolution microscopy method, e.g. Figure 1 The specific steps are as follows:

[0055] Step 1: Build a single-molecule polarization measurement module.

[0056] like Figure 2 As shown, the single-molecule polarization measurement module 1 includes a 50:50 polarization beam splitter cube 11, a reflector 21, a reflector 22, a reflector 23, a lens 31 and a lens 32. The single-molecule fluorescence imaged by the single-molecule localization microscope is focused by the lens 31 and then split into two beams of light with mutually perpendicular polarizations by the 50:50 polarization beam splitter cube 11. The first beam of light is reflected by the reflector 21 and the reflector 22 and then enters one half of the EMCCD through the lens 32. The second beam of light is reflected by the reflector 23 and then enters the other half of the EMCCD through the lens 32. The polarization beam splitter cube 11 can split the fluorescence of a single molecule according to polarization. Figure 2 As shown, the arrangement order and number of the reflectors and lenses can also form two different detection channels in other ways, so that two beams of light with mutually perpendicular polarizations can pass through the two different detection channels and finally be imaged in two different half areas of the EMCCD camera.

[0057] To facilitate construction, a dual-channel imaging splitter was directly used as the basic framework, and a 50:50 polarization beam splitter cube was installed in the insert module of the imaging splitter to form a single-molecule polarization measurement module.

[0058] Principle: The fluorescence emitted by a single molecule is split into two beams with mutually perpendicular polarizations by a 50:50 polarization beam splitter cube installed in a dual-channel imaging splitter. The two beams pass through two different detection channels and are finally imaged in two different half-areas of the EMCCD camera.

[0059] Specific operation method: Use the adjustment button on the imaging separator to adjust the size and shape of the images of the two detection channels on the EMCCD camera so that the images of the two detection channels divide the entire EMCCD screen equally.

[0060] Step 2: Perform single-molecule imaging on the sample. Use the above single-molecule polarization measurement module to split the single-molecule fluorescence into two beams and simultaneously image them in different half areas of the EMCCD.

[0061] Step 3: Perform single molecule identification and center calculation on the collected single molecule images.

[0062] Single-molecule recognition:

[0063] Single molecules in images were localized using an algorithm written in MATLAB (The MathWorks, Inc., Natick, MA). The four-step process for locating single molecules was as follows: 1) obtaining the average intensity of a single frame (using all pixels); 2) searching for pixels in the image with fluorescence intensities greater than 1.2 to 1.3 times the average intensity of the single frame; 3) obtaining the fluorescence intensity of a 5x5 region centered on these pixels and surrounding each pixel within a 5x5 region; and 4) calculating the average fluorescence intensity within the 5x5 region. Only regions with an average fluorescence intensity greater than 1.1 times the average fluorescence intensity of the single frame were selected for the next step of calculating the center of the single molecule.

[0064] Calculation of single molecule centers:

[0065] For each located single molecule, a two-dimensional Gaussian function is used to fit its intensity distribution, and the center of the fitted two-dimensional Gaussian function is used as the center of the single molecule. The two-dimensional Gaussian function used is as follows:

[0066]

[0067] Where A represents the peak height, (x0, y0) are the coordinates of the center of the Gaussian distribution, and σ is the standard deviation along the X and Y axes. The fitting is performed using the MATLAB nonlinear least squares fitting function lsqcurvefit.

[0068] Step 4: Construct a conversion matrix to map the positions of single molecules in two different channels.

[0069] Single-molecule polarization measurements require alignment of the positions of the same single molecule after imaging through two different detection channels. The single-molecule center is determined by two-dimensional Gaussian fitting, and the coordinates of the n single-molecule centers are summarized as follows:

[0070] X=(x1,x2,…,xn)X′=(x1′,x2′,…,xn′)

[0071] Y=(y1,y2,…,yn)Y′=(y1′,y2′,…,yn′)

[0072] Where x1, x2, …, xn and y1, y2, …, yn are the center coordinates of a single molecule in one detection channel, x1′, x2′, …, xn′ and y1′, y2′, …, yn′ are the center coordinates of the single molecule in another detection channel, and the relationship between (X, Y) and (X′, Y′) is as follows:

[0073]

[0074] Where M is the rotation matrix, f is the scaling factor, and B is the translation vector. We can determine M, f, and B by fitting using the MATLAB nonlinear least squares function lsqcurvefit.

[0075] The constructed conversion matrix is used to map the single molecule positions of two different channels (x i ,y i )value.

[0076] The expected (x j ,y j ) value and the experimentally measured (x i ,y i ) values, and the average of the differences in the X and Y axes is calculated as the alignment accuracy in the X and Y axes. The alignment accuracy can reach several nanometers.

