Multi-modal super-resolution imaging probe, imaging structure, imaging method and storage medium

Abstract

The invention discloses a multi-mode super-resolution imaging probe, which belongs to the technical field of optical microscopic imaging, and comprises a charge donor dye, a conjugate electron withdrawing acceptor and a conjugate linking group, the invention further discloses an imaging structure and an imaging method of the multi-mode super-resolution imaging probe and a storage medium. The multi-mode super-resolution imaging probe generates random light-induced flicker under single-wavelength exciting light wide-field illumination to perform random optical reconstruction super-resolution imaging, and meanwhile, stimulated radiation loss super-resolution imaging or ground state depletion super-resolution imaging is realized under short-wavelength laser irradiation and annular long-wavelength laser irradiation modulation. Through modulation of an illumination mode, super-resolution imaging of three modes can be realized respectively.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical microscopic imaging, and in particular relates to a multimodal super-resolution imaging probe, an imaging structure, an imaging method and a storage medium. Background Art

[0002] Super-resolution imaging refers to imaging technology that can break the resolution limit of optical microscopes, including single-molecule localization super-resolution imaging and point diffusion engineering super-resolution imaging.

[0003] Single-molecule localization super-resolution imaging, especially random optical reconstruction super-resolution imaging, is based on two single-molecule fluorescence imaging technologies, STORM and PALM. By using special fluorescent dyes or markers in the sample, it causes a random light-emission and extinguishing process. In this process, each marker will randomly emit a fluorescence signal and be recorded by the camera. By statistically analyzing these fluorescence signals, the spatial position information of each marker can be obtained. By combining these position information, a high-resolution image can be reconstructed. Due to the limited types of fluorescent probes that can be used for random optical reconstruction super-resolution imaging, it is mainly used in wide-field imaging microscopes and is difficult to apply to living cell imaging.

[0004] Point spread function engineering super-resolution imaging, by modulating the laser illumination mode to change the point spread function of the imaging system, in order to achieve imaging technology beyond the traditional optical resolution limit, including stimulated emission depletion super-resolution imaging and ground state depletion super-resolution imaging. Stimulated emission depletion super-resolution imaging, based on the principle of stimulated emission, introduces a special beam of depletion light, which is superimposed with the excitation light on the sample. The excitation light puts the fluorescent molecules in the sample in an excited state, while the depletion light causes the molecules in the edge area of ​​the excited fluorescent molecules to return to the ground state through the stimulated emission process, thereby suppressing the fluorescence emission in these areas. Only the fluorescent molecules in the central area can spontaneously radiate fluorescence to form a smaller luminous point, thereby achieving imaging resolution technology beyond the diffraction limit. Ground state depletion super-resolution imaging, when the sample is irradiated with excitation light, the labeled molecules absorb light energy and From the ground state to the excited state, a high-intensity loss light is then introduced, which overlaps with the excitation light in space and acts on the labeled molecules in the excited state. Due to the effect of the loss light, these excited-state labeled molecules will quickly return to the ground state by stimulated radiation and no longer emit fluorescence. Under the action of the loss light, only a small amount of labeled molecules that are not irradiated by the loss light and are still in the ground state can emit fluorescence and be captured by the detector to achieve super-resolution imaging of the sample. For small molecule fluorescent probe imaging, both stimulated emission depletion super-resolution imaging and ground state depletion super-resolution imaging require super-resolution imaging under high-power lasers, and the imaging resolution is proportional to the laser power. However, the higher the laser power, the more severe the photobleaching of the fluorescent probe and the stronger the phototoxicity to the cells. Therefore, it is also difficult to apply to living cell imaging.

[0005] In summary, there is currently no multimodal super-resolution imaging probe that can simultaneously meet the requirements of random optical reconstruction super-resolution imaging, stimulated emission depletion super-resolution imaging, and ground state depletion super-resolution imaging.

[0006] In view of this, a multimodal super-resolution imaging probe, an imaging structure, an imaging method and a storage medium are designed to solve the above problems. Summary of the invention

[0007] To solve the problems raised in the above background technology, the present invention provides a multimodal super-resolution imaging probe, an imaging structure, an imaging method and a storage medium, which have the characteristics of being able to realize super-resolution imaging of three modes respectively by modulation of the illumination mode.

