A method of molecular resolution microscopy based on two-color single molecule localization

Abstract

The application discloses a kind of molecular level resolution microscopy MITI (Molecular-level Imaging based on Two-color Imaging) based on two-color single molecule positioning. The application uses the method of two-color imaging, and the positioning points of the originally indistinguishable fluorescent molecules are successfully distinguished by the two-color method, and the different positioning point sets are distributed into the corresponding fluorescent channels to be distinguished, and then the center position of the fluorescent molecule positioning point distribution and the positioning accuracy are improved again by fitting the distribution of the fluorescent molecule positioning points again, so as to improve the resolution. The application can achieve a spatial resolution of up to 2 nm, providing an important means for analyzing molecular-scale biological structures and interaction processes.

Description

Technical Field

[0001] This invention belongs to the field of super-resolution microscopy, specifically relating to a molecular-level resolution microscopy method based on two-color single-molecule localization. Background Technology

[0002] Over the past two decades, super-resolution microscopy has become a key tool for revealing complex biological structures and processes, and has seen widespread development and application. However, while traditional single-molecule localization techniques can break the optical diffraction limit, their resolution is still limited to scales below 10 nm. SMLM (single-molecule localization microscopy) is insufficient for resolving complex biological processes at the molecular scale, i.e., several nanometer scales, such as interactions between biomolecules and proteins. In recent years, to meet the requirement of resolving biological processes at 10 nm, several studies have proposed different methods to improve the resolution of optical microscopes to several nanometers, ushering in the era of nanomicroscopy.

[0003] DNA-PIANT (DNA point accumulation in nanoscale topography) sequence imaging has been highly effective in distinguishing the distribution of two fluorescent molecules, achieving angstrom-level localization accuracy. However, DNA-PIANT requires the use of single-stranded DNA encoding modified molecules to access the target site of interest. This labeling method is more challenging than directly labeling the target site with fluorescent molecules, and not all biomolecules are suitable for DNA oligonucleotide labeling. Furthermore, DNA oligonucleotides do not cross cell membranes, and the imaging solution needs to be constantly replaced during imaging to maintain the number of fluorescent molecules. Most importantly, using DNA-PIANT sequence imaging to distinguish the localization distribution of fluorescent molecules requires constant replacement and washing of the imaging solution, increasing imaging time and potentially causing more severe drift. Therefore, this method presents an extremely high challenge for imaging at the molecular scale.

[0004] Many methods have improved resolution by combining them with single-molecule localization. Numerous microscopy techniques, such as combining structured light illumination with single-molecule localization, have effectively improved the localization accuracy of SMLMs, enabling them to achieve resolutions in the 1-5 nm range. However, achieving even higher resolutions requires more complex optical and control systems or more sophisticated labeling methods.

[0005] Therefore, how to achieve true molecular-level resolution imaging based on traditional single-molecule localization imaging systems remains a challenging and urgent scientific problem to be solved. Summary of the Invention

[0006] To address the problems of the existing technologies mentioned above, this invention proposes a molecular-level resolution microscopy based on two-color single-molecule localization. Without introducing an exceptionally complex optical system or special marking methods, the microscopic capabilities are enhanced to the molecular level through two-color single-molecule localization technology.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A molecular-level resolution microscopy method based on two-color single-molecule localization, which uses two-color imaging to further improve resolution based on the traditional single-molecule localization method SMLM, is characterized by the following specific steps:

[0009] 1) Two-color marking of the target object:

[0010] To achieve two-color imaging, two different fluorescent molecules must be used to label the target. These need to be fluorescent molecules suitable for use in SMLM (Simultaneous Microwave Imaging), including typical fluorescent dyes such as Cy5, Cy3, and Alexa 647; and light-controlled fluorescent proteins such as PcStar, PAmCherry1, and ffDronpa. The target is labeled with either the fluorescent dye or the fluorescent protein at both ends, ensuring that the two fluorescent molecules have different excitation and emission wavelengths.

[0011] 2) Two-color single-molecule localization super-resolution imaging

[0012] After obtaining the two-color labeled sample, signals from two fluorescent molecules are collected using a single-molecule-based super-resolution optical microscope (SMILE). SMILE-based SMILE microscopes are typically equipped with four laser wavelengths: 405 nm, 488 nm, 561 nm, and 647 nm, to activate and excite most types of fluorescent molecules. Two-color imaging can be performed in two modes: parallel dual-channel imaging and sequential fluorescence imaging. In parallel dual-channel imaging, the two corresponding excitation lasers are activated simultaneously, and a lower-intensity 405 nm laser is used to make the molecular densities in the two channels as close as possible. Signals from both channels are collected simultaneously for subsequent processing. In sequential imaging, the fluorescence signal at the longer excitation wavelength is collected first, followed by the fluorescence signal at the shorter wavelength, while a low-intensity 405 nm laser is used to control the fluorescent molecule density during imaging. Sufficient fluorescence signals are collected for subsequent processing.

