An ultrahigh spatiotemporal resolution detection imaging method and system

Abstract

The application discloses a kind of ultra-high space-time resolution detection imaging method and system, it is related to optical technology field, including the following steps: to multi-wavelength short pulse laser photon space-time dynamic modulation, make single pulse local irradiation field photon number in single photon / few photon state;The patterned photon distribution microshrink after photon space-time dynamic modulation is carried out on the sample containing fluorescent molecule under few photon irradiation two / multi-photon excitation, collects two / multi-photon fluorescence;Through high space-time resolution detection means, obtain the spectral information, life information and spatial position information of two / multi-photon fluorescence, reconstruct the fluorescence information in field of view, complete the entire field of view ultra-resolution imaging and positioning.The application can realize large field of view, high frame frequency, ultra-resolution fluorescence detection imaging, for solving the major scientific problem of "optical diffraction limit", realize ultra-resolution fluorescence microscopy technology innovation provides opportunity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical technology, more particularly to a super-high space-time resolution detection imaging method and system. BACKGROUND

[0002] Super-resolution fluorescence microscopy, which breaks the diffraction limit of light by using the spatiotemporal effects of light interaction with fluorescent molecules, has made optical microscopy enter the nanometer era, and even can observe subcellular ultra-fine structures of 10 nm scale, and has been widely used in the field of life science and medical research. One of the super-resolution fluorescence microscopy techniques, stimulated emission depletion (STED) microscopy, uses two lasers, excitation light and depletion light, and cooperates with specially designed fluorescent molecules to achieve super-resolution nanoscale imaging by relying on the transient transfer between the ground state and the excited state of the fluorescent molecules. Due to the need to use high depletion light power, it will cause photobleaching and phototoxicity to fluorescent molecules and cells in practical application, thus greatly limiting its usable range. STED imaging is based on point scanning, and the imaging speed and field of view cannot achieve the corresponding effect of wide-field imaging. Another type of super-resolution fluorescence microscopy includes photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), which achieve super-resolution imaging by imaging and locating single molecules of fluorescent groups with light switching function. PALM / STORM requires the use of two lasers of different wavelengths, one of which is a low-energy laser that irradiates cells to activate a few fluorescent molecules distributed sparsely, and then the other laser is used to excite to obtain a fluorescence image and precisely locate these fluorescent single molecules, thus breaking the optical diffraction limit and achieving super-resolution imaging. The outstanding advantage of PALM / STORM technology is its extremely high spatial resolution. Single-molecule localization super-resolution microscopy technology uses simple fluorescence switching to achieve ultra-high precision fluorescence imaging at the nanometer scale of subcellular structures. Two-color STORM can image the microtubule network and grid-like indentation organization in fixed mammalian cells, with a resolution of 20 nm. However, these technologies are random and each fluorescent molecule is excited one by one to complete the ultra-high resolution imaging: PALM technology records the signal of each fluorescent molecule until bleaching, and calculates its precise position by point spread function, and this process needs to be reconstructed repeatedly to obtain and locate the positions of tens of thousands of fluorescent molecules one by one, and finally the positions of all molecules are superimposed to form a complete high-resolution image. At the same time, the PALM technology has low photon yield of fluorescent proteins, large amount of calculation and long time consumption, and it is difficult to real-time distinguish the dynamic time scale of the life activities and interactions of organelles in cells, and there is a risk of phototoxicity; while STORM technology controls the bright-dark state transformation of fluorescent molecules through multiple cycles, and improves the positioning accuracy by Gaussian fitting, and relies on fixed sample labeling, which may introduce background noise.

[0003] The existing mainstream super-resolution microscopic imaging still originates from the traditional wave optics theory. The STED technology uses the traditional wave optics method combined with the molecular photochemical characteristics to realize the control of the point spread function. The PALM / STORM technology uses weak light to activate the molecules. Since the activated molecules are sparsely distributed at a distance greater than the optical diffraction limit, the super-resolution microscopic imaging is realized by observing the fluorescence positioning emitted under strong light excitation. The light activation and excitation are also based on the traditional wave optics method. After Planck introduced the concept of quantum, Einstein proposed the light quantum hypothesis based on the quantum concept to explain the photoelectric effect, Dirac proposed the light emission and absorption quantum theory, in 1931 For the first time, the quantum mechanics method was introduced to the electron energy level in the atom, and the two-photon absorption effect was theoretically predicted. However, so far, although the two-photon absorption effect has been widely used in two-photon microscopy, three-dimensional micro-nano processing, and combined with the STED super-resolution microscopic technology, the super-resolution scanning positioning imaging technology has been developed, but the related research is still based on the semi-classical theory using the traditional wave optics method to process the light field.

[0004] The two-photon absorption effect refers to the physical effect that a molecule in the ground state simultaneously absorbs two photons with energy lower than the transition energy band of the molecule to jump from the ground state to the excited state. When the energy of the two photons is different, it is non-degenerate two-photon absorption, and when the energy of the two photons is the same, it is degenerate two-photon absorption. Its process can be described by a quantum electrodynamics Feynman diagram. The molecule absorbs a photon to reach a virtual intermediate state energy level matching its energy, and absorbs another photon within its lifetime to jump to an excited state. The lifetime of the virtual intermediate state energy level follows the Heisenberg uncertainty principle, which is about several femtoseconds. Therefore, two-photon nonlinear absorption has significant time and space constraints, especially under the excitation of extremely weak light field with low photon density.