[0077] Step 5: Measure the single-molecule fluorescence intensity in the two channels, calculate the polarization value, and group the single-molecule localization based on the difference in polarization values.

[0078] The polarization value is calculated according to the following formula:

[0079]

[0080] Where P represents the polarization value, I p and I sRepresents the single molecule fluorescence intensity in the two detection channels, and g represents the system correction factor, which is used to correct the difference in photon collection efficiency between the two detection channels. In order to measure g, the light emitted by the fluorescent molecules needs to be unpolarized light, ensuring that the fluorescence intensity entering the two detection channels is the same when the fluorescence passes through the polarization beam splitter cube. To simulate this condition, we use TetraSpeck suspended in 92% glycerol. TM The fluorescent beads can be rotated rapidly on the scale of exposure time (10 milliseconds) to measure their fluorescence intensity in two detection channels.

[0081] Grouping of single-molecule localization:

[0082] The polarization of each single-molecule localization in each frame was calculated, and single-molecule localizations with polarization fluctuations within 10%, position deviations less than 20 nm, and appearing in consecutive frames were defined as coming from the same fluorescent molecule, and these single-molecule localizations were grouped into the same group.

[0083] Step 6: Calculate the single-molecule positioning distribution center and MIPI positioning accuracy for the grouped single-molecule positioning.

[0084] Calculation of single-molecule localization distribution center:

[0085] The single-molecule localization distribution obtained from multiple independent measurements of the target position is Gaussian. Therefore, we use a two-dimensional Gaussian function (Formula 1) to fit the localization distribution from the same fluorescent molecule to obtain the center and standard deviation (σ SMLM Here, we use a two-dimensional Gaussian function fitting method rather than a weighted average method. This method is simpler and avoids calculating the localization accuracy of each single molecule, while still achieving the same or better resolution. The fitting is performed using the MATLAB nonlinear least squares fitting function lsqcurvefit.

[0086] Calculation of MIPI positioning accuracy:

[0087] MIPI positioning accuracy (σ MIPI ) is calculated using the following formula:

[0088]

[0089] Step 7: Perform super-resolution image reconstruction to obtain molecular-scale resolution images.

[0090] Reconstruction of MIPI images:

[0091] For each fluorescent molecule, 200 data points are used to generate a σ with the center of the fluorescent molecule as the center and a standard deviation of σ. MIPI Two-dimensional Gaussian distribution.

[0092] Step 8: Evaluation of MIPI resolution.

[0093] On the reconstructed molecular scale resolution image, the discrete structures or vertical linear structures are marked, the intensity distribution profile along the marked line is obtained, and the distance between the discrete structures or the half-height full width of the linear structure is measured as the resolution of MIPI, and compared with the theoretical resolution of 2.35xσ MIPI Compare.

[0094] MIPI resolution effect of numerical simulation ( Figure 3 ):

[0095] Numerical simulation setup: Fluorescent molecules were arranged in a 4×4 square matrix, with a distance of 4 nm between adjacent fluorescent molecules. The number of localizations per single molecule was 300, with a localization accuracy of 10 nm. A two-dimensional Gaussian distribution was generated using the MATLAB mvnrnd function. Single-molecule distribution fitting: The center of the single-molecule distribution was calculated using the method described in "Specific Implementation Methods."

Claims

1. A molecular scale resolution optical super-resolution microscopy method, characterized in that: include: Step 1: Build a single-molecule polarization measurement module; Step 2: Perform single-molecule imaging on the sample. Use the single-molecule polarization measurement module to split the single-molecule fluorescence into two beams and simultaneously image them on different halves of the EMCCD. Step 3: Perform single molecule identification and center calculation on the collected single molecule images; Step 4: Construct a conversion matrix to map the positions of single molecules in two different channels; Step 5: Measure the single-molecule fluorescence intensity in the two channels, calculate the polarization value, and group the single-molecule localization based on the difference in polarization values; Step 6: Calculate the single-molecule localization distribution center and MIPI localization accuracy for the grouped single-molecule localization; Step 7: Perform super-resolution image reconstruction to obtain molecular-scale resolution images.

2. The molecular scale resolution optical super-resolution microscopy method according to claim 1, characterized in that: The single-molecule polarization module includes a polarization beam splitting unit and two detection channels, wherein the polarization beam splitting unit splits the single-molecule fluorescence into two beams of light with mutually perpendicular polarizations according to the polarization of the single molecule, and the two detection channels respectively introduce the two beams of light into different half-areas of the EMCCD.