[0008] Another object of the present invention is to provide an imaging structure, an imaging method and a storage medium of a multimodal super-resolution imaging probe.

[0009] To achieve the above object, the present invention provides the following technical solution: a multimodal super-resolution imaging probe, comprising: a charge donor dye, a conjugated electron-withdrawing acceptor and a conjugated linking group, wherein:

[0010] Charge donor dye, the donor is a short-wavelength organic dye;

[0011] A conjugated electron-withdrawing acceptor is conjugated with a charge-donating dye via a conjugated double bond, and the acceptor is a group having a conjugated electron-withdrawing ability;

[0012] The conjugated linking group connects the charge donor dye and the conjugated electron-withdrawing acceptor through conjugation to form a complete conjugated system.

[0013] Furthermore, the short-wavelength organic dyes include coumarin, fluorescein, naphthylimide, BODIPY, rhodamine, perylene imide and cyanine dyes.

[0014] Furthermore, the group having conjugated electron-withdrawing ability includes indole quaternary ammonium salt, benzindole quaternary ammonium salt, pyridinium quaternary ammonium salt, quinoline quaternary ammonium salt, dimethyl dicyanopyran group and metal complex.

[0015] Furthermore, the conjugated linking group includes a carbon-carbon double bond, a carbon-carbon triple bond, pyridine, a benzene ring and a naphthalene ring.

[0016] The multimodal super-resolution imaging structure of the multimodal super-resolution imaging probe comprises:

[0017] The imaging structure of the multimodal super-resolution imaging probe for random optical reconstruction super-resolution imaging includes: a first light source component, a first object carrier component, a first optical path component and a first receiving terminal, wherein:

[0018] The first light source assembly comprises:

[0019] Short-wavelength laser, generating short-wavelength excitation light;

[0020] The first carrier assembly includes:

[0021] A stage, carrying a sample that has been labeled with a multimodal super-resolution imaging probe;

[0022] The first optical path component includes:

[0023] The first quarter-wave plate adjusts the polarization of the short-wavelength excitation light;

[0024] A first polarizer, adjusting the polarization of the short-wavelength excitation light;

[0025] A first beam expander collimator is used to expand the spot diameter of the short-wavelength excitation light and collimate the short-wavelength excitation light beam;

[0026] The first half-wave plate adjusts the polarization of the short-wavelength excitation light;

[0027] A beam splitter that splits the short-wavelength excitation light into two;

[0028] The second quarter-wave plate adjusts the polarization of the short-wavelength excitation light;

[0029] A second polarizer, adjusting the polarization of the short-wavelength excitation light;

[0030] A second beam expander collimator is used to expand the spot diameter of the short-wavelength excitation light and collimate the short-wavelength excitation light beam;

[0031] A first reflector, reflecting short-wavelength excitation light;

[0032] A second reflector reflects short-wavelength excitation light;

[0033] The objective lens focuses the laser onto the sample on the stage and collects the fluorescence signal reflected by the sample;

[0034] Concave / flat mirror, collecting the fluorescence signal reflected upward from the sample;

[0035] A first dichroic mirror transmits the excitation light and reflects the fluorescence signal;

[0036] The first lens focuses the fluorescent signal;

[0037] The first receiving terminal comprises:

[0038] The first electron multiplying charge-coupled device, which detects photons quickly and with high sensitivity;

[0039] A computer receives the image collected by the first electron multiplying charge coupled device and performs data processing and analysis;

[0040] The imaging structure of the multimodal super-resolution imaging probe for stimulated emission depletion super-resolution imaging includes: a second light source component, a second object carrier component, a second optical path component and a second receiving terminal, wherein:

[0041] The second light source assembly comprises:

[0042] a first light source assembly;

[0043] Long wavelength laser, generating long wavelength excitation light;

[0044] The second carrier assembly includes:

[0045] a first loading assembly;

[0046] The second optical path components include:

[0047] A first optical path component;

[0048] The third quarter-wave plate adjusts the polarization of the long-wavelength excitation light;

[0049] The third polarizer adjusts the polarization of the long-wavelength excitation light;

[0050] A third beam expander collimator is used to expand the spot diameter of the long-wavelength excitation light and collimate the short-wavelength excitation light beam;

[0051] The third half-wave plate adjusts the polarization of the long-wavelength excitation light;