[0005] Therefore, how to break through the "optical diffraction limit" in traditional microscopic imaging, re-examine the two-photon absorption effect from the perspective of light quantum characteristics, realize efficient large-field patterned two-photon absorption excitation fluorescence detection and positioning under extremely weak light flux irradiation, and realize super-high resolution microscopic imaging, which breaks the long-standing contradiction between high resolution and high efficiency in traditional two-photon microscopic imaging technology, is a problem that those skilled in the art need to solve. SUMMARY

[0006] Therefore, the present application provides a kind of super-high time-space resolution detection imaging method and system, solve the problems existing in the background art.

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

[0008] A kind of super-high time-space resolution detection imaging method, comprising the following steps:

[0009] S1, photonically and temporally dynamically modulating a multi-wavelength short pulse laser to make the photon number in a single pulse localized irradiation field be in a single photon / low photon state;

[0010] S2, micro-fabricating the patterned photon distribution after the photonically and temporally dynamic modulation to a sample containing fluorescent molecules for two- / multi-photon excitation under low photon irradiation, and collecting two- / multi-photon fluorescence;

[0011] S3, obtaining spectral information, lifetime information and spatial position information of the two- / multi-photon fluorescence by high spatiotemporal resolution detection means, reconstructing fluorescence information in the field of view, and completing super-resolution imaging and positioning of the entire field of view.

[0012] Optionally, in S1, the multi-wavelength short pulse laser is photonically and temporally dynamically modulated, specifically as follows:

[0013] From the viewpoint of optical quanta, the laser Gaussian beam is shaped into a flat-top beam, and then independent discrete parallel excitation points with a distance greater than the diffraction limit of the imaging system are designed and modulated by the pixels of a spatial light modulator, so as to realize the spatiotemporal dynamic modulation of photon energy, number, time domain and space domain.

[0014] Optionally, the two- / multi-photon excitation under low photon irradiation in S2 is divided into the following different cases:

[0015] In the process of degenerate two-photon excitation, the same wavelength pulses after the photonically and temporally dynamic modulation are projected onto a sample containing dispersed fluorescent molecules, and two photons simultaneously acting on a molecular scale spatial range is a necessary condition for the occurrence of the degenerate two-photon excitation process;

[0016] In the process of non-degenerate two-photon excitation, two single photon / low photon pulses of different wavelengths after the photonically and temporally dynamic modulation are combined and projected onto a sample containing dispersed fluorescent molecules, and two photons simultaneously acting on a molecular scale spatial range is a necessary condition for the occurrence of the two-photon excitation process, and the double-beam pulse delay is adjusted by an optical element.

[0017] Optionally, in S3, the spectral information, lifetime information and spatial position information of the two- / multi-photon fluorescence are obtained, specifically as follows:

[0018] The single molecule fluorescence is detected with high spatiotemporal resolution, and the time and space information of the two- / multi-photon excitation and fluorescence is obtained; a large number of discrete point surface excitations with a distance greater than the optical diffraction limit are formed by the pixel pattern design of the photonically and temporally dynamic modulation, and the two- / multi-photon fluorescence of single molecules and its distribution map are obtained; and high spatiotemporal resolution dynamic microscopic imaging is obtained by switching the discrete point positions of the surface excitation.

[0019] An ultrahigh spatiotemporal resolution detection imaging system comprises:

[0020] A multi-wavelength short pulse laser module is used to generate laser pulses with different wavelengths and different photon numbers.

[0021] A photon spatiotemporal dynamic modulation module is used to modulate the multi-wavelength short pulse laser to obtain a discrete single-photon / few-photon excitation point array.

[0022] A large field of view projection imaging module is used to project a designed patterned discrete single-photon / few-photon excitation point array light field into a microscopic sample.

[0023] A light-matter interaction module is used to focus the discrete single-photon / few-photon excitation point array to the microscopic sample for two-photon excitation.

[0024] A fluorescence detection and signal acquisition and processing module is used to collect fluorescence signals in the microscopic sample, and detect fluorescence spectrum, fluorescence lifetime and single-molecule fluorescence imaging according to the fluorescence signals.

[0025] Optionally, the photon spatiotemporal dynamic modulation module comprises a cascaded liquid crystal spatial light modulator and a digital micromirror device.

[0026] The cascaded liquid crystal spatial light modulator is used to modulate the visible and near-infrared waveband, and is combined with a light source system to select wavelengths, thereby constituting a large field of view discrete point synchronous excitation light source with coordinated modulation of photon energy, number, delay time and spatial position.

[0027] The digital micromirror device is used to modulate the double-color double-beam light field after beam alignment and pulse synchronization, thereby realizing large field of view discrete point synchronous excitation.

[0028] Optionally, the light-matter interaction module comprises a motion platform and a microscope stage, wherein the motion platform is arranged above the microscope stage, and the microscopic sample to be measured is placed on the motion platform.

[0029] Optionally, the fluorescence detection and signal acquisition and processing module comprises a weak light fluorescence spectrum detection unit, a weak light fluorescence lifetime detection unit and a fluorescence imaging unit.

[0030] The weak light fluorescence spectrum detection unit is used to explore the time and spectral response characteristics of two-photon fluorescence under few-photon conditions through a PMT detector and a spectrometer.

[0031] The weak light fluorescence lifetime detection unit is used to measure the two-photon fluorescence lifetime through a single-photon counting APD and a time-dependent single-photon counting method, thereby obtaining two-photon fluorescence lifetime information.

[0032] A fluorescence imaging unit is used for collecting and processing the fluorescence signal by using an EMCCD, and super-resolution imaging of the whole field of view is realized by multiple exposure superposition in a structured light field scanning process.

