WO2020037527A1 - Super-resolution imaging method and apparatus, and terminal device - Google Patents

Abstract

A super-resolution imaging method and apparatus, and a terminal device. The method comprises: exciting a fluorescent probe having a dual emission peak on a biological sample by means of an excitation light, and acting an affinity reagent onto the excited fluorescent probe (S101); irradiating, by means of an activated light, the fluorescent probe acted by the affinity reagent to obtain a dual-channel fluorescence intensity image (S102); respectively acquiring a short wavelength signal having a short wavelength emitting peak and a long wavelength signal having a long wavelength emitting peak in the dual-channel fluorescence intensity image (S103); selecting a fluorescence intensity image of the short wavelength signal and that of the long wavelength signal from the same frame of dual-channel fluorescence intensity image according to the respectively acquired short wavelength signal and long wavelength signal, and respectively calculating a fluorescence intensity ratio of the images to obtain N proportional fluorescence images, N being a positive integer (S104); reconstructing the proportional fluorescence image to obtain a super-resolution image by means of a STORM super-resolution imaging method (S105); and coloring the super-resolution image according to the N proportional fluorescence images to obtain a proportional super-resolution image (S106). According to the method, the proportional super-resolution image can be obtained for reflecting sample parameters at a fluorescent probe mark in the biological sample.

Description

Super-resolution imaging method, device and terminal equipment Technical field

The present invention relates to the field of image processing technology, and in particular, to a super-resolution imaging method, device, and terminal device.

Background technique

Fluorescence microscopy is widely used in cell microbiology imaging. Among them, super-resolution localized imaging is a representative super-resolution fluorescence imaging technology. This technology combines single-molecule imaging with high-precision molecular positioning algorithms on the basis of an optical reconstruction microscope to achieve an ultra-high spatial resolution of 20-30nm to observe the ultrastructure of cells. The key to this technique is to use an excited fluorescent probe to randomly bind to the affinity reagent in the surrounding environment, causing its fluorescence to decay into the dark state, and then using another wavelength or activating light of the same wavelength to make the affinity reagent from the fluorescent molecule The random drop off has the ability to emit light again, so that the fluorescent molecules change randomly between luminous and dark states, and then continuously record multiple frames of fluorescent molecules, perform a single molecule positioning algorithm to determine the center position, and finally reconstruct super-resolution fluorescence from the image image.

However, at present, optical reconstruction microscopes can only perform super-resolution imaging on the structure of biological samples, and cannot perform functional super-resolution imaging or super-resolution imaging research on quantification of sample parameters.

Summary of invention

technical problem

In the prior art, an optical reconstruction microscope can only perform super-resolution imaging on the structure of a biological sample, and cannot perform functional super-resolution imaging or super-resolution imaging research on quantification of sample parameters.

Problem solution

Technical solutions

A first aspect of the embodiments of the present invention provides a super-resolution imaging method, including:

Fluorescent probes with double emission peaks on biological samples are excited by excitation light, and affinity reagents are applied to the excited fluorescent probes;

By activating light and irradiating the fluorescent probe after the action of the affinity reagent, a dual-channel fluorescence intensity image is formed;

Respectively collecting a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the dual-channel fluorescence intensity image;

Select N frames of two-channel fluorescence intensity images, and select the fluorescence intensity images of the short-wavelength signals in the two-channel fluorescence intensity images of the same frame according to the separately acquired short-wavelength signals and the long-wavelength signals. And the fluorescence intensity image of the long-wavelength signal, and calculating the fluorescence intensity ratios thereof, to obtain N proportional fluorescence images, where N is a positive integer;

STORM (Stochastic Optical Reconstruction Microscopy, Random Optical Reconstruction Microscope) super-resolution imaging method is used to reconstruct the proportional fluorescence image to obtain a super-resolution image;

Color the super-resolution image according to N number of the proportional fluorescence images to obtain a proportional-type super-resolution image.

With reference to the first aspect of the present invention, in a first embodiment of the first aspect of the present invention, the super-resolution imaging method further includes:

Establishing a function model of the environmental parameter of the fluorescent probe and the ratio of the fluorescent intensity;

The establishing a function model of the environmental parameter of the fluorescent probe and the ratio of the fluorescent intensity includes:

Placing the fluorescent probe with a double emission peak in a probe solution test environment, and exciting the fluorescent probe through excitation light;

Changing a first parameter of the test environment of the probe solution to obtain a steady-state fluorescence emission spectrum of the fluorescent probe under different first parameters;

According to the double emission peak in the steady-state fluorescence emission spectrum, a fluorescence intensity test ratio is calculated, and a function model of the first parameter and the fluorescence intensity test ratio is established.

With reference to the first aspect of the present invention and the second embodiment of the first aspect of the present invention, the forming a dual-channel fluorescence intensity image by activating the light and irradiating the fluorescent probe after the action of the affinity reagent includes:

Obtaining the short-wavelength signal according to a first structure in the fluorescent probe;

Obtaining the long-wavelength signal according to a second structure in the fluorescent probe;

When the excitation light irradiates the fluorescent probe, the first structure or the second structure emits fluorescence;

When the activating light acts on the fluorescent probe after the affinity reagent acts, the first structure and the second structure alternately emit light according to the binding state with the affinity reagent to form the dual-channel fluorescence intensity image.