[0052] A spatial light modulator converts the long-wavelength excitation light into a ring laser with a weak center and strong outer ring;

[0053] A second dichroic mirror reflects the ring laser;

[0054] A third dichroic mirror reflects the short-wavelength excitation light so as to combine it with the ring laser to form a composite laser beam;

[0055] Scanning galvanometer, deflecting the composite laser beam;

[0056] Scanning tube lens, adjusting and focusing the composite laser beam;

[0057] Scanning lens to adjust the composite laser beam;

[0058] a fourth dichroic mirror, reflecting the composite laser beam so as to allow it to enter the objective lens;

[0059] The second lens focuses the fluorescent signal returning along the original path;

[0060] Electric pinhole to optimize imaging quality;

[0061] a fifth dichroic mirror, reflecting the fluorescence signal;

[0062] The second receiving terminal comprises:

[0063] a first receiving terminal;

[0064] Photomultiplier tubes collect fluorescent photons, amplify fluorescent signals, and output electrical signals to computers for data processing and analysis;

[0065] The imaging structure of the multimodal super-resolution imaging probe for ground state depletion super-resolution imaging includes: a third light source component, a third object carrier component, a third optical path component and a third receiving terminal, wherein:

[0066] The third light source assembly comprises:

[0067] a second light source assembly;

[0068] The third carrier assembly includes:

[0069] a second loading assembly;

[0070] The third optical path components include:

[0071] A second optical path component;

[0072] The third receiving terminal includes:

[0073] a second receiving terminal;

[0074] The second electron multiplying charge coupled device detects the number of photons quickly and with high sensitivity, and transmits the data to a computer for data processing and analysis.

[0075] Furthermore, an optical anti-drift component is also included, including:

[0076] Near-infrared laser to produce drift-resistant laser;

[0077] The sixth dichroic mirror reflects the anti-drift laser;

[0078] The seventh dichroic mirror reflects the anti-drift laser and receives the reflected signal of the anti-drift laser;

[0079] A third reflecting mirror reflects a reflected signal of the anti-drift laser;

[0080] The four-quadrant detector receives the reflected signal of the anti-drift laser, processes and analyzes it, and performs drift correction.

[0081] The multimodal super-resolution imaging method of the multimodal super-resolution imaging structure of the multimodal super-resolution imaging probe comprises the following steps:

[0082] S1: Multimodal super-resolution imaging probes are used to label cell samples and perform pre-processing for imaging;

[0083] S2: Assume that two laser beams are used, a short-wavelength Gaussian laser is split into two laser beams, and a long-wavelength ring laser has weak central light and strong outer light;

[0084] S3: A short-wavelength laser beam is shaped and expanded for wide-field illumination. The multimodal super-resolution imaging probe generates random scintillation. The random scintillation signal generated by the multimodal super-resolution imaging probe is recorded. Multiple emission signals are collected and the image is reconstructed through the reconstruction algorithm to obtain a super-resolution image, that is, random optical reconstruction super-resolution imaging is realized.

[0085] S4: A short-wavelength laser beam is shaped and combined with a ring laser to excite the multimodal super-resolution imaging probe to an excited state. The ring laser irradiation produces stimulated radiation and radiationless excitation, and the spontaneous emission fluorescence signal in the center of the remaining ring area is collected. The image is reconstructed through a reconstruction algorithm to obtain a super-resolution image, that is, stimulated emission loss super-resolution imaging is realized;

[0086] S5: A long-wavelength ring laser irradiates the ground state fluorescent molecules to the excited state, depleting the ground state. A short-wavelength Gaussian laser lags behind the ring laser for a period of time to excite the fluorescent molecules in the central area of ​​the ring laser to produce fluorescence emission. The remaining spontaneous radiation fluorescence signal in the central area of ​​the ring area is collected, and the image is reconstructed through a reconstruction algorithm to obtain a super-resolution image, that is, to achieve ground state depletion super-resolution imaging.

[0087] The storage medium includes: storing computer-readable computer instructions, which are used to enable the computer to execute the operations of collecting images, storing data, and processing and analyzing image data in the multimodal super-resolution imaging method of the multimodal super-resolution imaging structure of the multimodal super-resolution imaging probe.