[0033] Compared with the prior art, the application provides an ultrahigh space-time resolution detection imaging method and system, which clarifies the quantum space-time statistical law and space-time mechanism of photon-molecule interaction of two-photon absorption effect of different fluorescent molecules under the condition of few photons, establishes a photon space-time dynamic modulation method to provide a scientific experiment basis, and lays a foundation of scientific theory and core key technology for developing a large field of view high frame rate two-photon single molecule fluorescence super-resolution imaging technology. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0035] Figure 1 A schematic diagram of the ultrahigh space-time resolution detection imaging system provided by the application is shown in the figure.

[0036] Figure 2 A design diagram of the ultrahigh space-time resolution detection imaging system provided by the application is shown in the figure.

[0037] Figure 3 A working principle diagram of the weak light fluorescence lifetime detection provided by the application is shown in the figure.

[0038] Figure 4 A multi / single photon fluorescence excitation degree distribution diagram provided by the application is shown in the figure.

[0039] Figure 5 A distribution curve of fluorescent photons with spatial position provided by the application is shown in the figure.

[0040] Figure 6 A discrete point position control principle and resolution limit criterion principle diagram of surface excitation provided by the application is shown in the figure.

[0041] Figure 7 A two-photon discrete point fluorescence surface excitation and detection principle diagram provided by the application is shown in the figure. DETAILED DESCRIPTION

[0042] Clearly, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present application.

[0043] The present application relates to a kind of weak light flux irradiation under the realization of high efficiency large field of view patterned two-photon absorption excitation fluorescence detection and positioning method, realize super high resolution microscopic imaging, break the contradiction between high resolution and high efficiency which exists long-term in traditional two-photon microscopic imaging technology.Therefore, the present application is from the basic quantum theory of two-photon absorption effect, combine the latest achievements in current light field regulation, single-photon detection, super-resolution microscopic imaging etc.Technical field, develop photon space-time dynamic modulation, super-resolution single molecule fluorescence excitation and detection imaging technology, through the research on quantum statistical law and space-time mechanism of light and matter interaction, provide scientific basis for breaking the major scientific problem of optical diffraction limit, develop new principle, new method and new technology of optical super-resolution fluorescence microscopic technique for front research of life science.

[0044] Specifically, the embodiment of the present application discloses a kind of super high space-time resolution detection imaging method, comprising the following steps:

[0045] S1, photon space-time dynamic modulation is carried out to multi-wavelength short pulse laser, so that the number of photons in single pulse local irradiation field (single pixel) is in single-photon / low-photon state;

[0046] S2, the patterned photon distribution after photon space-time dynamic modulation is microscopically shrunk to sample containing fluorescent molecules to carry out two-photon / multi-photon excitation under low-photon irradiation, and two-photon / multi-photon fluorescence is collected;

[0047] S3, through high space-time resolution detection means, the spectral information, lifetime information and spatial position information of two-photon / multi-photon fluorescence are acquired, the fluorescence information in field of view is reconstructed, and the super-resolution imaging and positioning of entire field of view are completed.

[0048] The existing super-resolution fluorescence microscopic imaging technology has a restrictive relationship between spatial resolution, time resolution and field of view, and it is difficult to realize large field of view, high frame frequency and super-resolution microscopic imaging.In the embodiment, two-photon excitation in focal point is realized by using the above-mentioned photon preparation method, discrete multi-focus large-area scanning excitation is formed by controlling high-frequency switching pixels of spatial light modulator, large field of view and high frame frequency fluorescence excitation are realized, super-diffraction single molecule discrete excitation is realized based on the space-time constraint effect of two-photon fluorescence excitation, and large field of view, high frame frequency and super-resolution fluorescence detection imaging are realized.

[0049] Furthermore, in S1, photonic spatiotemporal dynamic modulation is performed on multi-wavelength short-pulse lasers, specifically as follows:

[0050] From the perspective of photons, this method uses photons as the control object. After shaping the laser Gaussian beam into a flat-top beam, it utilizes millions of pixels of a spatial light modulator to design and modulate hundreds of thousands of independent discrete parallel excitation points with distances between them greater than the diffraction limit of the imaging system. This enables large-field-of-view discrete-point excitation and single-molecule fluorescence detection. By using the spatial light modulator to dynamically modulate the spatial position of the discrete excitation points at a high frame rate, programmable large-field-of-view discrete-point high-frame-rate scanning excitation is achieved, enabling dynamic and rapid super-resolution fluorescence imaging. This embodiment integrates multiple spatial light modulators in series, enabling spatiotemporal dynamic modulation of multiple parameters such as photon energy, quantity, time domain, and spatial domain. This is the most important technical innovation and unique feature of this solution.

[0051] Unlike existing light field manipulation techniques based on traditional wave optics, the "photon spatiotemporal dynamic modulation" in this embodiment uses photons, the basic elements constituting the light field, as the manipulation object. It uses a pixelated spatial light modulator to modulate the extremely weak light field of multi-wavelength ultrashort pulse lasers, thereby achieving dynamic modulation of measurable parameters such as the energy, quantity, time, and spatial position of photons within a single pixel and single pulse.

[0052] Furthermore, the two / multiphoton excitation under few-photon irradiation in S2 can be divided into the following different cases:

[0053] During degenerate two-photon excitation, a pulse of the same wavelength after spatiotemporal dynamic modulation of photons is projected onto a sample containing dispersed fluorescent molecules. The simultaneous action of two photons on a molecular-scale spatial range is a necessary condition for the degenerate two-photon excitation process to occur.

[0054] In non-degenerate two-photon excitation, two single-photon / minor-photon pulses of different wavelengths, after being spatiotemporally modulated by photons, are combined and projected onto a sample containing dispersed fluorescent molecules. The simultaneous action of two photons on a molecular-scale spatial range is a necessary condition for the non-degenerate two-photon excitation process to occur. The delay of the two-beam pulses is adjusted by optical elements.