With reference to the second embodiment of the first aspect of the present invention, in the third embodiment of the first aspect of the present invention, the dual-channel fluorescence intensity image includes a fluorescence intensity image of a short-wavelength signal and a fluorescence intensity image of a long-wavelength signal;

The fluorescence intensity image of the short-wavelength signal corresponds to the light emission process of the first structure in the fluorescent probe;

The fluorescence intensity image of the long-wavelength signal corresponds to the light emission process of the second structure in the fluorescent probe.

With reference to the first aspect of the present invention, in a fourth embodiment of the first aspect of the present invention, in the acquiring the two-channel fluorescence intensity images, the short-wavelength signal of the short-wavelength emission peak and the long-wavelength signal of the long-wavelength emission peak include :

Separating the excitation light from the short-wavelength signal and the long-wavelength signal through a first dichroic mirror;

After the short-wavelength signal and the long-wavelength signal are separated by a second dichroic mirror, the short-wavelength signal is sent to a reflecting mirror through a first filter, and the reflecting mirror transmits the filtered short-wavelength signal to A first EMCCD; the long-wavelength signal passes through a second filter, and the filtered long-wavelength signal is transmitted to the first EMCCD, thereby collecting the dual-channel fluorescence intensity image. ;

Different regions of the first EMCCD receive the filtered short-wavelength signal and the filtered long-wavelength signal, respectively.

With reference to the first aspect of the present invention, in a fifth embodiment of the first aspect of the present invention, the separately collecting a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the fluorescence emission spectrum includes:

Separating the excitation light from the short-wavelength signal and the long-wavelength signal through a first dichroic mirror;

After the short-wavelength signal and the long-wavelength signal are separated by a second dichroic mirror, the short-wavelength signal is sent to a reflecting mirror through a first filter, and the reflecting mirror transmits the filtered short-wavelength signal to A first EMCCD; the long-wavelength signal passes through a second filter, and the filtered long-wavelength signal is transmitted to a second EMCCD;

The first EMCCD and the second EMCCD respectively receive the filtered short-wavelength signal and the filtered long-wavelength signal, thereby collecting the dual-channel fluorescence intensity image. .

With reference to the first to fifth embodiments of the first aspect of the present invention, in the sixth embodiment of the first aspect of the present invention, the selected N-frame two-channel fluorescence intensity images are obtained according to the separately acquired short-wavelength signals and For the long-wavelength signal, in the two-channel fluorescence intensity image of the same frame, the fluorescence intensity image of the short-wavelength signal and the fluorescence intensity image of the long-wavelength signal are selected, and the fluorescence intensity ratios thereof are calculated to obtain N Ratio fluorescent images, where N is a positive integer, including:

Selecting the N-th fluorescence intensity image of the short-wavelength signal according to the dual-channel fluorescence intensity image and the separately collected short-wavelength signal, and calculating the emission peak fluorescence intensity thereof;

Selecting the Nth long-wavelength signal fluorescence intensity image according to the two-channel fluorescence intensity image and the separately acquired long-wavelength signal, and calculating the emission peak fluorescence intensity thereof;

The calculation formula for calculating the fluorescence intensity ratio is:

,

Wherein, IBN is the emission peak fluorescence intensity of the long-wavelength signal, and IAN is the emission peak fluorescence intensity of the short-wavelength signal.

A second aspect of the present invention provides a super-resolution imaging device, including:

An excitation module for exciting a fluorescent probe having a double emission peak on a biological sample by using excitation light, and applying an affinity reagent to the excited fluorescent probe;

A fluorescence intensity image acquisition module, configured to obtain a dual-channel fluorescence intensity image by activating light and irradiating a fluorescent probe after the action of an affinity reagent;

A signal acquisition module configured to separately acquire a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the dual-channel fluorescence intensity image;

A proportional fluorescence image calculation module is configured to select N frames of two-channel fluorescence intensity images, and select the selected one of the two-channel fluorescence intensity images in the same frame according to the separately acquired short-wavelength signals and the long-wavelength signals. The fluorescence intensity image of the short-wavelength signal and the fluorescence intensity image of the long-wavelength signal, and respectively calculating a fluorescence intensity ratio thereof to obtain N proportional fluorescence images, where N is a positive integer, where N is a positive integer;

An image reconstruction module, configured to reconstruct the proportional fluorescence image by using the STORM super-resolution imaging method to obtain a super-resolution image;

An image acquisition module is configured to color the super-resolution image according to N of the proportional fluorescence images to obtain a proportional-type super-resolution image.

A third aspect of the embodiments of the present invention provides a terminal device, including a memory, a processor, and a computer program stored in the memory and executable on the processor. When the processor executes the computer program, the first implementation is implemented as above. Aspects of the provided method steps.

A fourth aspect of the embodiments of the present invention provides a computer-readable storage medium. The computer-readable storage medium stores a computer program. When the computer program is executed by a processor, the steps of the method provided by the first aspect are implemented. .

The beneficial effects of the invention

Beneficial effect

The super-resolution imaging method proposed in the embodiment of the present invention uses a fluorescent probe with dual emission peaks to label a biological sample. After exciting the fluorescent probe, a super-resolution image is reconstructed by the STORM super-resolution imaging method. After excitation, it can generate a fluorescent signal with double emission peaks, that is, a short-wavelength signal with a short-wavelength emission peak and a long-wavelength signal with a long-wavelength emission peak. Therefore, it can be formed when a nucleophile and an activating light act on this fluorescent probe. The two-channel fluorescence intensity image is then separately collected by the optical signal acquisition device. The short-wavelength signal with a short-wavelength emission peak and the long-wavelength signal with a long-wavelength emission peak can be calculated based on the two-channel fluorescence intensity image at the same time. And the ratio of the fluorescence intensity with a long-wavelength signal emission peak to obtain a proportional fluorescence image. Finally, the super-resolution image is colored according to the proportional fluorescence image to obtain a proportional-type super-resolution image. The proportional super-resolution image obtained by the super-resolution imaging method provided by the embodiment of the present invention can not only reflect the structure of the biological sample, but also reflect the sample parameters at the fluorescent probe mark in the biological sample by the ratio of the fluorescence intensity, so that it can be based on Sample parameters, functions for analyzing biological samples.