[0088] Compared with the prior art, the present invention has the following beneficial effects:

[0089] 1. The multimodal super-resolution imaging probe of the present invention generates random photoinduced scintillation under wide-field illumination of single-wavelength excitation light to perform random optical reconstruction super-resolution imaging. At the same time, it also satisfies the requirements of stimulated emission depletion super-resolution imaging or ground state depletion super-resolution imaging under short-wavelength laser irradiation and modulation of annular long-wavelength laser irradiation. By modulating the illumination mode, three modes of super-resolution imaging can be achieved respectively.

[0090] 2. The multimodal super-resolution imaging probe of the present invention can share the optical system for illumination in the three super-resolution imaging processes, and couples the random optical reconstruction super-resolution imaging detection with the stimulated emission depletion super-resolution imaging or ground state depletion super-resolution imaging illumination modes, thereby hopefully obtaining higher-resolution images. It has the characteristics of simple optical system and system integration as a whole, and can minimize the damage of the light source to cells and probes under the synergy of low-power laser, thereby realizing multimodal super-resolution imaging.

[0091] 3. The present invention selects fluorescent probes with different properties to give them special photophysical properties, which can meet the needs of various super-resolution imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 Schematic diagram of the structure of the multimodal super-resolution imaging probe of the present invention;

[0093] Figure 2 Schematic diagram of absorption and emission spectra of the probe of the present invention;

[0094] Figure 3 This is a schematic diagram of the imaging structure of the multimodal super-resolution imaging probe of the present invention;

[0095] Figure 4 It is a schematic diagram of the imaging method flow of the imaging structure of the multimodal super-resolution imaging probe of the present invention;

[0096] Figure 5 The schematic diagram is a schematic diagram of the excitation, internal conversion, fluorescence emission and non-radiative luminescence of the probe of the present invention;

[0097] Figure 6 Schematic diagram of intensity distribution of the excitation laser and the ambient laser of the present invention;

[0098] In the figure: 1. Charge donor dye; 2. Conjugated electron-withdrawing acceptor; 3. Conjugated linking group;

[0099] 101, short wavelength laser; 102, first quarter wave plate; 103, first polarizer; 104, first beam expander collimator; 105, first half wave plate; 106, beam splitter; 107, second quarter wave plate; 108, second polarizer; 109, second beam expander collimator; 110, first reflector; 111, second reflector; 112, objective lens; 113, stage; 114, concave / plane reflector; 115, first dichroic mirror; 116, first lens; 117, first electron multiplying charge coupled device; 118, computer;

[0100] 201, long wavelength laser; 202, third quarter wave plate; 203, third polarizer; 204, third beam expander collimator; 205, third half wave plate; 206, spatial light modulator; 207, second dichroic mirror; 208, third dichroic mirror; 209, scanning galvanometer; 210, scanning tube mirror; 211, scanning lens; 212, fourth dichroic mirror; 213, second lens; 214, electric pinhole; 215, fifth dichroic mirror; 216, photomultiplier tube;

[0101] 301. A second electron multiplying charge coupled device;

[0102] 401. Near-infrared laser; 402. Sixth dichroic mirror; 403. Seventh dichroic mirror; 404. Third reflecting mirror; 405. Four-quadrant detector. DETAILED DESCRIPTION

[0103] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0104] See attached Figure 1 , a multimodal super-resolution imaging probe, comprising: a charge donor dye 1, a conjugated electron-withdrawing acceptor 2 and a conjugated linking group 3, wherein:

[0105] Charge donor dye 1, the donor is a short-wavelength organic dye;

[0106] The conjugated electron-withdrawing acceptor 2 is conjugated with the charge-donating dye 1 through a conjugated double bond, and the acceptor is a group having a conjugated electron-withdrawing ability;

[0107] The conjugated linking group 3 connects the charge donor dye 1 and the conjugated electron-withdrawing acceptor 2 through conjugation to form a complete conjugated system.

[0108] Specifically, short wavelength organic dyes include coumarin, fluorescein, naphthylimide, BODIPY, rhodamine, peryleneimide, and cyanine dyes.

[0109] Specifically, the group having conjugated electron withdrawing ability includes indole quaternary ammonium salt, benzindole quaternary ammonium salt, pyridinium quaternary ammonium salt, quinoline quaternary ammonium salt, dimethyl dicyanopyran group and metal complex.