[0013] 3) MITI Implementation Method

[0014] After obtaining signals from two fluorescent molecules, super-resolution data for single-molecule localization was obtained using standard SMLM data analysis methods. This included the coordinates of the localization points of the fluorescent molecules, their intensity, and localization accuracy. Two-color imaging was then used to distinguish the localization information of adjacent, previously indistinguishable fluorescent molecules. Furthermore, by fitting the positional information of the fluorescent molecules in the two fluorescent molecule channels separately, the center position of the fluorescent molecule was obtained. The final MITI localization accuracy is determined by both the original SMLM localization accuracy and the number of localization points of the fluorescent molecules.

[0015] Since the location distribution of fluorescent molecules largely conforms to a Gaussian distribution, a Gaussian function is used to fit this distribution. The parameters of the Gaussian function are derived from the coordinates of the point set and the localization accuracy. The Gaussian function takes the following form:

[0016]

[0017] Where (x0, y0) are the coordinates of the center point of the Gaussian distribution, A in this function can be considered a constant, B is the background intensity, and considering that there is no background when fitting the center of the localization point, B can be taken as 0, σ 2 This represents the variance of the Gaussian function in the x and y directions. We fit the Gaussian function using maximum likelihood estimation to obtain the center coordinates of the localization point set corresponding to the MITI image. The final localization accuracy of the MITI image can be expressed by the following relationship: Decision, in which σ SMLM This represents the localization accuracy of single-molecule localization imaging, where K is the total number of localization points. Fitting images from two channels using this method improves localization accuracy; the final localization accuracy is directly proportional to the number of localization points, K.

[0018] Finally, the resolution-enhanced images from the two channels are merged to obtain a molecular-level resolution image, thus achieving MITI imaging.

[0019] After obtaining the fluorescent molecular signals of the two channels in step 2), the first step is to align the two fluorescent molecular channels. A 100nm fluorescent microsphere is used to image each of the two channels, and the position of the fluorescent microsphere is moved with an appropriate step size (determined by the imaging area) to fill the entire imaging region. The center position of the fluorescent microsphere is extracted, and a correction matrix between the two channels is established and applied to the subsequent alignment of the fluorescent channels.

[0020] The method for obtaining the single-molecule point localization accuracy in step 3) is usually obtained by fitting the point spread function of the diffraction-limited image with a Gaussian distribution using the least squares method, where the root mean square error is the localization accuracy of the single-molecule point, and the relationship between the localization accuracy of the SMLM and the point spread function of the diffraction-limited function is given by equation (2):

[0021]

[0022] Where s is the standard deviation of the point spread function of the diffraction-limited image, a is the pixel size in the image (considering the system magnification), N is the total number of photons measured from the fluorescent molecule, and b is the number of background photons collected from the fluorescent molecule. Therefore, in order to obtain higher localization accuracy of SMLM, when designing SMLM experiments, the highest possible diffraction-limited resolution and photon collection efficiency should be achieved, while minimizing background noise.

[0023] Compared with the prior art, the advantages of the present invention are:

[0024] This invention utilizes a traditional single-molecule localization super-resolution microscopy system, combining dual-color imaging technology with single-molecule localization imaging to distinguish fluorescent molecules with different emission wavelengths through two imaging channels. By fitting the distribution of localization points of the two fluorescent molecules separately, the center position and localization accuracy (MITI) of the fluorescent molecule localization points are obtained. The localization accuracy of the fitted MITI is further improved compared to that of SMLM images, increasing the imaging resolution to a maximum of 2 nm, achieving molecular-level resolution. This is used to resolve the structures and interactions of biomolecules below 10 nm. Furthermore, this method does not require the introduction of complex optical systems or special labeling methods, enabling molecular-level resolution on existing single-molecule localization super-resolution microscopy systems. It significantly improves the spatial resolution of single-molecule localization-based super-resolution microscopy. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the molecular-level resolution microscopy method MITI based on two-color SMLM of the present invention.

[0026] Figure 2 This is a simulation diagram illustrating the relationship between the number of fluorescent molecules and the resolution of MITI according to the present invention.

