Super-resolution microscopy method and system for optical super-vibration excitation and array detection and reception

Abstract

The present invention discloses a super-resolution microscopy method and system integrating optical supervibration excitation and array detection and reception. By modulating the illumination beam in terms of amplitude, phase, and polarization, an optical supervibration field composed of a central focal spot and a sidelobe annulus is obtained; dual-mode modulation illumination of the central focal spot and the sidelobe annulus is realized through two-dimensional scanning of the beam to excite fluorescence signals in different spatial frequency band ranges; an array detector is used to receive the modulated fluorescence signals; according to the spatio-temporal modulation characteristics of the fluorescence signals at the excitation and detection ends, two images generated by dual-mode modulation illumination are reconstructed through an algorithm, and finally, a super-resolution image corresponding to optical supervibration modulation excitation is reconstructed. The present invention uses low-intensity dual-mode optical supervibration excitation, and the fluorescence linear effect provides image contrast, which is more suitable for long-term, multi-color, fast, and super-resolution live cell imaging.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical microscopy imaging, and particularly relates to a super-resolution microscopy method and system integrating optical super-vibration excitation and array detection and reception. Background Art

[0002] Cells are the smallest units of all organisms on earth and have very delicate and complex functional units. Analyzing the fine structure and operation mechanism of cells through microscopy imaging plays a very important role in promoting the development of cell biology, tumor cytology, etc. Super-resolution microscopy technology can break through the constraints of the diffraction limit and provide new opportunities for people to explore the life information of cells at the molecular level. In particular, in order to study the internal structure and function of living cells at the molecular level, there is an urgent need to develop super-resolution microscopy imaging technology with long-term, multi-color, fast, three-dimensional and super-resolution performance.

[0003] However, existing super-resolution microscopy technologies based on fluorescence nonlinear effects (stimulated emission depletion microscopy STED / single molecule localization microscopy SMLM) still face many challenges in live cell imaging. For example, strong light illumination is likely to induce phototoxicity and photobleaching, resulting in a decrease in resolution, cell death, etc.; it is necessary to control the fluorescence dye to "switch" repeatedly to obtain a sufficient number of photons, resulting in a relatively slow imaging speed; special fluorescence dyes with "switch" characteristics are not easily penetrated through the cell membrane and it is difficult to label internal targets of cells; the labeling method of special fluorescence dyes affects the spatial resolution, etc.

[0004] In addition, for super-resolution microscopy technology based on structured illumination (SIM), the use of the mixing effect can achieve a two-fold improvement in spatial resolution (about 100 nm). Only low-intensity light illumination is required, which can be compatible with standard sample production, and fast, multi-color, long-term, etc. live cell imaging can be realized using conventional fluorescence dyes. However, the relatively low spatial resolution limits the application range of SIM in live cell imaging. Although nonlinear SIM can obtain higher spatial resolution by using fluorescence saturation excitation or fluorescence dyes with "switch" characteristics, it also has the limitations of the above-mentioned super-resolution microscopy technologies based on fluorescence nonlinear effects.

[0005] In addition, theoretically, optical superoscillation can obtain an infinitesimal super-diffraction-limited focal spot in the far field, which is a super-resolution microscopy method with great development potential. Currently, the modes based on optical superoscillation microscopy imaging are mainly divided into two types: (1) using an optical superoscillation lens to achieve super-resolution imaging; (2) using a super-diffraction light spot to achieve super-resolution imaging. For mode (1), the optical superoscillation lens can simplify the structure of the microscopy imaging system, but it often faces the optimization design of complex structures and the high-precision requirements of micro-nano structure processing. For mode (2), the optical superoscillation focal spot can be flexibly obtained or controlled through beam regulation, and it is easy to be compatible with other microscopy imaging methods and microscopy optical path systems. However, the compression of the optical superoscillation central focal spot will lead to a decrease in the light intensity of the central focal spot and an increase in the light intensity of the sidebands and sidelobes, which is likely to introduce a large background noise in the microscopy imaging system. It can be seen that the method of only single-control of the optical superoscillation central focal spot scale to achieve super-resolution imaging restricts the practical application of optical superoscillation in live cell super-resolution microscopy imaging.