[0055] In this embodiment, two modulated photons of different energies are simultaneously subjected to multi-point discrete single-photon / few-photon irradiation across the entire field of view to excite the two-photon absorption effect of a single molecule, highlighting the particle nature of the (optical) quantum process excited by two photons. This breakthrough in principle overcomes the optical diffraction limit and has significant innovation in the research paradigm.

[0056] In the embodiment, weak light excitation is adopted to avoid the damage of strong light excitation of STED super-resolution fluorescence microscopy to cells; the random single molecule activation / excitation mode of PALM / STORM super-resolution fluorescence microscopy is improved to a programmable control single molecule excitation mode, which can greatly improve the positioning speed of single molecule super-resolution imaging and realize high-speed dynamic super-resolution imaging of ultra-fine structures of living cells; by using two-photon effect, visible and near-infrared wavelength photons are used to realize effective excitation of spontaneous fluorescence molecules in living tissues and cells in ultraviolet or deep ultraviolet band, and dynamic super-resolution imaging of spontaneous fluorescence molecules of living tissues and subcellular structures can be realized.

[0057] Further, in S3, the spectral information, lifetime information and spatial position information of the two / multi-photon fluorescence are acquired, specifically:

[0058] Single photon counters, EMCCDs, high-sensitivity spectrometers and photomultiplier tubes (PMTs) are used to detect single molecule fluorescence with high temporal and spatial resolution, acquire the time and spatial information of two / multi-photon excitation and fluorescence, form a large number of discrete point surfaces with excitation point spacing greater than the optical diffraction limit by pixel patterning design of photon temporal and spatial dynamic modulation, and obtain the two / multi-photon fluorescence of single molecules and its distribution map, which provides experimental data for studying the quantum temporal and spatial statistical rules of photon-molecule interaction; when biological samples such as living cells are used, high temporal and spatial resolution dynamic microscopic imaging is obtained by high-speed switching of the discrete point positions of surface excitation.

[0059] Based on the above research, the ultra-high temporal and spatial resolution detection imaging system developed in the embodiment mainly consists of a multi-wavelength short pulse laser module 116, a photon temporal and spatial dynamic modulation module 117, a large field of view projection imaging module 118, a light-matter interaction module 120, a fluorescence detection and signal acquisition and processing module 119, as shown in Figure 1 , specifically:

[0060] The multi-wavelength short pulse laser module 116 includes a laser 101, a pulse number controller 102 and a single pulse photon number controller 103, and is used to generate laser pulses 301 with different wavelengths and different photon numbers 302;

[0061] The photon temporal and spatial dynamic modulation module 117 includes a spatial light modulator 104 and a first beam splitter 105, and is used to modulate the multi-wavelength short pulse laser to obtain a discrete single photon / low photon excitation point array;

[0062] The large field of view projection imaging module 118 includes a large field of view microscopic objective 109, an infinite field of view imaging cylinder lens 107 and a reflector 108, and is used to project the designed patterned discrete single photon / low photon excitation point array light field into the microscopic sample 111;

[0063] The light and matter interaction module 120 is used for focusing the discrete single photon / low photon excitation point array to the sample 111 for two-photon excitation. The sample 111 to be detected is located on the high-precision motion platform 114 and the microscope stage 115.

[0064] The fluorescence detection and signal acquisition and processing module 119 includes a single photon counter, an EMCCD, a high-sensitivity spectrometer, a photomultiplier tube (PMT), and other detectors 113 and a second beam splitter 106, which are used to collect the fluorescence signal in the sample 111. According to the fluorescence signal, the fluorescence spectrum, the fluorescence lifetime, and the single-molecule fluorescence imaging are detected. The computer 204 analyzes the detected fluorescence signal and reconstructs the spatially resolved image.

[0065] Referring to Figure 1 and Figure 3 The laser emitted by the laser 101 passes through the pulse number controller 102 and the single-pulse photon number controller 103, and is used as the entire multi-wavelength short pulse laser module 116. The pulse number controller 102 and the single-pulse photon number controller 103 can be controlled by the computer 204 programming. The single pulse 301 and the photon number 302 in the single pulse can be controlled. Then, the photon space-time dynamic modulation module 117 modulates the excitation light beam by the spatial light modulator 104 (which can be a liquid crystal light spatial modulator SLM or a digital micromirror DMD). The first beam splitter 105 satisfies the turning and emission of the light path. After passing through the second beam splitter 106, the light beam is incident to the large-field projection imaging module 118. The light beam is formed into a reduced patterned light field by the infinity imaging tube lens 107, the reflector 108, and the large-field microscope objective 109. Then, the light beam is irradiated to the light and matter interaction module 120 composed of the high-precision motion platform 114 and the microscope stage 115. The fluorescence detection and signal acquisition and processing module 119 collects the fluorescence 112 generated by the incident light beam 110 irradiated to the light and matter interaction module 120 by the detector 113.

[0066] Although the fluorescence super-resolution imaging technology based on fluorescence molecule labeling such as STED / PALM / STORM has been commercialized and applied to the research of biophotonics, its applicability is seriously dependent on the labeling of specific fluorescence molecules to biological samples, the corresponding specific excitation wavelength laser device and the specific illumination method, and the disadvantages in imaging field, frame rate, universality and other aspects limit its technical practicability, especially in the application of bio-vivo imaging. By selecting and matching the near-infrared and visible wavelength, the non-degenerate two-photon effect effective excitation wavelength is pushed to the deep ultraviolet band, and the self-fluorescence molecules such as nicotinamide adenine dinucleotide and folate adenine dinucleotide in the living tissue are excited, realizing the single-molecule super-resolution fluorescence imaging without fluorescence molecule labeling, thereby developing into a universal super-resolution imaging technology. Two-photon excitation not only has a larger absorption coefficient, a wider absorption spectrum and a more flexible light source combination, but also has a higher spatial resolution. The ultra-high space-time resolution detection imaging system uses a photon space-time dynamic modulation module 117 to realize large field of view high frame rate two-photon single-molecule fluorescence super-resolution imaging by synergistic modulation of the energy, number, delay time and spatial position of the excitation photons, combined with fluorescence spectrum, fluorescence lifetime measurement and single-molecule fluorescence imaging detection imaging technology.