Brief description of the drawings

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flowchart of a super-resolution imaging method provided by Embodiment 1 of the present invention; FIG.

FIG. 2 is a detailed implementation flowchart of S103 in FIG. 1 provided in Embodiment 2 of the present invention; FIG.

3 is a schematic diagram of alternate light emission of a fluorescent probe provided in Embodiment 2 of the present invention;

FIG. 4 is a detailed implementation flowchart of S104 in FIG. 1 provided in Embodiment 3 of the present invention; FIG.

5 is a schematic diagram of a device for implementing the steps in FIG. 2;

FIG. 6 is an implementation effect diagram of a super-resolution imaging method provided by Embodiment 4 of the present invention; FIG.

FIG. 7 is a schematic structural diagram of a super-resolution imaging device provided by Embodiment 6 of the present invention.

The realization of the purpose, functional characteristics and advantages of the present invention will be further explained with reference to the embodiments and the drawings. .

The best embodiment for carrying out the invention

Best Mode of the Invention

Example one

As shown in FIG. 1, an embodiment of the present invention provides a super-resolution imaging method, which can be applied to super-resolution positioning imaging of an optical reconstruction microscope, which includes:

S101. Exciting a fluorescent probe having a double emission peak on a biological sample through excitation light, and applying an affinity reagent to the excited fluorescent probe.

In the above step S101, after the fluorescent probe having dual emission peaks is excited, two emission peaks will be displayed in the fluorescent emission spectrum of the fluorescent probe, that is, a short-wavelength emission peak and a long-wavelength emission peak.

In specific applications, before the excitation light acts on the biological sample, it should also pass through optical processing elements, such as lenses, field diaphragms, tube lenses, objective lenses, etc., so that the excitation light can be uniformly and concentratedly illuminated on the biological sample. Thereby, the fluorescent probe is excited.

S102. A dual-channel fluorescence intensity image is formed by irradiating the fluorescent probe after the action of the affinity reagent with activating light.

In the above step S102, the affinity reagent is not bound to the probe. When the affinity reagent covers the structure in the fluorescent probe, the structure does not emit light, and when the affinity reagent falls off the structure, the structure has the light-emitting ability again.

In the embodiment of the present invention, the affinity reagent is reversible, that is, under certain conditions, the affinity reagent can be transferred between the structures in the fluorescent probe.

In specific applications, the activation light and the excitation light may have the same frequency or different frequencies, which are not specifically limited in the embodiments of the present invention.

S103. Collect a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the dual-channel fluorescence intensity image, respectively.

In the above step S103, since the fluorescent probe after the action of the reagent is still a fluorescent probe having a double emission peak, the fluorescence emission spectrum obtained in step S103 still has a double emission peak. When activating light irradiates the fluorescent probe after the action of the affinity reagent, the affinity reagent can be transferred from one structure in the fluorescent probe to the other structure in the fluorescent probe, and the two structures will emit light alternately; In the light-emitting structure, a short-wavelength signal with a short-wavelength emission peak and a long-wavelength signal with a long-wavelength emission peak can be obtained in the obtained fluorescence emission spectrum.

In specific applications, collecting signals in the fluorescence emission spectrum can be achieved by any spectral analysis instrument or a combination of optical components, such as EMCCD, which is not specifically limited in the embodiments of the present invention.

S104. Select an N-frame dual-channel fluorescence intensity image, and select the fluorescence of the short-wavelength signal in the dual-channel fluorescence intensity image of the same frame according to the separately acquired short-wavelength signal and the long-wavelength signal. The intensity image and the fluorescence intensity image of the long-wavelength signal, and the fluorescence intensity ratios thereof are calculated respectively to obtain N proportional fluorescence images, where N is a positive integer.

In the above step S104, in the two-channel fluorescence intensity image of the same frame, the fluorescence intensity images corresponding to the short-wavelength signal and the long-wavelength signal are selected, and the fluorescence intensity ratios thereof are calculated to obtain N ratio fluorescence images, which is equivalent to a period of time. Multi-frame images of fluorescence probe flicker are continuously recorded inside, and the N-th proportional fluorescence image is the N-th dual-channel fluorescence intensity image, which can reflect the fluorescence intensity images corresponding to the short-wavelength signal and the long-wavelength signal at the same time, and The N-th ratio fluorescence image and the N + 1-th ratio fluorescence image may be two adjacent frames of images or two non-adjacent frames of images.

The embodiment of the present invention also provides detailed implementation steps for calculating the fluorescence intensity ratio in the above step S104, which includes:

Selecting the N-th fluorescence intensity image of the short-wavelength signal according to the dual-channel fluorescence intensity image and the separately collected short-wavelength signal, and calculating the emission peak fluorescence intensity thereof;

Selecting the Nth long-wavelength signal fluorescence intensity image according to the two-channel fluorescence intensity image and the separately acquired long-wavelength signal, and calculating the emission peak fluorescence intensity thereof;

The calculation formula for calculating the fluorescence intensity ratio is:

,

Wherein, IBN is the emission peak fluorescence intensity of the long-wavelength signal, and IAN is the emission peak fluorescence intensity of the short-wavelength signal.