[0006] In summary, whether it is the fluorescence super-resolution microscopy technology using fluorescence nonlinear effects or structured illumination, or the far-field super-resolution imaging achieved by controlling the optical superoscillation central focal spot, there are still many constraints in long-term, multi-color, fast, three-dimensional and super-resolution live cell imaging for existing methods. Summary of the Invention

[0007] The object of the present invention is to provide a super-resolution microscopy method and system integrating optical superoscillation excitation and array detection and reception in view of the deficiencies of the prior art. The present invention obtains super-resolution microscopy images through light field regulation and mixing reception based on fluorescence linear effects, solves problems such as strong light illumination, special fluorescent dye labeling, and strong background noise, and is more suitable for long-term, multi-color, fast and super-resolution microscopy imaging of live cells.

[0008] The object of the present invention is achieved by the following technical solutions: A super-resolution microscopy method integrating optical superoscillation excitation and array detection and reception includes the following steps:

[0009] 1) The excitation light emitted by the light source module is filtered and transmitted by the wavelength selection module; the output beam enters the beam control module, and the beam control module regulates the amplitude, phase, and polarization parameters of the beam; the regulated output beam forms an optical superoscillation with the size of an Airy disk inside the test sample through the microscopic imaging module, which consists of a central focal spot and a sidelobe annulus; the fluorescence signal excited by the optical superoscillation is received by the microscopic imaging module and enters the beam conversion module through a dichroic mirror; the fluorescence signal is filtered and beam-converted and then coupled into the fiber array at the input end of the array detector; the optical signals collected by each channel of the array detector are converted into electrical signals and input into a computer for processing; the test sample is fixed on a three-dimensional scanning platform to achieve large-scale lateral movement of the sample and high-precision one-dimensional movement in the axial direction; by combining two-dimensional beam scanning, three-dimensional movement of the sample, and selection of wavelength, pinhole, and filter, the system obtains a data array group of the fluorescence signals modulated and excited inside the sample.

[0010] 2) The data obtained by the array detector at the same detection moment are grouped together. The data obtained by each detection channel at the same scanning position are processed according to the arrangement structure of the input end of the fiber array and the spatial positions of each fiber relative to the optical axis of the detection optical path. The fluorescence signal of the scanning point corresponding to the modulation excitation of the central focal spot is obtained through translation and superposition; the initial measurement image corresponding to the modulation excitation of the central focal spot is obtained according to the timing and spatial position of the scanning point, and then the super-resolution microscopic image corresponding to the modulation excitation of the central focal spot is obtained by using the deconvolution algorithm.

[0011] 3) The data obtained by the array detector at the same detection position are grouped together. The image data obtained by each detection channel at different moments are processed according to the arrangement structure of the input end of the fiber array and the spatial positions of each fiber relative to the optical axis of the detection optical path. The initial measurement image corresponding to the modulation excitation of the sidelobe annulus focal spot is obtained through filtering and superposition, and then the super-resolution microscopic image corresponding to the modulation excitation of the sidelobe annulus focal spot is obtained by using the deconvolution algorithm.

[0012] 4) The super-resolution images obtained in step 2) and step 3) are weighted and superimposed to obtain the initial measurement image of the optical superoscillation modulation excitation. The deconvolution algorithm is used to stitch the super-resolution images of different frequency bands corresponding to the modulation excitation of the central focal spot and the sidelobe annulus, and finally the super-resolution image obtained by the optical superoscillation modulation excitation is reconstructed.

[0013] Preferably, in step 1), the following optical superoscillation is obtained through modulation of beam parameters (amplitude, phase, and polarization): the full width at half maximum of both the central focal spot and the sidelobe annulus is less than the diffraction limit, and the peak light intensities of the two are basically equal; a central circular focal spot and at least one annular spot are included within the diffraction limit; the central focal spot and the annulus form a dual-mode modulation illumination mode.