[0027] Figure 3 An example of the MITI microscopy according to the present invention is an MITI image of single-stranded DNA labeled at both ends. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0029] like Figure 1As shown, a molecular-level resolution microscopy method based on two-color single-molecule localization includes first designing two fluorescent molecules to label both ends of the target analyte, wherein the distance between the two different fluorescent molecules is 2≤2.35σ. SMLM This distance is indistinguishable by SMLM. To verify whether MITI can distinguish them, target objects labeled with different fluorescent molecules at both ends are then arranged laterally, where distance 1 is the distance from fluorescent molecule 1 of one target object to fluorescent molecule 1 of another target object, and distance 1 > 2.35σ. SMLM At this point, two targets with fluorescent molecules arranged in this way are imaged. Channel 1 collects the signal of fluorescent molecule 1, and channel 2 collects the signal of fluorescent molecule 2. Due to the resolution limitations of single-molecule localization super-resolution imaging, when distance 1 is designed to be 40 nm, the localization points of two adjacent fluorescent molecules 1 can be distinguished and will not be indistinguishable due to overlap. Similarly, when the distance between two fluorescent molecules 2 is 40 nm, the localization points of the two adjacent fluorescent molecules can be distinguished. However, it can be imagined that if channel 1 and channel 2 are not imaged separately, the localization points of fluorescent molecules 1 and 2 at distance 2 will overlap and cannot be distinguished. After generating the corresponding SMLM images from the histogram distribution of these points in each of the two channels, the center position of these localization points can be fitted to obtain the center position of the fluorescent molecules in the final MITI image. Since the center position and localization accuracy of the fluorescent molecules are obtained by refitting after distinguishing the localization points of the fluorescent molecules, the localization accuracy of the original SMLM is further improved to the localization accuracy of MITI, which can significantly improve the localization accuracy of the image. After obtaining MITI images in two separate channels, the MITI images from the two channels are merged to obtain a higher resolution MITI image. The MITI image can resolve distances as small as 2 nm, which SMLM cannot resolve. This method, which assigns signals from different fluorescent molecules to different channels, fits them individually to improve localization accuracy, and finally merges them to obtain a higher-precision image, successfully improves the imaging resolution of dual-color single-molecule localization to the molecular level.

[0030] To illustrate the relationship between the number of localizations K and the resolution of the MITI image, based on Figure 2 The simulation results are visible.

[0031] exist Figure 2 When K=1, it is a super-resolution image of traditional SMLM. According to... Figure 1The principle of the MITI method is explained by assigning different fluorescent molecule signals to different channels for analysis. First, the distance between two adjacent identical fluorescent molecules is set to 30 nm, which is the limit of resolution for a traditional SMLM super-resolution image. Then, the distance between two adjacent different fluorescent molecules is set to 2 nm, which is the molecular-level resolution that MITI aims to achieve. The bottom image shows the image after merging the two channels. When K=1, the images of the two channels almost completely overlap and are indistinguishable. When the localization number K is increased to 50, the resolution of the fluorescent molecule images in the two channels is significantly improved. The 30 nm distance, which could barely be separated under traditional SMLM resolution, can now be completely separated, but the merged image of the two channels still largely overlaps and is indistinguishable. When the localization number K is increased again to 100, the fluorescent molecules in the merged two channels begin to show distinguishable outlines, but in reality, there is still a large amount of overlap, making them indistinguishable. When the localization number K is increased to 150, the fluorescent molecules in the merged two channels can be completely distinguished, proving that when the localization number is increased to above 150, the imaging resolution of MITI can reach 2 nm. The relationship between the localization number K and the MITI resolution satisfies the formula:

[0032]

[0033] Therefore, the simulation results provide a theoretical basis for the relationship between the number of localizations K and the resolution of MITI, and also confirm that... Figure 1 The proposed approach is correct.

[0034] according to Figure 1 and Figure 2 The proposed ideas and theoretical verification, in Figure 3 In this study, a single-stranded DNA sample labeled with fluorescent dyes Cy3 and Cy5, resolving at both ends, was designed. The single-stranded DNA was 6 bases (bp) long. Given that one base is approximately 0.314 nm long, a 6 bp single-stranded DNA sample is approximately 2 nm long. First, wide-field imaging of the sample yielded a diffraction-limited image, clearly showing that the wide-field microscopy could not resolve the 6 bp single-stranded DNA sample. Further, single-molecule localization reconstruction was used to obtain a stochastic optical microscopy (STORM) super-resolution image. At a resolution of 30 nm, it was also clearly impossible to resolve the sample information, as the two fluorescent molecules completely overlapped. Finally, based on the present invention, molecular-level resolution microscopy based on dual-color single-molecule localization (MITI), the 6 bp sample (approximately 2 nm long), Cy3-6bp-Cy5, was successfully resolved in the MITI image.

[0035] The specific steps for imaging the Cy3-6 bp-Cy5 sample are as follows:

[0036] Since STORM super-resolution images are reconstructed from many frames of wide-field images through single-molecule localization, MITI is an image enhancement image obtained by refitting the set of localization points of fluorescent molecules in the single-molecule localization super-resolution image. Therefore, the imaging process required by the MITI method is consistent with that of STORM.

[0037] First, a STORM imaging buffer solution must be prepared before each imaging session.

[0038] After diluting the sample to an appropriate concentration, it was mixed with STORM imaging buffer, and fluorescent microspheres of a certain concentration were added to prepare for the subsequent drift correction process.