[0067] 1) Single-photon / low-photon excitation two-photon quantum space-time mechanism research method

[0068] The few-photon / single-photon excitation referred to in this embodiment refers to the number of photons per unit area for excitation by a single pulse is from a single photon to several thousand photons, and the spot diameter under tight focusing condition is in the range of hundreds of nanometers. After attenuating the ultra-short pulse laser source into an extremely weak light field, the photon space-time dynamic modulation module 117 is used to amplitude modulate a single pixel of the spatial light modulator 104 to realize single-pulse few-photon / single-photon excitation. The modulated photons of different energies are collectively irradiated to the same spot area to excite two-photon single-molecule fluorescence. Through photon space-time dynamic modulation technology, the excitation photons are modulated in terms of photon energy, photon number, delay time, spatial position and other physical parameters. Single-molecule excitation spectrum, emission fluorescence spectrum, fluorescence lifetime and single-molecule fluorescence super-resolution positioning imaging detection means are used to obtain the relevant experimental data of the interaction between two-photon effect photons and molecules. Reference Figure 4 For the fluorescence imaging technology of a large number of photon excitation, the photon distribution in the light field satisfies the classical light intensity fluctuation distribution 402, and the instantaneous position 401 of the large number of fluorescence molecules excited thereby is limited by the optical diffraction limit. For the fluorescence imaging technology of few-photon or single-photon excitation proposed by the present application, the spatial position of a single photon is determined, and the random occurrence probability in the light field conforms to the Gaussian light intensity fluctuation distribution 404, so the instantaneous position 403 of the fluorescence molecule excited by single / few photons is determined, and the range of the fluorescence molecule detection position is extremely small, far less than the diffraction limit, seeFigure 4 Comparison between the middle 403 and 401. As Figure 5 The statistical results of single / few photon excitation fluorescence show that, by adjusting the number of pulses and the number of photons in a single pulse, the number of excitations decreases from 501 to 502 within a certain radius interval Δr; when the number of photons in a single pulse is adjusted, the number of photons within the radius 505 decreases rapidly from the central peak 503 to half of the central value 504, showing a significant spatial confinement effect.

[0069] Using single-molecule super-resolution localization imaging algorithm, the uncertainty of two / multi-photon fluorescence emission in time and spatial position is determined. Through the quantum space-time statistical rules and space-time mechanism of photon-molecule interaction, the excitation time and spatial position of the projection fluorescence molecules are accurately reconstructed, breaking the technical bottleneck of existing super-resolution microscopy that time and spatial resolution cannot be compatible, and developing large field of view, ultra-high space-time resolution, high-speed fluorescence microscopy imaging technology.

[0070] 2) Development of ultra-high space-time resolution detection imaging system

[0071] The ultra-high space-time resolution detection imaging system mainly includes a multi-wavelength short pulse laser module 116, a photon space-time dynamic modulation module 117, a large field of view projection imaging module 118, a light-matter interaction module 120, a fluorescence detection and signal acquisition processing module 119, as shown in Figure 1 .

[0072] First, the photon space-time dynamic modulation module 117 is used to modulate the extremely weak pulse light field after attenuation to obtain the programmable discrete point plane excitation photons required by the experimental research. Specifically, the photon space-time dynamic modulation module 117 includes two sets of cascaded liquid crystal spatial light modulators (SLM) or digital micro-mirror devices (DMD);

[0073] The cascaded liquid crystal spatial light modulator is used to modulate the visible and near-infrared waveband, combined with the light source system for wavelength selection, to form a large field of view discrete point synchronous excitation point light source 201 for the coordinated modulation of photon energy, number, delay time and spatial position, providing a multi-parameter tunable excitation light source for related experiments on the photon-molecule interaction rules of two-photon effect using the light-matter interaction module 120;

[0074] The digital micro-mirror device is used to modulate the double-color double-beam light field after beam alignment and pulse synchronization, realizing synchronous excitation of the large field of view discrete point synchronous excitation point light source 201. Using the high refresh rate of DMD up to 20 kHz, high-frequency large field of view discrete point synchronous excitation is realized, providing an efficient excitation light source for the development of large field of view, ultra-high space-time resolution, high-speed fluorescence microscopy imaging technology.

[0075] Secondly, the photon micro-structured by the photon space-time dynamic modulation is projected onto the sample 202 containing fluorescent molecules by the large field of view projection imaging module 118 for two-photon excitation. The spatially discrete two-photon fluorescence 203 is collected by the fluorescence detection and signal acquisition processing module 119 and the light and matter interaction module 120, and then transmitted to the fluorescence detection and signal acquisition processing module 119 for detection and imaging. In the process of detection and imaging, the three-dimensional position drift of the mechanical device such as the translation stage is inevitable. A position alignment system is developed by using the method of real-time feedback of multiple marker points of the sample stage to correct the position drift.