[0014] A super-resolution microscopic system integrating optical superoscillation excitation and array detection and reception, comprising:

[0015] A light source module that provides the excitation light required for the excitation of different fluorescent dyes.

[0016] A wavelength selection module that sequentially or time-division selects different excitation wavelengths according to different fluorescent dyes.

[0017] A beam control module that collimates the excitation beam into a parallel beam and controls the amplitude, phase, and polarization distribution of the beam.

[0018] A dichroic mirror that splits and transmits the excitation beam and the fluorescence signal beam.

[0019] A microscopic imaging module that realizes optical super-vibration two-dimensional fast scanning modulation excitation and fluorescence signal reception; includes a two-dimensional galvanometer scanning device, a scanning lens, a tube lens, and a microscopic objective lens.

[0020] A sample stage that fixes and moves the sample to be measured.

[0021] A light field conversion module that filters background stray light and controls the coupling of the fluorescence signal into the fiber array.

[0022] An array detector that converts the fluorescence signal output by the fiber array into an electrical signal.

[0023] A computer that collects and processes multi-channel sample information, displays the reconstructed super-resolution image, and issues control signals for scanning the sample, as well as control signals for wavelength, pinhole, and filter selection.

[0024] Preferably, the light source module is one of a combination of multiple semiconductor lasers, a white light laser, or a supercontinuum laser.

[0025] Preferably, the wavelength selection module uses an AOTF (acousto-optic tunable filter) to select the required excitation wavelength according to the fluorescent dye, and a single-mode polarization-maintaining fiber is connected to the AOTF to provide the input and output of the illumination beam.

[0026] Preferably, the beam modulation module includes a collimating lens, a half-wave plate, amplitude, phase, and polarization modulators.

[0027] Preferably, the light field conversion module includes a collimating lens, a filter wheel, a pinhole wheel, and a beam conversion lens group composed of doublet lenses.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] 1) By using optical super-vibration dual-mode modulation to excite super-resolution information, it is possible to avoid the limitations of achieving super-resolution based on fluorescence nonlinear effects or based on optical super-vibration single-center focal spot excitation, be compatible with the standard sample production method of ordinary fluorescent dye labeling, and more easily achieve multi-color live cell imaging;

[0030] 2) Through the parallel detection mode of the array detector, more mixing signals excited by the central focal spot and the sidelobe annulus modulation can be obtained, while improving the signal-to-noise ratio of the system measurement, which helps to achieve low-intensity optical superoscillation modulation excitation and makes it easier to realize long-term live cell imaging.

[0031] 3) By demodulating the fluorescence signals temporally and spatially modulated at the excitation and detection ends through an algorithm, the fluorescence excitation light field can be digitally decomposed into two parts: the central focal spot and the sidelobe annulus, making it easier to demodulate the aliased ultra-high spatial frequency information and reconstruct the super-resolution image. Description of the Drawings

[0032] Figure 1 is the structural diagram of the system of the present invention;

[0033] Figure 2 is the optical path structure of the present invention;

[0034] Figure 3 is the signal processing flow chart of the method of the present invention;

[0035] In the figure, 1. Light source module; 2. Wavelength selection module; 3. Beam modulation module; 4. Dichroic mirror; 5. Microscopic imaging module; 6. Light field conversion module; 7. Array detector; 8. Computer; 9. Sample stage; 10. Single-mode polarization-maintaining fiber; 11. AOTF; 12. Single-mode polarization-maintaining fiber; 13. Collimating lens; 14. Half-wave plate; 15. Amplitude, phase and polarization modulator; 16. Two-dimensional galvanometer scanning device; 17. Scanning lens; 18. Tube lens; 19. Microscopic objective lens; 20. Filter wheel; 21. Converging lens; 22. Pinhole wheel; 23. Lens; 24. Lens; 25. Fiber array; 26. Sample. Detailed Embodiments

[0036] The present invention will be further described below with reference to the accompanying drawings.