In specific applications, a larger number of proportional fluorescent images (for example, 1,000 proportional fluorescent images) are needed as a basis for image reconstruction.

An embodiment of the present invention further provides a super-resolution imaging method for establishing a function model of the environmental parameter of the fluorescent probe and the ratio of the fluorescent intensity;

The establishing a function model of the environmental parameter of the fluorescent probe and the ratio of the fluorescent intensity includes:

Placing the fluorescent probe with a double emission peak in a probe solution test environment, and exciting the fluorescent probe through excitation light;

Changing a first parameter of the test environment of the probe solution to obtain a steady-state fluorescence emission spectrum of the fluorescent probe under different first parameters;

According to the double emission peak in the steady-state fluorescence emission spectrum, a fluorescence intensity test ratio is calculated, and a function model of the first parameter and the fluorescence intensity test ratio is established.

In specific applications, a function model of the first parameter and the fluorescence intensity test ratio, that is, the correspondence between the fluorescence intensity ratio and the sample parameter at the fluorescent probe label in the biological sample; for example, if the first parameter is the solution concentration, then When this probe is applied to a biological sample including a solution, the fluorescence intensity ratio can reflect the concentration of the solution at the probe mark of the biological sample.

S105. Reconstruct the proportional fluorescence image by using a STORM (Stochastic Optical Reconstruction Microscopy) method to obtain a super-resolution image.

In specific applications, the STORM super-resolution imaging method uses a random optical reconstruction microscope to continuously record multiple frames of fluorescent molecules, performs a single-molecule positioning algorithm to determine the center position, and finally reconstructs a super-resolution fluorescent image from the image.

S106. Color the super-resolution image according to the N fluorescence images to obtain a proportional super-resolution image.

The super-resolution imaging method proposed in the embodiment of the present invention uses a fluorescent probe with dual emission peaks to label a biological sample. After exciting the fluorescent probe, a super-resolution image is reconstructed by the STORM super-resolution imaging method. After excitation, it can generate a fluorescent signal with double emission peaks, that is, a short-wavelength signal with a short-wavelength emission peak and a long-wavelength signal with a long-wavelength emission peak. Therefore, it can be formed when a nucleophile and an activating light act on this fluorescent probe The two-channel fluorescence intensity image is then collected separately by the optical signal acquisition device. The short-wavelength signal with a short-wavelength emission peak and the long-wavelength signal with a long-wavelength emission peak can be calculated from the two-channel fluorescence intensity image at the same time And the ratio of the fluorescence intensity with a long-wavelength signal emission peak to obtain a proportional fluorescence image. Finally, the super-resolution image is colored according to the proportional fluorescence image to obtain a proportional-type super-resolution image. The proportional super-resolution image obtained by the super-resolution imaging method provided by the embodiment of the present invention can not only reflect the structure of the biological sample, but also reflect the sample parameters at the fluorescent probe mark in the biological sample by the ratio of the fluorescence intensity, so that it can be based on Sample parameters, functions for analyzing biological samples.

Invention Examples

Embodiments of the invention

Example two

As shown in FIG. 2, an embodiment of the present invention exemplarily shows a detailed implementation process of S102 in the first embodiment, where step S102 is:

S102. A dual-channel fluorescence intensity image is obtained by irradiating the fluorescent probe after the action of the affinity reagent with activating light.

The detailed implementation process includes:

S1021. Obtain the short-wavelength signal according to a first structure in the fluorescent probe.

S1022. Obtain the long-wavelength signal according to a second structure in the fluorescent probe.

In the above steps S1021 and S1022, the wavelength of the activation light is the same as the wavelength of the excitation light, wherein the short-wavelength emission peak is caused by the first structure in the probe, and the long-wavelength emission peak is caused by the second structure in the probe. And the fluorescence emission spectra of the two emission peaks do not completely overlap.

S1023. When the excitation light irradiates the fluorescent probe, the first structure or the second structure emits fluorescence.

S1024. When the activating light acts on the fluorescent probe after the affinity reagent acts, the first structure and the second structure alternately emit light according to a binding state with the affinity reagent to form the dual channel. Image of fluorescence intensity.

In the above steps S1023 and S1024, when the fluorescent probe is irradiated with the laser, both the first structure and the second structure in the fluorescent probe may absorb the excitation light energy and transition to the excited state to emit fluorescence, that is, the laser irradiates the fluorescent probe. In the case of needles, the first structure may emit fluorescence or the second structure may emit fluorescence.

In the embodiment of the present invention, if the first structure of one of the fluorescent probes emits fluorescence after the excitation light is irradiated, the first structure is an energy acceptor and the second structure is an energy donor; The energy provided by the body causes the energy acceptor to emit fluorescence, and the energy donor emits weak fluorescence due to the loss of energy. When the light is illuminated by the fluorescent probe after the affinity reagent is activated, the energy donor no longer provides energy to the energy acceptor. So that the energy acceptor does not emit fluorescence, and the energy donor emits fluorescence because it does not lose energy. Due to the randomness of the binding of the affinity reagent and the fluorescent probe, in the biological sample, the fluorescence emitted by the first structure and the second structure of each fluorescent probe is also random.