[0110] Specifically, the conjugated linking group 3 includes a carbon-carbon double bond, a carbon-carbon triple bond, pyridine, a benzene ring, and a naphthalene ring.

[0111] See attached Figure 2 , A represents the absorption spectrum - the absorption peak of the short-wavelength fluorescent dye, B represents the absorption spectrum - the absorption peak of the long-wavelength dye, C represents the emission spectrum - the fluorescence emission peak of the short-wavelength dye, and D represents the emission spectrum - the fluorescence emission peak of the long-wavelength dye (no fluorescence or weak fluorescence emission);

[0112] The multimodal super-resolution imaging probe is a fluorescent emission dye with a dual dye conjugated structure, that is, a composite fluorescent dye of a short-wavelength and a long-wavelength complex, which has at least an absorption peak, a fluorescence emission peak, and a spectral characteristic of no fluorescence emission when excited at the long-wavelength absorption peak.

[0113] See attached Figure 3 , the multimodal super-resolution imaging structure of the multimodal super-resolution imaging probe includes:

[0114] The imaging structure of the multimodal super-resolution imaging probe for random optical reconstruction super-resolution imaging includes: a first light source component, a first object carrier component, a first optical path component and a first receiving terminal, wherein:

[0115] The first light source assembly comprises:

[0116] A short-wavelength laser 101 generates short-wavelength excitation light;

[0117] The first carrier assembly includes:

[0118] The stage 113 carries the sample labeled by the multimodal super-resolution imaging probe;

[0119] The first optical path component includes:

[0120] A first quarter wave plate 102, for adjusting the polarization of the short wavelength excitation light;

[0121] A first polarizer 103, for adjusting the polarization of the short-wavelength excitation light;

[0122] A first beam expander collimator 104, which expands the spot diameter of the short-wavelength excitation light and collimates the short-wavelength excitation light beam;

[0123] A first half-wave plate 105, for adjusting the polarization of the short-wavelength excitation light;

[0124] A beam splitter 106 splits the short wavelength excitation light into two;

[0125] A second quarter wave plate 107, for adjusting the polarization of the short wavelength excitation light;

[0126] A second polarizer 108, for adjusting the polarization of the short-wavelength excitation light;

[0127] The second beam expander collimator 109 expands the spot diameter of the short-wavelength excitation light and collimates the short-wavelength excitation light beam;

[0128] A first reflector 110 reflects short-wavelength excitation light;

[0129] The second reflector 111 reflects the short-wavelength excitation light;

[0130] The objective lens 112 focuses the laser onto the sample on the stage 113 and collects the fluorescence signal reflected by the sample;

[0131] A concave / plane reflector 114 collects the fluorescence signal reflected upward by the sample;

[0132] A first dichroic mirror 115 transmits the excitation light and reflects the fluorescence signal;

[0133] A first lens 116, focusing the fluorescent signal;

[0134] The first receiving terminal comprises:

[0135] A first electron multiplying charge coupled device 117 detects the number of photons quickly and with high sensitivity;

[0136] The computer 118 receives the image collected by the first electron multiplying charge coupled device 117 and performs data processing and analysis;

[0137] The imaging structure of the multimodal super-resolution imaging probe for stimulated emission depletion super-resolution imaging includes: a second light source component, a second object carrier component, a second optical path component and a second receiving terminal, wherein:

[0138] The second light source assembly comprises:

[0139] a first light source assembly;

[0140] A long wavelength laser 201 generates long wavelength excitation light;

[0141] The second carrier assembly includes:

[0142] a first loading assembly;

[0143] The second optical path components include:

[0144] A first optical path component;

[0145] A third quarter wave plate 202, for adjusting the polarization of the long wavelength excitation light;

[0146] A third polarizer 203, for adjusting the polarization of the long-wavelength excitation light;

[0147] The third beam expander collimator 204 expands the spot diameter of the long-wavelength excitation light and collimates the short-wavelength excitation light beam;

[0148] The third half-wave plate 205 adjusts the polarization of the long-wavelength excitation light;

[0149] A spatial light modulator 206 converts the long-wavelength excitation light into a ring-shaped laser with a weak center and a strong outer ring;

[0150] A second dichroic mirror 207 reflects the ring laser;

[0151] The third dichroic mirror 208 reflects the short-wavelength excitation light to combine it with the ring laser to form a composite laser beam;