[0037] Reference Figure 1 , an integrated super-resolution microscopy system for optical superoscillation excitation and array detection and reception in this embodiment includes a light source module 1, a wavelength selection module 2, a beam modulation module 3, a dichroic mirror 4, a microscopic imaging module 5, a beam conversion module 6, an array detector 7, a computer 8, and a sample stage 9. Among them, the wavelength selection module 2 includes a single-mode polarization-maintaining fiber 10, an AOTF 11, and a single-mode polarization-maintaining fiber 12. The beam modulation module 3 internally includes a collimating lens 13, a half-wave plate 14, an amplitude, phase and polarization modulator 15. The microscopic imaging module 5 includes a two-dimensional galvanometer scanning device 16, a scanning lens 17, a tube lens 18, and a microscopic objective lens 19. The beam conversion module 6 includes a filter wheel 20, a converging lens 21, a pinhole wheel 22, and a beam conversion lens group; the beam conversion lens group consists of a lens 23 and a lens 24 to form a telecentric optical system.

[0038] Among them, as Figure 2 shown, the light source module 1 is one of a combination of multiple semiconductor lasers with different central wavelengths, a white light laser, or a supercontinuum light source. The excitation light emitted by the light source module 1 is coupled into the single-mode polarization-maintaining fiber 10 at the input end of the wavelength selection module 2. According to the type of fluorescent dye, the AOTF 11 filters and selects corresponding time-division or sequential different excitation light wavelengths to provide an excitation light source for obtaining a microscopic image of a multi-color fluorescent dye-labeled living cell; the selected excitation light wave is introduced into the optical beam modulation module 3 through the single-mode polarization-maintaining fiber 12 at the output end of the wavelength selection module 2. The collimating lens 13 collimates the input beam into a parallel beam; the half-wave plate 14 controls the vibration direction of the incident linearly polarized parallel beam and controls the efficiency of beam parameter regulation; the amplitude, phase, and polarization modulator 15 controls the phase, amplitude, and polarization distribution of the beam by a computer respectively; the specific control parameters are determined by the vector diffraction theory of optical superoscillation for optical field regulation, so that the optical superoscillation consists of a central focal spot and a sidelobe annulus. The parallel beam with modulated beam parameters is introduced into the microscopic imaging module 5 through the dichroic mirror 4. The two-dimensional galvanometer scanning device 16 controls the two-dimensional fast scanning of the optical superoscillation in the sample; the scanning lens 17 and the tube lens 18 constitute a telecentric optical system to control the aberration-free entry of the scanning beam into the microscopic objective lens 19 with an infinite conjugate distance; the microscopic objective lens 19 converts the scanning parallel beam into a scanned optical superoscillation spot. The dual-mode modulation of the central focal spot and the sidelobe annulus of the optical superoscillation excites a fluorescent signal, and the backscattered fluorescent signal is received by the microscopic imaging module 5; the fluorescent signal is introduced into the beam conversion module 6 through the dichroic mirror 4. The filter wheel 20 filters out the background stray light other than the target fluorescent signal by motor control according to the excitation light selection timing; the converging lens 21 focuses the fluorescent signal; the pinhole wheel 22 filters out the background fluorescent signal excited by the personalized optical superoscillation sideband and controls the intensity of the detectable fluorescent signal; the beam conversion lens group controls the coupling of the fluorescent signal into the fiber array 25 at the input end of the array detector 7; each signal channel of the array detector 7 converts the received fluorescent signal into an electrical signal, and the electrical signal is input into the computer 8 after amplification and conditioning.

[0039] The computer 8 collects, processes, and displays the electrical signals measured by the system, and issues control signals for wavelength selection, beam scanning, sample stage movement, filter wheel, and pinhole wheel; through two-dimensional beam scanning and one-dimensional or three-dimensional movement of the sample 26, the array detector obtains a data array corresponding to the two-dimensional or three-dimensional information of the measured sample.