3. The molecular scale resolution optical super-resolution microscopy method according to claim 2, characterized in that: The polarization beam splitting unit is a 50:50 polarization beam splitting cube.

4. The molecular scale resolution optical super-resolution microscopy method according to claim 1, characterized in that: In step 2, the method for performing single-molecule imaging on the sample is: performing single-molecule imaging on the sample using single-molecule localization microscopy to obtain single-molecule fluorescence.

5. The molecular scale resolution optical super-resolution microscopy method according to claim 1, characterized in that: In step 3, the method for performing single molecule identification and center calculation on the collected single molecule image includes: An algorithm written in MATLAB was used to locate single molecules in the image. The process involved four steps: 1) obtaining the average intensity of all pixels in a single frame; 2) finding pixels in the image with fluorescence intensities 1.2-1.3 times the average intensity of the single frame; 3) centering these pixels, selecting 5x5 pixels adjacent to each pixel to obtain the fluorescence intensity within the 5x5 region; 4) calculating the average fluorescence intensity within the 5x5 region, and selecting regions with an average fluorescence intensity greater than 1.1 times the average fluorescence intensity of the single frame for the next step of calculating the center of the single molecule. Calculation of single molecule centers: For each located single molecule, a two-dimensional Gaussian function is used to fit its average fluorescence intensity distribution. The center of the fitted two-dimensional Gaussian function is used as the center of the single molecule. The two-dimensional Gaussian function f(x, y) used is as follows: Where A represents the peak height, (x0, y0) is the coordinate of the center of the Gaussian distribution, σ is the standard deviation along the X-axis and Y-axis, and (x, y) is the pixel position coordinate.

6. The molecular scale resolution optical super-resolution microscopy method according to claim 1, characterized in that: In step 4, the method of constructing a conversion matrix to map the positions of single molecules in two different channels includes: The single molecule center is determined by two-dimensional Gaussian fitting, and the coordinates of n single molecule centers are as follows: X=(x1,x2,…,xn)X′=(x1′,x2′,…,xn′) Y=(y1,y2,…,yn)Y′=(y1′,y2′,…,yn′) Where x1, x2, …, xn and y1, y2, …, yn are the center coordinates of a single molecule in one detection channel, x1′, x2′, …, xn′ and y1′, y2′, …, yn′ are the center coordinates of the single molecule in another detection channel, and the relationship between (X, Y) and (X′, Y′) is as follows: Among them, M represents the rotation matrix, f represents the scaling factor, and B represents the translation vector. M, f, and B can be determined by fitting using the MATLAB nonlinear least squares fitting function lsqcurvefit. The constructed conversion matrix is used to map the single molecule positions of two different channels (x i ,y i )value.

7. The molecular scale resolution optical super-resolution microscopy method according to claim 1, characterized in that: In step five, the single-molecule fluorescence intensity in the two channels is measured, the polarization value is calculated, and the single-molecule localization is grouped according to the difference in polarization values. The polarization value is calculated according to the following formula: Where P represents the polarization value, I p and I s Represent the single-molecule fluorescence intensity in the two detection channels, respectively, and g represents the system correction factor, which is used to correct the difference in photon collection efficiency between the two detection channels; Grouping of single-molecule localization: The polarization of each single-molecule localization in each frame was calculated, and single-molecule localizations with polarization fluctuations within 10%, position deviations less than 20 nm, and appearing in consecutive frames were defined as coming from the same fluorescent molecule, and these single-molecule localizations were grouped into the same group.

8. The molecular scale resolution optical super-resolution microscopy method according to claim 5, characterized in that: In step six, Calculation of single-molecule localization distribution center: The single-molecule localization distribution obtained by multiple independent measurements of the target position is Gaussian. The two-dimensional Gaussian function described in formula (1) is used to fit the localization distribution from the same fluorescent molecule to obtain the center and standard deviation σ of the localization distribution. SMLM , where the nonlinear least squares fitting function lsqcurvefit of MATLAB is used for fitting; Calculation of MIPI positioning accuracy: MIPI positioning accuracy σ MIPI Use the following formula to calculate:

9. The molecular scale resolution optical super-resolution microscopy method according to claim 8, characterized in that: In step seven, the method for performing super-resolution image reconstruction to obtain a molecular-scale resolution image includes: For each fluorescent molecule, 200 data points are used to generate a σ with the center of the fluorescent molecule as the center and a standard deviation of σ. MIPI Molecular-scale resolution image of a two-dimensional Gaussian distribution.

10. The molecular scale resolution optical super-resolution microscopy method according to claim 2, characterized in that: The sample is a high-density fluorescent-labeled sample.

Images