As shown in FIG. 3, it is a schematic diagram of alternately emitting light by a fluorescent probe provided by an embodiment of the present invention. First, the energy receptor emits light after the excitation light is irradiated; when the affinity reagent is not bound to the fluorescent probe, the energy receptor no longer absorbs the energy of the energy donor and the excitation light, and no longer emits fluorescence, due to the reversibility of the affinity reagent And, under the irradiation of activation light, the energy donor reversely absorbs the activation light energy to emit fluorescence, and then based on this, it randomly circulates to achieve the purpose of the fluorescent probe to alternately emit light.

In the embodiment of the present invention, the two-channel fluorescence intensity images described in the above steps S1021 to S1024 include a fluorescence intensity image of a short wavelength signal and a fluorescence intensity image of a long wavelength signal; the fluorescence intensity image of the short wavelength signal corresponds to The light emission process of the first structure in the fluorescent probe; the fluorescence intensity image of the long-wavelength signal corresponds to the light emission process of the second structure in the fluorescent probe.

The super-resolution imaging method proposed in the embodiment of the present invention uses a fluorescent probe with dual emission peaks to label a biological sample, and under the action of activating light irradiation and the reversible action of an affinity reagent, a single fluorescent probe molecule in the biological sample emits two randomly alternately. Different wavelengths of fluorescence.

Example three

As shown in FIG. 4, an embodiment of the present invention exemplarily illustrates a detailed implementation process of step S103 in the foregoing first embodiment, where step S103 is:

S103. Collect a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the dual-channel fluorescence intensity image, respectively.

The detailed implementation process includes:

S1031. Separate the excitation light from the short-wavelength signal and the long-wavelength signal through a first dichroic mirror.

In the above step S1031, the dichroic mirror almost completely transmits light of a certain wavelength and almost completely reflects light of other wavelengths.

In specific applications, after the excitation light acts on the biological sample, the light emitted by the fluorescent probe on the biological sample includes a short-wavelength signal, a long-wavelength signal, and a small amount of excitation light. Wavelength signals are separated from long-wavelength signals.

S1032. After the short-wavelength signal and the long-wavelength signal are separated by a second dichroic mirror, the short-wavelength signal is sent to a reflecting mirror through a first filter, and the reflecting mirror separates the filtered short-wavelength signal. Transmitting to a first EMCCD (ElectBon-MultiplyinACCD); the long-wavelength signal passes through a second filter, and the filtered long-wavelength signal is transmitted to the first EMCCD.

In the above step S1032, the light wave signal is also separated by a dichroic mirror, and the separated short-wavelength signal and long-wavelength signal are filtered by a filter and sent to the same EMCCD.

In specific applications, single emission wavelengths are susceptible to environmental noise interference, which will result in an increase in the proportion of system noise in the imaging results and blurring of reconstructed images. To avoid or mitigate this effect, algorithmic noise reduction is required during subsequent results processing. Before image reconstruction, filtering it can simplify the post-processing algorithm in the super-resolution imaging method based on optical reconstruction microscope.

S1033. Different regions of the first EMCCD receive the filtered short-wavelength signal and the filtered long-wavelength signal, respectively, so as to collect the dual-channel fluorescence intensity image.

In step S1033, the size of the short-wavelength signal or the long-wavelength signal should be smaller than the maximum pixel size of the EMCCD; More than 256 * 256.

In specific applications, the use of different areas of an EMCCD to receive optical path signals separately does not require synchronous control.

As shown in FIG. 5, an embodiment of the present invention also provides a schematic structural diagram of a device for implementing steps S1031 to S1032.

The reference numerals are as follows: 51. laser; 52. lens group; 53. field diaphragm; 54. tube mirror; 55. biological sample; 56. objective lens; 57. first dichroic mirror; 58. reflector; 59. Second dichroic mirror; 510. first filter; 511. second filter; 512. EMCCD.

In the embodiment of the present invention, the lens group is used to converge or diverge the light beam; the field diaphragm is used to appropriately adjust the beam size.

In the embodiment of the present invention, two identical EMCCDs can also be used to receive the short-wavelength signal and the long-wavelength signal, respectively. The detailed implementation process is as follows:

Separating the excitation light from the short-wavelength signal and the long-wavelength signal through a first dichroic mirror;

After the short-wavelength signal and the long-wavelength signal are separated by a second dichroic mirror, the short-wavelength signal is sent to a reflecting mirror through a first filter, and the reflecting mirror transmits the filtered short-wavelength signal to A first EMCCD; the long-wavelength signal passes through a second filter, and the filtered long-wavelength signal is transmitted to a second EMCCD;

The first EMCCD and the second EMCCD respectively receive the filtered short-wavelength signal and the filtered long-wavelength signal, thereby collecting the dual-channel fluorescence intensity image.

In specific applications, using two identical EMCCDs to separately collect two signal lights under the same conditions can achieve data collection in a larger pixel area, but the requirements for the high synchronization of the two EMCCDs require synchronous control.

In the super-resolution imaging method provided by the embodiment of the present invention, after the signal light is separated into two channels by a dichroic mirror, part of the stray light is filtered by a filter, and then the two signal lights are collected by EMCCD, thereby improving the anti-noise of biological samples The ability to resist environmental media inhomogeneity and system instability, improve imaging signal-to-noise ratio, and simplify the process of later data processing.

Embodiment 4

The embodiment of the present invention exemplarily describes the implementation process of the super-resolution imaging methods provided in the first to third embodiments.