[0040] Within an illumination area the size of an Airy disk in optical superoscillation, adjacent fluorescent dye-labeled points may be simultaneously excited. What the array detector 7 actually receives is a mixed-frequency signal generated by different fluorescence-labeled positions. Using an algorithm based on the time-division modulation (relative to the same labeled position) imaging characteristics, the mixed-frequency information from different spatial position points (relative to the same measurement time) is distinguished. Among them, the central focal spot modulation excitation is similar to the Gaussian spot illumination + array detector technology, while the sidelobe annulus modulation illumination is similar to the multi-focus parallel structured illumination and the array detector reception. Through the Richardson-Lucy (RL) deconvolution algorithm, the microscopic images corresponding to the two modulation excitation models are reconstructed respectively. Given that the signal spectral ranges loaded by the two-mode modulation excitations are inconsistent, the RL multi-dimensional deconvolution method will be used to stitch the information of the two frequency bands, and finally the super-resolution image obtained by the optical superoscillation modulation excitation is reconstructed.

[0041] See Figure 3 , a super-resolution microscopy method integrating optical superoscillation excitation and array detection reception in this embodiment includes the following processes:

[0042] 1) According to the scanning imaging time and the spatial positions of the corresponding signal channels of the array detector in the optical path of the detection system, the numerical array measured by the array detector is divided into two categories: at the same detection time, the measurement data of all signal channels are classified as the information obtained by the central focal spot modulation excitation; at the same detection position, the measurement data of all signal channels are classified as the information obtained by the sidelobe annulus modulation excitation.

[0043] 2) For the case of central focal spot modulation excitation, the optical fiber on the optical axis of the optical path of the detection system in the fiber array is used as the central detection channel. The signals obtained by other detection channels at each scanning position are translated and summed towards the central detection channel to obtain the sample information corresponding to the scanning position point, and the initial image is obtained according to the scanning time sequence.

[0044] 3) For the case of sidelobe annulus modulation illumination, the microscopic images obtained by each channel of the array detection are filtered, summed, and normalized to obtain the initial image of the sample.

[0045] 4) According to the blind deconvolution method described by the following formula, image reconstruction is performed:

[0046]

[0047] Among them, F and F -1 respectively represent the Fourier and inverse Fourier operations; * represents the complex conjugate; k is the number of iterations; the optical transfer function OTF of the microscopic imaging system can be obtained through confocal microscopy theory. When Iteration stops when it is lower than the set threshold, and the reconstructed images corresponding to sidelobe annulus modulation and central focal spot modulation illumination can be obtained respectively and

[0048] 5) Use the following multi-dimensional RL deconvolution method to stitch the reconstructed images corresponding to the excitation of the central focal spot and the sidelobe annulus modulation

[0049] The initial estimated object is obtained by the following formula

[0050]

[0051] In the formula, max() represents taking the maximum value of the image light intensity

[0052] Then substitute I obj-c and I obj-s in turn according to the following steps, and iterate to reconstruct the super-resolution image

[0053]

[0054] When is lower than the set threshold, iteration stops, and the reconstructed super-resolution image will be obtained