[0152] Scanning galvanometer 209, deflecting the composite laser beam;

[0153] Scanning tube lens 210, adjusting and focusing the composite laser beam;

[0154] A scanning lens 211, for adjusting the composite laser beam;

[0155] The fourth dichroic mirror 212 reflects the composite laser beam so as to enter the objective lens 112;

[0156] The second lens 213 focuses the fluorescent signal returning along the original path;

[0157] Electric pinhole 214 to optimize imaging quality;

[0158] A fifth dichroic mirror 215 reflects the fluorescent signal;

[0159] The second receiving terminal comprises:

[0160] a first receiving terminal;

[0161] The photomultiplier tube 216 collects fluorescent photons, amplifies the fluorescent signal, and outputs the electrical signal to the computer 118 for data processing and analysis;

[0162] The imaging structure of the multimodal super-resolution imaging probe for ground state depletion super-resolution imaging includes: a third light source component, a third object carrier component, a third optical path component and a third receiving terminal, wherein:

[0163] The third light source assembly comprises:

[0164] a second light source assembly;

[0165] The third carrier assembly includes:

[0166] a second loading assembly;

[0167] The third optical path components include:

[0168] A second optical path component;

[0169] The third receiving terminal includes:

[0170] a second receiving terminal;

[0171] The second electron multiplying charge coupled device 301 detects the number of photons quickly and with high sensitivity, and transmits the data to the computer 118 for data processing and analysis.

[0172] Specifically, it also includes an optical anti-drift component, including:

[0173] A near-infrared laser 401 generates an anti-drift laser;

[0174] A sixth dichroic mirror 402 reflects the anti-drift laser;

[0175] The seventh dichroic mirror 403 reflects the anti-drift laser and receives the reflected signal of the anti-drift laser;

[0176] A third reflecting mirror 404 reflects a reflection signal of the anti-drift laser;

[0177] The four-quadrant detector 405 receives the reflected signal of the anti-drift laser, processes and analyzes it, and performs drift correction.

[0178] See attached Figure 4 The multimodal super-resolution imaging method of the multimodal super-resolution imaging structure of the multimodal super-resolution imaging probe comprises the following steps:

[0179] S1: Multimodal super-resolution imaging probes are used to label cell samples and perform pre-processing for imaging;

[0180] S2: Assume that two laser beams are used, a short-wavelength Gaussian laser is split into two laser beams, and a long-wavelength ring laser has weak central light and strong outer light;

[0181] S3: A short-wavelength laser beam is shaped and expanded for wide-field illumination. The multimodal super-resolution imaging probe generates random scintillation. The random scintillation signal generated by the multimodal super-resolution imaging probe is recorded. Multiple emission signals are collected and the image is reconstructed through the reconstruction algorithm to obtain a super-resolution image, that is, random optical reconstruction super-resolution imaging is realized.

[0182] S4: A short-wavelength laser beam is shaped and combined with a ring laser to excite the multimodal super-resolution imaging probe to an excited state. The ring laser irradiation produces stimulated radiation and radiationless excitation, and the spontaneous emission fluorescence signal in the center of the remaining ring area is collected. The image is reconstructed through a reconstruction algorithm to obtain a super-resolution image, that is, stimulated emission loss super-resolution imaging is realized;

[0183] S5: A long-wavelength ring laser irradiates the ground state fluorescent molecules to the excited state, depleting the ground state. A short-wavelength Gaussian laser lags behind the ring laser for a period of time to excite the fluorescent molecules in the central area of ​​the ring laser to produce fluorescence emission. The remaining spontaneous radiation fluorescence signal in the central area of ​​the ring area is collected, and the image is reconstructed through a reconstruction algorithm to obtain a super-resolution image, that is, to achieve ground state depletion super-resolution imaging.