[0076] The research on the fluorescence emission characteristics of fluorescent molecules is generally carried out by using a spectrometer and a fluorescence lifetime analyzer. However, these analysis methods still face the following key difficulties in the two-photon fluorescence characterization based on photon space-time dynamic modulation: (a) low response sensitivity, usually >100 photons / count for visible light, which cannot meet the needs of single-photon level quantum space-time mechanism research; (b) low spatial resolution, which is limited by the diffraction limit and cannot characterize the fluorescence emission characteristics of nanoscale. Compared with the traditional degenerate two-photon fluorescence under high light intensity, the two-photon fluorescence signal excited by single pulse and few photons / single photon is weaker. Therefore, the fluorescence detection and signal acquisition processing module 119 designed in the embodiment includes: a weak light fluorescence spectrum detection unit, a weak light fluorescence lifetime detection unit, and a fluorescence imaging unit.

[0077] The weak light fluorescence spectrum detection unit is used to explore the time and spectral response characteristics of two-photon fluorescence under few-photon conditions by using a PMT detector with high time resolution characteristics and a spectrometer;

[0078] The weak light fluorescence lifetime detection unit is used to measure the two-photon fluorescence lifetime by using a single-photon counting APD and a time-correlated single-photon counting method (TCSPC), and obtain two-photon fluorescence lifetime information;

[0079] The fluorescence imaging unit is used to collect and process the fluorescence signal by using an EMCCD with high signal-to-noise ratio, and realize super-resolution imaging of the entire field of view by multiple exposure superposition in the process of structured light field scanning. A deep learning-based pattern reconstruction algorithm is developed to further improve the resolution of the image, realizing the leap from wide-field high-resolution imaging to super-resolution imaging. At the same time, combined with the user-optimized human-computer interface, all modules work cooperatively.

[0080] The ultra-high space-time resolution detection and imaging system is integrated and designed as shown in Figure 2The light source system is integrated with the space light modulator 104 to generate a two-photon fluorescence excitation spectrum, an emission spectrum, and a fluorescence lifetime characteristic by photon space-time dynamic modulation. The experimental detection means are provided for verifying and analyzing the action mechanism of core physical quantities such as photon energy, space, and time of two-photon excitation of fluorescent molecules, and energy levels and material characteristics such as virtual state lifetime of the molecules. The scientific principles and key technical foundations are provided for developing a large-field-of-view, ultra-high space-time resolution, high-speed fluorescence microscopy technology.

[0081] A) Weak light spectrum detection

[0082] In order to study the two-photon fluorescence excitation process under the condition of few photons or even single photons, it is necessary to perform spectrum measurement on the weak fluorescence signal. An EMCCD and a high-sensitivity spectrometer are used to detect and measure the weak light, and the spectral resolution is better than 0.2 nm. For the equivalent absorption and emission bands of two photons, the spectrometer uses a cryogenic detector to suppress dark current noise and effectively improve the signal-to-noise ratio (1000:1). The output dark count corresponding to the dark current is less than 100 counts / second. In addition, the traditional CCD is designed to allow light sources to shine from the imaging area, that is, a front-illuminated CCD. The photosensitive area of the front-illuminated CCD is at the front end of the silicon plate, so that most of the light sources are absorbed or reflected when passing through the silicon plate. Therefore, the quantum efficiency is limited to 40%, and the sensor cannot detect ultraviolet light. The EMCCD with single-photon response is used in this embodiment, based on the area array back-illumination technology, light is introduced into the PN junction from the back, improving the quantum conversion efficiency, especially the quantum conversion efficiency of the ultraviolet band, with the advantages of low noise, deep ultraviolet sensitivity, high quantum efficiency (peak 95%), etc., providing a guarantee for the detection of weak fluorescence in the ultraviolet emission band in the experiment.

[0083] B) Weak light fluorescence lifetime detection

[0084] In this embodiment, a single-photon counting APD is combined with a time-dependent single-photon counting method (TCSPC) to measure the two-photon fluorescence lifetime, so as to obtain the two-photon fluorescence lifetime information. The working principle is as follows Figure 3The laser 101 is driven by a synchronization signal source, and the sample is excited by high-repetition pulsed laser. The number of laser pulses 301 and the number of photons within a pulse 302 are controllable and adjustable. In each pulse cycle, the fluorescence molecules are excited to emit a photon at most, and the fluorescence signal is detected by a photon detection device. Each photon counting signal falls into a corresponding time window in the time-amplitude converter (TAC) and records the time of photon occurrence. The above process is repeated in the next pulse cycle, and the distribution curve of fluorescence photons over time can be obtained by multiple counting statistics superposition, instead of a large number of photons directly incident on the excited fluorescence molecules. When a large number of photons excite fluorescence, the wave nature of light is manifested, and only the intensity distribution analysis of classical wave optics can be used; when a small number of photons or even a single photon excite fluorescence, the particle nature of light is manifested, and the probability distribution is obtained by using semi-classical theory analysis, such as Figure 4 As shown in Figure 5 The statistical results of multi / less photon excitation fluorescence show that when a large number of photons are excited, the excitation times decrease from 501 to 502 within a certain radius interval Δr; however, when a small number of photons are excited, the number of photons within the radius 505 rapidly decreases from the central peak value 503 to half of the central value 504.

[0085] C) Two-photon fluorescence super-resolution detection

[0086] In the process of imaging by degenerate two-photon fluorescence detection, a beam of excitation light is homogenized by a flat top to illuminate the entire field of view with uniform intensity, and a controllable discrete multi-focus array form of structured light field is generated by a digital micromirror system, and degenerate two-photon fluorescence excitation occurs at the same molecular position; in the process of imaging by non-degenerate two-photon fluorescence detection, a beam of excitation light is homogenized by a flat top to illuminate the entire field of view with uniform intensity, and another beam of wavelength excitation light is generated by a digital micromirror system in the form of a controllable discrete multi-focus array structured light field. In the wide field range, only the region where the spatial positions of the two excitation lights overlap will occur non-degenerate two-photon excitation.