Claims

1. A super-resolution microscopy method integrating optical super-vibration excitation and array detection and reception, characterized in that, it includes the following steps: 1) The excitation light emitted by the light source module enters the beam control module after being filtered by the wavelength selection module; The beam modulation module regulates the amplitude, phase, and polarization distribution of the beam; the regulated beam forms an optical super-vibration field composed of a central focal spot and a sidelobe annulus inside the test sample through a dichroic mirror and a microscopy imaging module; the fluorescence signal excited by the optical super-vibration modulation enters the beam conversion module through the microscopy imaging module and the dichroic mirror; the fluorescence signal is received by the array detector through the beam conversion module; the collected fluorescence signal is converted into an electrical signal and input into a computer for processing; by controlling the two-dimensional scanning of the beam and the movement of the sample, as well as the selection of wavelength, filter, and pinhole, the system obtains a two-dimensional / three-dimensional data array group; 2) The data obtained by the array detector at the same detection moment are grouped together, the data obtained by each detection channel are translated and superimposed to obtain the fluorescence signal of the scanning point corresponding to the central focal spot modulation excitation; according to the timing and spatial position of the scanning point, the initial measurement image corresponding to the central focal spot modulation excitation is obtained, and then the super-resolution microscopy image corresponding to the central focal spot modulation excitation is obtained by using the deconvolution algorithm; 3) The data obtained by the array detector at the same detection position are grouped together, the image data obtained by each detection channel at different times are filtered and superimposed to obtain the initial measurement image corresponding to the sidelobe annulus modulation excitation, and then the super-resolution microscopy image corresponding to the sidelobe annulus modulation excitation is obtained by using the deconvolution algorithm; 4) The super-resolution images obtained in step 2) and step 3) are weighted and superimposed to obtain the initial measurement image of the optical super-vibration modulation excitation. The deconvolution algorithm is used to stitch the super-resolution images of different spatial frequency bands of the central focal spot and the sidelobe annulus modulation excitation, and finally the super-resolution image obtained by the optical super-vibration modulation excitation is reconstructed.

2. The super-resolution microscopy method integrating optical super-vibration excitation and array detection and reception according to claim 1, characterized in that, in step 1), the full width at half maximum of both the central focal spot and the sidelobe annulus is less than the diffraction limit, and the peak light intensities of the two are basically equal; within the diffraction limit range, it includes a central circular focal spot and at least one annular spot; the central focal spot and the sidelobe annulus constitute a dual-mode modulation illumination.

3. A super-resolution microscopy system integrating optical super-vibration excitation and array detection and reception, characterized in that, for implementing the super-resolution microscopy method of integrating optical super-vibration excitation and array detection and reception according to claim 1 or 2, including: A light source module, providing the excitation light required for exciting different fluorescent dyes; A wavelength selection module, sequentially or time-division selecting different excitation wavelengths according to different fluorescent dyes; A beam modulation module, collimating the excitation beam into a parallel beam and controlling the amplitude, phase, and polarization distribution of the beam; A dichroic mirror, splitting and transmitting the excitation beam and the fluorescence signal beam; A microscopy imaging module, realizing two-dimensional fast scanning modulation excitation of optical super-vibration and reception of fluorescence signals; including a two-dimensional galvanometer scanning device, a scanning lens, a tube lens, and a microscopy objective; A sample stage, fixing and moving the sample to be measured; A light field conversion module, filtering background stray light and controlling the coupling of fluorescence signals into the fiber array; An array detector that converts the fluorescence signals output from the fiber optic array into multi-channel electrical signals respectively; A computer that collects and processes multi-channel sample information, displays the reconstructed super-resolution image, and issues scanning control signals, as well as control signals for wavelength, pinhole, and filter selection.

4. The super-resolution microscopy system integrating optical super-vibration excitation and array detection and reception according to claim 3, characterized in that, the light source module is one of a combination of multiple semiconductor lasers, a white light laser, or a supercontinuum laser.

5. The super-resolution microscopy system integrating optical super-vibration excitation and array detection and reception according to claim 3, characterized in that, the wavelength selection module uses an AOTF to select the required excitation wavelength according to the fluorescent dye; a single-mode polarization-maintaining fiber is connected to the AOTF to achieve the input and output of the illumination beam.

6. The super-resolution microscopy system integrating optical super-vibration excitation and array detection and reception according to claim 3, characterized in that, the beam modulation module includes a collimating lens, a half-wave plate, amplitude, phase, and polarization modulators.

7. The super-resolution microscopy system integrating optical super-vibration excitation and array detection and reception according to claim 3, characterized in that, the light field conversion module includes a beam conversion lens group composed of a collimating lens, a filter wheel, a pinhole wheel, and a doublet lens.

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