[0184] See attached Figure 5 Under the irradiation of short-wavelength laser, the charge donor dye 1 is excited, and the charge donor dye 1 is excited to transition from the ground state (S0) to the excited state (Sn, n>2), and then internally converts to the second excited state (S2), and then transitions from the second excited state (S2) back to the ground state (S0) to emit fluorescence, which competes with the internal conversion from the second excited state (S2) to the first excited state (S1), and the process of transitioning from the first excited state (S1) back to the ground state (S0) has weak fluorescence or no fluorescence emission. The two processes occur randomly, that is, after the short-wavelength laser excites the charge donor dye 1, the charge donor dye 1 produces the fluorescence of the short-wavelength dye. In the instantaneous process, it is a random blinking process, which meets the needs of random optical reconstruction super-resolution imaging;

[0185] GSD is the abbreviation of ground state depletion super-resolution imaging technology. Under the irradiation of long-wavelength ring laser, the laser new long-wavelength fluorescent dye, the new long-wavelength fluorescent dye is excited to jump from the ground state (S0) to the first excited state (S1), but there is no fluorescence emission when the first excited state (S1) jumps back to the ground state (S0), that is, no fluorescence emission will be generated in the ring area. This process will reduce the number of molecules in the ground state (S0), so that the ground state of the fluorescent dye molecules in the ring area is depleted. When irradiated with a short-wavelength laser, the new long-wavelength fluorescent dye is excited. , the new long-wavelength fluorescent dye is excited by electrons and transitions from the ground state (S0) to the excited state (Sn, n>2), then internally converts to the second excited state (S2), and then transitions from the second excited state (S2) back to the ground state (S0) to emit fluorescence, which competes with the internal conversion from the second excited state (S2) to the first excited state (S1), and the process of transitioning from the first excited state (S1) back to the ground state (S0) has weak fluorescence or no fluorescence emission, which produces the fluorescence of the short-wavelength dye, so that it can meet the needs of ground state depletion super-resolution imaging under the action of low-power laser;

[0186] STED is the abbreviation of stimulated emission depletion super-resolution imaging technology. Under short-wavelength laser irradiation, the charge donor dye 1 is excited. The charge donor dye 1 is excited to transition from the ground state (S0) to the excited state (Sn, n>2). The charge donor transfers the charge to the conjugated electron-withdrawing acceptor 2 through the conjugated linking group 3 to produce a new long-wavelength fluorescent dye. When the ring laser is irradiated at the absorption peak of the long-wavelength fluorescent dye, the fluorophore molecules in the ring area transition from the second excited state (S2) back to the ground state (S0). After the fluorescent dye returns to the ground state (S0), it is easy to absorb the ring laser photons to generate re-excitation to the first excited state (S1). However, there is no fluorescence emission when the first excited state (S1) transitions back to the ground state (S0), that is, no fluorescence emission is generated in the ring area due to re-absorption. This process will reduce the number of molecules in the ground state (S0), so that it can meet the requirements of stimulated emission depletion super-resolution imaging under the action of low-power laser.

[0187] The dye is excited at the short-wavelength absorption peak, generating short-wavelength fluorescence emission to compete with the long-wavelength non-fluorescence radiation process. By adjusting the mode or intensity of the irradiated laser, a fluorescence emission effect that meets the requirements is generated. At the same time, the laser irradiation intensity suitable for this type of fluorescent dye is relatively low, that is, under low-power laser irradiation, random optical reconstruction super-resolution imaging, stimulated emission depletion super-resolution imaging and ground state depletion super-resolution imaging can be achieved, which is beneficial to multimodal super-resolution imaging of living cells.

[0188] See attached Figure 1, A represents a linking group, including carbon-carbon single-bond carboxylic acids, benzoic acid and naphthoic acid, and B represents a targeting labeling group, including cell microfilaments and microtubules, mitochondria, endoplasmic reticulum, lysosomes and cell nuclei. Among them, cell microfilaments and microtubules targets include phalloidin and paclitaxel, mitochondrial targets include positively charged groups such as triphenylphosphine quaternary phosphonium salts, endoplasmic reticulum targets include glibenclamide and hydrophobic fatty chains, lysosomal targets include polyamine groups such as piperidine and morpholine, and cell nucleus targets include water-soluble positively charged groups such as polyamines.

[0189] Multimodal super-resolution imaging probes are covalently linked to different functional targets to construct labeling and imaging probes for different organelle structures, thereby achieving super-resolution imaging of different target structures in living cells.

[0190] The storage medium includes: storing computer instructions readable by a computer 118, wherein the computer instructions are used to enable the computer 118 to execute operations of acquiring images, storing data, and processing and analyzing image data in a multimodal super-resolution imaging method of a multimodal super-resolution imaging structure of a multimodal super-resolution imaging probe.