As shown in FIG. 6, after a short-wavelength signal with a short-wavelength emission peak and a long-wavelength signal with a long-wavelength emission peak are separately collected, data processing is performed based on the two signals to obtain a proportional fluorescence image. The data processing process can be represented by FIG. 6, where IB + IA is expressed as an unseparated long-wavelength signal and a short-wavelength signal. After the super-resolution imaging method provided in the third embodiment, the data are separated into optical long-wavelength signals IB and Short-wavelength signal IA; when the fluorescence intensity ratio of emission peaks is calculated after the acquisition is completed, the fluorescence intensity map of the two signals at the same time is used as the ratio, that is,

To obtain a proportional fluorescence image, and finally reconstruct a large number of proportional images by algorithm to obtain a super-resolution image and color the marked points with pseudo color to obtain a proportional-type super-resolution image, in which different colors represent the ratio

The size of the value.

Example 5

The embodiment of the present invention exemplarily illustrates the practical application of the super-resolution imaging methods provided in the first to third embodiments. In the practical application, how to reflect the ratio of the sample parameters at the fluorescent probe markers in the biological sample to the proportional super-resolution image is used to analyze the function of the biological sample according to the sample parameters.

The embodiment of the present invention provides an example of super-resolution imaging using the super-resolution imaging methods provided in the first to fourth embodiments to realize a quantitative study of the microenvironment viscosity value of a labeled sample.

In the embodiment of the present invention, the fluorescent probe provided is a viscosity-sensitive fluorescent probe. When the environmental viscosity changes, the steady-state emission spectrum also changes accordingly. The short-wavelength emission peak is caused by the structure A in the probe, the long-wavelength emission peak is caused by the structure B in the probe, and there is no overlap region between the two fluorescence emission spectra, IB is the fluorescence intensity of the emission peak of structure B, and IA is the structure A Emission peak fluorescence intensity.

In specific applications, first construct a function of IB / IA as a function of viscosity, that is, measure the steady-state emission spectra of fluorescent probes in different viscosity solutions, and separately process the steady-state spectra at different viscosities with IB / IA. , Get the value of IB / IA under different viscosities, and then plot the value of each proportional point and fit a linear or non-linear function to get the fitting function relationship formula of IB / IA to viscosity.

In practical applications, fluorescent probes are used to label biological samples, lasers are used for excitation, and imaging is performed in a super-resolution imaging system to obtain a large number of proportional fluorescent images. In the later data processing, the ratio of the two-channel proportional fluorescence image IB to IA is firstly calculated for each frame, and then a large number of fluorescence intensity ratio images are algorithmically reconstructed to obtain a super-resolution image of the structure of the labeled organism, and the pseudo-color pairs are used to Each pixel of the marker structure is colored. The value of each pixel in the image is the value of IB / IA. Different colors represent the size of the IB / IA ratio, and the size of the IB / IA ratio can be expressed in the fitting function relationship of the solution test result. Correspondingly, the viscosity values under different ratios are found, that is, the difference in pseudo-colors represents the difference in the distribution of viscosity values in the microenvironment. At the same time, the reconstructed super-resolution image shows an ultra-fine structure diagram of the labeled structure. Super-resolution Imaging for Quantitative Study of Ambient Viscosity Values.

The embodiment of the present invention also provides an example of super-resolution imaging using the super-resolution imaging methods provided in the first to third embodiments to realize the quantitative study of the mitochondrial membrane protein of the labeled sample.

In the embodiment of the present invention, the fluorescent probe provided is a fluorescent probe that can specifically label mitochondrial membrane proteins. When a solution or a biological test is performed, after the membrane protein and the probe specifically bind in the environment, the steady-state emission spectrum will also be Corresponding changes. The short-wavelength emission peak is caused by the structure A in the probe, and the long-wavelength emission peak is caused by the structure B in the probe. There is no serious overlap region between the two fluorescence emission spectra to avoid crosstalk between the two fluorescence emission. IB is the structure B. Emission peak fluorescence intensity, IA is the fluorescence intensity of emission peak of structure A.

In specific applications, first construct a function equation of the value of IB / IA with respect to the content or concentration of mitochondrial membrane protein, that is, measure the steady-state emission spectra of fluorescent probe 2 in solutions with different mitochondrial membrane protein content or concentration. The IB / IA treatment is performed on the steady-state spectrum at the content or concentration, respectively, to obtain the value of IB / IA at different mitochondrial membrane protein content or concentration, and then the proportional value is plotted against the content or concentration and fitted with a linear or non-linear function. The fitted function relationship of IB / IA to mitochondrial membrane protein content or concentration was obtained.

In practical applications, fluorescent probes are used to label biological mitochondrial membrane proteins, excitation is performed using excitation light, and imaging is performed in a super-resolution imaging system to obtain a large number of fluorescent images. In the later data processing, the ratio of each frame of the two-channel fluorescence intensity image IB to IA is firstly compared, and then a large number of proportional fluorescence images are reconstructed by algorithm to obtain a super-resolution image of the structure of the labeled organism, and the labels are marked in pseudo-color. Each pixel of the structure is colored. The value of each pixel in the image is the value of IB / IA. Different colors represent the size of the IB / IA ratio, and the size of the IB / IA ratio can be in the fitting function relationship of the solution test result. Correspondingly find the protein content value at different ratios, that is, the difference in pseudo-colors represents the difference in the distribution of the protein content value in the microenvironment. At the same time, the reconstructed super-resolution image shows an ultra-fine structure map of the labeled structure. Super-resolution Imaging of Quantitative Study of Mitochondrial Membrane Protein Content Values

Example Six

As shown in FIG. 7, an embodiment of the present invention provides a super-resolution imaging device 70, including:

An excitation module 71, configured to excite a fluorescent probe having a double emission peak on a biological sample through excitation light, and apply an affinity reagent to the excited fluorescent probe;

A fluorescence intensity image acquisition module 72, configured to obtain a dual-channel fluorescence intensity image by activating light and irradiating a fluorescent probe after the action of an affinity reagent;

A signal acquisition module 73, configured to separately acquire a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the dual-channel fluorescence intensity image;

A proportional fluorescence image calculation module 74 is configured to select N frames of two-channel fluorescence intensity images, and select the two-channel fluorescence intensity images in the same frame according to the separately acquired short-wavelength signals and the long-wavelength signals. Calculate the fluorescence intensity ratio of the short-wavelength signal fluorescence image and the long-wavelength signal fluorescence intensity image, respectively, to obtain N proportional fluorescence images, where N is a positive integer, where N is a positive integer;

An image reconstruction module 75, configured to reconstruct the proportional fluorescence image by using a STORM super-resolution imaging method to obtain a super-resolution image;

An image acquisition module 76 is configured to color the super-resolution image according to N number of the proportional fluorescence images to obtain a proportional-type super-resolution image.

In specific applications, the above-mentioned fluorescence intensity image acquisition module 73 includes:

A short-wavelength emission peak acquisition unit, configured to obtain the short-wavelength emission peak according to a first structure in the fluorescent probe;

A long-wavelength emission peak acquisition unit, configured to obtain the long-wavelength emission peak according to a second structure in the fluorescent probe;

The first structure is an energy acceptor, and the second structure is an energy donor;

A dual-channel fluorescence intensity image acquisition unit is configured to apply the activated light to the processed fluorescent probe to cause the first structure and the second structure to emit light alternately to obtain the dual-channel fluorescence intensity image.

An embodiment of the present invention further provides a terminal device including a memory, a processor, and a computer program stored in the memory and executable on the processor. When the processor executes the computer program, the implementation is implemented as in Embodiments 1 to 3. Each step in the super-resolution imaging method described in.

An embodiment of the present invention also provides a storage medium. The storage medium is a computer-readable storage medium, and a computer program is stored thereon. When the computer program is executed by a processor, the implementation is as described in Embodiments 1 to 3. Steps in a super-resolution imaging method.

The above-mentioned embodiments are only used to illustrate the technical solutions of the present invention, but are not limited thereto. Although the foregoing embodiments describe the present invention in detail, those skilled in the art should understand that they can still implement the foregoing embodiments. Modifications to the recorded technical solutions, or equivalent replacements of some of the technical features thereof; and these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the present invention Within the scope of the invention.

Industrial applicability

The super-resolution imaging method proposed by the present invention uses a fluorescent probe with dual emission peaks to label a biological sample. After the fluorescent probe is excited, a super-resolution image is reconstructed by the STORM super-resolution imaging method. Able to generate fluorescent signals with dual emission peaks, that is, short-wavelength signals with short-wavelength emission peaks and long-wavelength signals with long-wavelength emission peaks, so dual channels can be formed when this fluorescent probe is acted on by a nucleophile and activating light The fluorescence intensity image is then collected by an optical signal acquisition device. The short-wavelength signal with a short-wavelength emission peak and the long-wavelength signal with a long-wavelength emission peak can be calculated from the two-channel fluorescence intensity image at the same time. The ratio of the fluorescence intensity of the long-wavelength signal emission peak is used to obtain a proportional fluorescence image. Finally, the super-resolution image is colored according to the proportional fluorescence image to obtain a proportional-type super-resolution image. The proportional super-resolution image obtained by the super-resolution imaging method provided by the embodiment of the present invention can not only reflect the structure of the biological sample, but also reflect the sample parameters at the fluorescent probe mark in the biological sample by the ratio of the fluorescence intensity, so that it can be based on Sample parameters, functions for analyzing biological samples.

Claims (10)

1. A super-resolution imaging method, comprising:

   Exciting a fluorescent probe having a double emission peak on a biological sample by exciting light, and applying an affinity reagent to the excited fluorescent probe;

   By activating light and irradiating the fluorescent probe after the action of the affinity reagent, a dual-channel fluorescence intensity image is formed;

   Respectively collecting a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the dual-channel fluorescence intensity image;

   Select N frames of two-channel fluorescence intensity images, and select the fluorescence intensity images of the short-wavelength signals in the two-channel fluorescence intensity images of the same frame according to the separately acquired short-wavelength signals and the long-wavelength signals. And the fluorescence intensity image of the long-wavelength signal, and calculating the fluorescence intensity ratios thereof, to obtain N proportional fluorescence images, where N is a positive integer;

   Reconstructing the proportional fluorescence image using a STORM super-resolution imaging method to obtain a super-resolution image;

   Color the super-resolution image according to N number of the proportional fluorescence images to obtain a proportional-type super-resolution image.
2. The super-resolution imaging method according to claim 1, wherein the super-resolution imaging method further comprises:

   Establishing a function model of the environmental parameter of the fluorescent probe and the ratio of the fluorescent intensity;

   The establishing a function model of the environmental parameter of the fluorescent probe and the ratio of the fluorescent intensity includes:

   Placing the fluorescent probe with a double emission peak in a probe solution test environment, and exciting the fluorescent probe through excitation light;

   Changing a first parameter of the test environment of the probe solution to obtain a steady-state fluorescence emission spectrum of the fluorescent probe under different first parameters;