[0039] In dual-color imaging, time-series imaging was used, employing a 647nm laser to excite Cy5 fluorescent molecules while simultaneously using a low-intensity 405nm laser to maintain Cy5 fluorescent molecules at a suitable density.

[0040] After collecting the signal from Cy5 fluorescent molecules, the 647nm laser was turned off, and the Cy3 fluorescent molecules were excited using a 561nm laser. At the same time, a low concentration of 405nm laser was used to maintain the Cy3 fluorescent molecules at a suitable concentration.

[0041] After collecting sufficient fluorescent molecule signals from both channels, STORM image reconstruction can be performed to obtain the coordinate information, fluorescence intensity information, and localization accuracy information of the fluorescent molecules in the corresponding channels.

[0042] First, fluorescent beads are used to correct the color difference between the two channels and then the channels are aligned.

[0043] After color difference correction is completed, the positional changes of the fluorescent spheres in each frame are analyzed, and the fluorescent spheres are used as a standard for drift correction.

[0044] Note that drift correction is even more important for sequential imaging due to the longer imaging time required. Therefore, dual-color parallel imaging is preferred when performing MITI imaging.

[0045] After color difference correction and drift correction, the coordinate information of fluorescent molecules is extracted and the center position of the fluorescent molecule positioning point set is fitted using the least squares method with a Gaussian function. The positioning accuracy of the MITI image is obtained by the relationship between the number of positioning points K and the positioning accuracy of MITI. Finally, the fluorescent molecule information of the two channels is reconstructed and merged to obtain the MITI image.

[0046] Finally, it should be noted that the purpose of disclosing the embodiments is to help further understand the present invention. However, those skilled in the art will understand that various substitutions and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the present invention should not be limited to the content disclosed in the embodiments, and the scope of protection of the present invention is defined by the claims.

Claims

1. A molecular-level resolution microscopy method based on two-color single-molecule localization, characterized in that: Includes the following steps: S1. Dual-color labeling of target: The target is labeled using two different fluorescent molecules, which have different excitation and emission bands and are suitable for single-molecule localization microscopy; S2. Two-color single-molecule localization super-resolution imaging: Using a super-resolution optical microscope equipped with at least four wavelength lasers, two-color labeled samples are imaged and the signals of two fluorescent molecules are collected. The imaging methods include dual-channel parallel imaging or sequential imaging. S3. MITI Implementation Method: The localization information of fluorescent molecules is obtained through standard single-molecule localization microscopy data analysis methods, including the coordinates of the localization point, the intensity of the fluorescent molecule, and the localization accuracy. The localization information of adjacent fluorescent molecules is distinguished by dual-color imaging. The position information of fluorescent molecules in two fluorescent molecule channels is fitted separately to obtain the center position of the fluorescent molecule, thereby achieving molecular-level resolution MITI imaging. The localization accuracy of single-molecule points is optimized by fitting the diffraction-limited image point diffusion function with a Gaussian distribution. The positioning information mentioned in step S3 also includes the distribution information of the positioning points of fluorescent molecules. The position distribution of the positioning points of fluorescent molecules is fitted by a Gaussian function, and the center coordinates of the positioning point set corresponding to the MITI image are obtained by the maximum likelihood estimation method. The positioning accuracy optimization method includes fitting the Gaussian-distributed point spread function of the diffraction-limited image using the least squares method, achieving a positioning accuracy that satisfies... The relationship is given by K, where K is the total number of localization sites for the same fluorescent molecule.

2. The molecular-level resolution microscopy method based on two-color single-molecule localization according to claim 1, characterized in that, It also includes channel alignment and positioning accuracy optimization: before performing MITI imaging, fluorescent microspheres of known size are used to image the two channels separately, and a correction matrix is ​​established to achieve precise alignment between the two fluorescent molecule channels.

3. The molecular-level resolution microscopy method based on two-color single-molecule localization according to claim 1, characterized in that, The two different fluorescent molecules mentioned in step S1 are selected from fluorescent dyes Cy3, Cy5, Alexa 647, or light-controlled fluorescent proteins PcStar, PAmCherry1, and ffDronpa.

4. The molecular-level resolution microscopy method based on two-color single-molecule localization according to claim 1, characterized in that, The super-resolution optical microscope described in step S2 is equipped with four wavelength lasers of 405 nm, 488 nm, 561 nm, and 647 nm, which are used to activate and excite different types of fluorescent molecules.

5. The molecular-level resolution microscopy method based on two-color single-molecule localization according to claim 2, characterized in that, The fluorescent microspheres are 100 nm in size. By moving the positions of the fluorescent microspheres to fill the entire imaging area, the center position of the fluorescent microspheres is extracted, and a correction matrix between the two channels is established.

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