[0087] The generation of discrete multi-foci is realized by a spatial light modulator 104, and the positional relationship of the two excitation light points cannot be smaller than the size of the diffraction limit, that is, the distance between the two excitation light points in the fluorescence molecule sample 202 is smaller than the Rayleigh diffraction limit 608 (0.61λ / NA, where λ is the wavelength of the excitation light source, and NA is the numerical aperture of the projection objective). Referring to Figure 6Case 1: By controlling the positions 601 of two pixels A and B in the spatial light modulator 104, or by changing the magnification 604 of the projection lens, the light field diffusion spots 607 of the corresponding images A' and B' in the fluorescent molecule sample 202 can be completely resolved. Case 2: By controlling the positions 602 of two pixels A and B in the spatial light modulator 104, or by changing the magnification 605 of the projection lens, the light field diffusion spots 608 of the corresponding images A' and B' in the fluorescent molecule sample 202 can be just resolved, corresponding to the Rayleigh diffraction limit distance. Case 3: By controlling the positions 603 of two pixels A and B in the spatial light modulator 104, or by changing the magnification 606 of the projection lens, the light field diffusion spots 609 of the corresponding images A' and B' in the fluorescent molecule sample 202 cannot be resolved. The image distance of the random discrete focal point designed in this invention is set according to Case 1 and Case 2.

[0088] Wide-field imaging is achieved using an array detector. Within each pixel, the photon density within a single pulse is reduced to a few photons. Under these low-photon conditions, the interaction between light and matter exhibits quantum properties; that is, the reduced number of photons within a pulse introduces uncertainty in the spatial location of the photon-molecule interaction. Therefore, for each pixel, the necessary condition for the two-photon excitation process to occur is that two photons must simultaneously act within a molecular-scale spatial range, thus exhibiting randomness.

[0089] This embodiment uses a schematic diagram of the two-photon fluorescence super-resolution detection principle ( Figure 7 As shown, fluorescence occurring at random locations within a pixel is precisely located by fitting the fluorescence signal acquired from the EMCCD. A controllable multifocal array is used to regulate the emission position of fluorescent molecules, activating a fluorescent molecule in a diffraction-limited region at a random location under a single laser pulse. This activator is then individually imaged and precisely located. Subsequently, a fluorescent molecule at another random location is excited during the next laser pulse, and the activation and localization are repeated. Because the multifocal array generated within a given field of view is sufficiently discrete, it ensures that only one non-degenerate two-photon fluorescence is excited within each pixel, preventing signal overlap between pixels. Through multiple iterations of activation and imaging, the positions of more fluorescent molecules are determined. The point spread function of the imaging system is obtained through statistical superposition of image sequences acquired via multiple pulse localization imaging, enabling the detection of imaging resolution. Super-resolution detection of two-photon fluorescence across the entire field of view is achieved through scanning the structured light field and multiple exposure superpositions.

[0090] D) Large field-of-view high-speed super-resolution imaging

[0091] Traditional two-photon fluorescence imaging requires extremely strong illumination to generate effective fluorescence emission. Therefore, most methods employ a focused light source to scan the sample point by point, resulting in significant problems such as low imaging speed and resolution limited by the diffraction limit. This embodiment utilizes the physical mechanism of two-photon absorption effect combined with photon time-space modulation to precisely control the excitation region, enabling effective two-photon fluorescence to occur even under conditions of few or even single photons. This facilitates the use of wide-field illumination to excite two-photon fluorescence.

[0092] During the excitation process, a spatial light modulator 104, which in this embodiment uses a DMD, is used to randomly generate an excitation spatial surface illumination array 702 based on a preset digital pattern 701. The distance 703 between the array focal points is less than or equal to the Rayleigh diffraction limit. To ensure that every pixel of the preset digital pattern 701 can be illuminated, a random generation algorithm traverses all pixels to form a set of multiple discrete digital layouts 704, which are loaded into the DMD to provide time-sequential illumination for the fluorescent molecule sample 202.

[0093] During the imaging process, a high-speed EMCCD array detector is used instead of the point detector commonly used in traditional two-photon fluorescence imaging, achieving wide-field imaging with a large field of view in a single pass, significantly improving imaging speed and facilitating the imaging of living organisms. Unlike random reconstruction techniques such as PALM and STORM, this technique uses the concept of digital surface projection to construct the illumination dot matrix. A developed dot matrix structured light field algorithm is used to generate a structured pattern, which is then imported into the DMD unit. A 4F optical system is used to transfer the illumination photon pattern on the DMD to the back focal plane of the microscope objective. Through the Fourier transform characteristics of the objective, the desired random discrete laser illumination points are generated on the front focal plane of the microscope objective. This scheme can fully utilize the effective numerical aperture of the microscope objective to obtain a high-resolution illumination structured light field, which is beneficial for improving imaging resolution. Simultaneously, the high frame rate switching of the illumination photon pattern using the DMD, through high-speed scanning with a multi-focal array, achieves large field of view, high frame rate, and super-resolution imaging.

[0094] Furthermore, unlike existing wide-field super-resolution imaging techniques such as PALM and STORM, the proposed method in this embodiment does not require specific fluorescent labels and can be applied to any endogenous or exogenous fluorescent sample. For fluorescent molecules with different two-photon absorption spectral positions, matching can be achieved by adjusting the wavelength of the illumination light, enabling the sample to undergo effective two-photon absorption and release of two-photon fluorescence signals, significantly expanding the universality of this technique.