   According to the double emission peak in the steady-state fluorescence emission spectrum, a fluorescence intensity ratio is calculated, and a function model of the first parameter and the fluorescence intensity ratio is established.
3. The super-resolution imaging method according to claim 1, wherein obtaining a dual-channel fluorescence intensity image by activating light and irradiating a fluorescent probe under the action of an affinity reagent comprises:

   Obtaining the short-wavelength signal according to a first structure in the fluorescent probe;

   Obtaining the long-wavelength signal according to a second structure in the fluorescent probe;

   When the excitation light irradiates the fluorescent probe, the first structure or the second structure emits fluorescence;

   When the activating light acts on the fluorescent probe after the affinity reagent acts, the first structure and the second structure alternately emit light according to the binding state with the affinity reagent to form the dual-channel fluorescence intensity image.
4. The super-resolution imaging method according to claim 3, wherein the dual-channel fluorescence intensity image comprises a fluorescence intensity image of a short-wavelength signal and a fluorescence intensity image of a long-wavelength signal;

   The fluorescence intensity image of the short-wavelength signal corresponds to the light emission process of the first structure in the fluorescent probe;

   The fluorescence intensity image of the long-wavelength signal corresponds to the light emission process of the second structure in the fluorescent probe.
5. The super-resolution imaging method according to claim 1, wherein in the acquiring the two-channel fluorescence intensity images, a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak respectively include:

   Separating the excitation light from the short-wavelength signal and the long-wavelength signal through a first dichroic mirror;

   After the short-wavelength signal and the long-wavelength signal are separated by a second dichroic mirror, the short-wavelength signal is sent to a reflecting mirror through a first filter, and the reflecting mirror transmits the filtered short-wavelength signal to A first EMCCD; the long-wavelength signal passes through a second filter, and the filtered long-wavelength signal is transmitted to the first EMCCD;

   Different regions of the first EMCCD receive the filtered short-wavelength signal and the filtered long-wavelength signal, respectively, so as to collect the dual-channel fluorescence intensity image.
6. The super-resolution imaging method according to claim 1, wherein said collecting the short-wavelength signal of a short-wavelength emission peak and the long-wavelength signal of a long-wavelength emission peak in the fluorescence emission spectrum separately comprises:

   Separating the excitation light from the short-wavelength signal and the long-wavelength signal through a first dichroic mirror;

   After the short-wavelength signal and the long-wavelength signal are separated by a second dichroic mirror, the short-wavelength signal is sent to a reflecting mirror through a first filter, and the reflecting mirror transmits the filtered short-wavelength signal to A first EMCCD; the long-wavelength signal passes through a second filter, and the filtered long-wavelength signal is transmitted to a second EMCCD;

   The first EMCCD and the second EMCCD respectively receive the filtered short-wavelength signal and the filtered long-wavelength signal, thereby collecting the dual-channel fluorescence intensity image.
7. The super-resolution imaging method according to any one of claims 1 to 6, wherein the selected N-frame two-channel fluorescence intensity images are based on the short-wavelength signal and the long-wavelength signal acquired separately, In the two-channel fluorescence intensity image of the same frame, the fluorescence intensity image of the short-wavelength signal and the fluorescence intensity image of the long-wavelength signal are selected, and the fluorescence intensity ratios thereof are calculated to obtain N ratio fluorescence images, where N is a positive integer including:

   Selecting the N-th fluorescence intensity image of the short-wavelength signal according to the dual-channel fluorescence intensity image and the separately collected short-wavelength signal, and calculating the emission peak fluorescence intensity thereof;

   Selecting the Nth long-wavelength signal fluorescence intensity image according to the two-channel fluorescence intensity image and the separately acquired long-wavelength signal, and calculating the emission peak fluorescence intensity thereof;

   The calculation formula for calculating the fluorescence intensity ratio is:

   

   ,

   Wherein, IBN is the emission peak fluorescence intensity of the long-wavelength signal, and IAN is the emission peak fluorescence intensity of the short-wavelength signal.
8. A super-resolution imaging device, comprising:

   An excitation module for exciting a fluorescent probe having a double emission peak on a biological sample by using excitation light, and applying an affinity reagent to the excited fluorescent probe;

   A fluorescence intensity image acquisition module, configured to obtain a dual-channel fluorescence intensity image by activating light and irradiating a fluorescent probe after the action of an affinity reagent;

   A signal acquisition module configured to separately acquire a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the dual-channel fluorescence intensity image;

   A proportional fluorescence image calculation module is configured to select N frames of two-channel fluorescence intensity images, and select the selected one of the two-channel fluorescence intensity images in the same frame according to the separately acquired short-wavelength signals and the long-wavelength signals. The fluorescence intensity image of the short-wavelength signal and the fluorescence intensity image of the long-wavelength signal, and respectively calculating a fluorescence intensity ratio thereof to obtain N proportional fluorescence images, where N is a positive integer, where N is a positive integer;

   An image reconstruction module, configured to reconstruct the proportional fluorescence image by using the STORM super-resolution imaging method to obtain a super-resolution image;

   An image acquisition module is configured to color the super-resolution image according to N of the proportional fluorescence images to obtain a proportional-type super-resolution image.
9. A terminal device, comprising a memory, a processor, and a computer program stored on the memory and operable on the processor, and is characterized in that when the processor executes the computer program, it implements claim 1 Each step in the super-resolution imaging method according to any one of 7 to 7.
10. A storage medium is a computer-readable storage medium on which a computer program is stored, wherein when the computer program is executed by a processor, the computer program according to any one of claims 1 to 7 is implemented. Steps in a super-resolution imaging method.

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