[0095] After obtaining the detection data of two-photon fluorescence imaging under the condition of few photons, a deep learning image reconstruction algorithm is used to establish a large data set from under-sampling low spatial resolution imaging to high sampling high spatial resolution imaging, and a neural network is trained by big data to establish the corresponding connection mapping relationship between the two, so as to realize a fast high spatial resolution image reconstruction algorithm, and realize wide field, high speed and high spatial resolution imaging.

[0096] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the difference from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0097] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detection and imaging with ultra-high spatiotemporal resolution, characterized in that, Includes the following steps: S1. Photon spatiotemporal dynamic modulation of multi-wavelength short-pulse lasers is performed to make the number of photons in the single-pulse local irradiation field in a single-photon / few-photon state. S2. The patterned photon distribution, which has been dynamically modulated by photons in time and space, is miniaturized onto a sample containing fluorescent molecules and subjected to two / multiphoton excitation under low-photon irradiation to collect two / multiphoton fluorescence. S3. By using high spatiotemporal resolution detection methods, the spectral information, lifetime information and spatial location information of two / multiphoton fluorescence are obtained, the fluorescence information within the field of view is reconstructed, and the super-resolution imaging and positioning of the entire field of view are completed. In S1, photonic spatiotemporal dynamic modulation of multi-wavelength short-pulse laser is performed, specifically as follows: From the perspective of photons, using photons as the control object, after shaping the laser Gaussian beam into a flat-top beam, the pixels of the spatial light modulator are designed and modulated to create independent, discrete, parallel excitation points with a distance between them greater than the diffraction limit scale of the imaging system, so as to achieve spatiotemporal dynamic modulation of photon energy, quantity, time domain and spatial domain multi-parameter coordination. In S3, the spectral information, lifetime information, and spatial location information of two / multiphoton fluorescence are obtained, specifically: High spatiotemporal resolution detection of single-molecule fluorescence is performed to obtain temporal and spatial information of two / multiphoton excitation and fluorescence; through pixel patterning design with photon spatiotemporal dynamic modulation, a large number of discrete point surface excitations with single-photon / few-photon pulse excitation points spacing greater than the optical diffraction limit are formed to obtain the two / multiphoton fluorescence of single molecules and their distribution map. By switching the discrete point positions of surface excitation, high spatiotemporal resolution dynamic microscopic imaging can be obtained.

2. The ultra-high spatiotemporal resolution detection and imaging method according to claim 1, characterized in that, Two / multiphoton excitation under few-photon irradiation in S2 can be divided into the following different cases: During degenerate two-photon excitation, a pulse of the same wavelength after spatiotemporal dynamic modulation of photons is projected onto a sample containing dispersed fluorescent molecules. The simultaneous action of two photons on a molecular-scale spatial range is a necessary condition for the degenerate two-photon excitation process to occur. In non-degenerate two-photon excitation, two single-photon / minor-photon pulses of different wavelengths, after being spatiotemporally modulated by photons, are combined and projected onto a sample containing dispersed fluorescent molecules. The simultaneous action of two photons on a molecular-scale spatial range is a necessary condition for the non-degenerate two-photon excitation process to occur. The delay of the two-beam pulses is adjusted by optical elements.

3. A high spatiotemporal resolution detection and imaging system, characterized in that, The application of the ultra-high spatiotemporal resolution detection and imaging method as described in any one of claims 1-2 includes: Multi-wavelength short-pulse laser module, used to generate laser pulses of different wavelengths and photon numbers; The photonic spatiotemporal dynamic modulation module is used to modulate multi-wavelength short-pulse lasers to obtain discrete single-photon / few-photon excitation lattices; The large field-of-view projection imaging module is used to project the patterned discrete single-photon / few-photon excitation lattice light field of the design onto the microscopic sample; The light-matter interaction module is used to focus discrete single-photon / few-photon excitation lattices onto a microscopic sample for two-photon excitation; The fluorescence detection and signal acquisition and processing module is used to collect fluorescence signals from microscopic samples and to detect fluorescence spectra, fluorescence lifetimes, and single-molecule fluorescence imaging based on the fluorescence signals.

4. The ultra-high spatiotemporal resolution detection and imaging system according to claim 3, characterized in that, The photonic spatiotemporal dynamic modulation module includes: a cascaded liquid crystal spatial light modulator and a digital micromirror device; A cascaded liquid crystal spatial light modulator is used for modulation in the visible and near-infrared bands. Combined with a light source system for wavelength selection, it forms a large field-of-view discrete point synchronous excitation light source with coordinated modulation of photon energy, quantity, delay time, and spatial position. Digital micromirror devices are used to modulate a dual-color, dual-beam optical field that has been aligned and pulse-synchronized to achieve synchronous excitation of discrete points in a large field of view.

5. The ultra-high spatiotemporal resolution detection and imaging system according to claim 3, characterized in that, The light-matter interaction module includes a motion platform and a microscope stage; the motion platform is positioned above the microscope stage, and the microscopic sample to be tested is placed on the motion platform.

6. The ultra-high spatiotemporal resolution detection and imaging system according to claim 3, characterized in that, The fluorescence detection and signal acquisition and processing module includes: a weak light fluorescence spectroscopy detection unit, a weak light fluorescence lifetime detection unit, and a fluorescence imaging unit; The weak-light fluorescence spectroscopy detection unit is used to explore the temporal and spectral response characteristics of two-photon fluorescence under few-photon conditions using a PMT detector and a spectrometer. The weak-light fluorescence lifetime detection unit is used to measure the two-photon fluorescence lifetime using single-photon counting APD and time-correlated single-photon counting methods to obtain two-photon fluorescence lifetime information. The fluorescence imaging unit is used to acquire and process fluorescence signals using an EMCCD, and achieves super-resolution imaging of the entire field of view through multiple exposures during structured light field scanning.

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