[0191] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A multimodal super-resolution imaging probe, characterized in that: include: A charge donor dye (1), a conjugated electron withdrawing acceptor (2) and a conjugated linking group (3), wherein: A charge donor dye (1), wherein the donor is a short-wavelength organic dye; A conjugated electron-withdrawing acceptor (2) is conjugated with the charge-donating dye (1) via a conjugated double bond, wherein the acceptor is a group having a conjugated electron-withdrawing ability; The conjugated linking group (3) connects the charge donor dye (1) and the conjugated electron-withdrawing acceptor (2) through conjugation to form a complete conjugated system.

2. The multimodal super-resolution imaging probe according to claim 1, characterized in that: The short wavelength organic dyes include coumarin, fluorescein, naphthylimide, BODIPY, rhodamine, peryleneimide and cyanine dyes.

3. The multimodal super-resolution imaging probe according to claim 1, characterized in that: The groups with conjugated electron-withdrawing ability include indole quaternary ammonium salts, benzindole quaternary ammonium salts, pyridinium quaternary ammonium salts, quinoline quaternary ammonium salts, dimethyl dicyanopyran groups and metal complexes.

4. The multimodal super-resolution imaging probe according to claim 1, characterized in that: The conjugated linking group (3) includes a carbon-carbon double bond, a carbon-carbon triple bond, pyridine, a benzene ring and a naphthalene ring.

5. The multimodal super-resolution imaging structure of the multimodal super-resolution imaging probe according to claims 1-4, characterized in that: include: The imaging structure of the multimodal super-resolution imaging probe for random optical reconstruction super-resolution imaging includes: a first light source component, a first object carrier component, a first optical path component and a first receiving terminal, wherein: The first light source assembly comprises: A short-wavelength laser (101) generates short-wavelength excitation light; The first carrier assembly includes: A stage (113) for carrying a sample labeled with a multimodal super-resolution imaging probe; The first optical path component includes: A first quarter wave plate (102) for adjusting the polarization of the short wavelength excitation light; A first polarizer (103) for adjusting the polarization of the short-wavelength excitation light; A first beam expander collimator (104) is used to expand the spot diameter of the short-wavelength excitation light and collimate the short-wavelength excitation light beam; A first half-wave plate (105) for adjusting the polarization of the short-wavelength excitation light; A beam splitter (106) is used to split the short wavelength excitation light into two; A second quarter wave plate (107) for adjusting the polarization of the short wavelength excitation light; A second polarizer (108) for adjusting the polarization of the short-wavelength excitation light; A second beam expander collimator (109) is used to expand the spot diameter of the short-wavelength excitation light and collimate the short-wavelength excitation light beam; A first reflector (110) reflects short-wavelength excitation light; A second reflector (111) reflects short-wavelength excitation light; An objective lens (112) focuses the laser onto the sample on the stage (113) and collects the fluorescence signal reflected by the sample; A concave / plane reflector (114) collects the fluorescence signal reflected upward by the sample; A first dichroic mirror (115) transmits the excitation light and reflects the fluorescence signal; A first lens (116) for focusing the fluorescent signal; The first receiving terminal comprises: A first electron multiplying charge coupled device (117) for detecting the number of photons quickly and with high sensitivity; A computer (118) receives the image collected by the first electron multiplying charge coupled device (117) and performs data processing and analysis; The imaging structure of the multimodal super-resolution imaging probe for stimulated emission depletion super-resolution imaging includes: a second light source component, a second object carrier component, a second optical path component and a second receiving terminal, wherein: The second light source assembly comprises: a first light source assembly; A long wavelength laser (201) generates long wavelength excitation light; The second carrier assembly includes: a first loading assembly; The second optical path components include: A first optical path component; A third quarter wave plate (202) for adjusting the polarization of the long wavelength excitation light; A third polarizer (203) for adjusting the polarization of the long-wavelength excitation light; A third beam expander collimator (204) is used to expand the spot diameter of the long-wavelength excitation light and collimate the short-wavelength excitation light beam; A third half-wave plate (205) for adjusting the polarization of the long-wavelength excitation light; A spatial light modulator (206) converts the long-wavelength excitation light into a ring-shaped laser with a weak center and a strong outer ring; A second dichroic mirror (207) reflects the ring laser; A third dichroic mirror (208) reflects the short-wavelength excitation light so as to combine it with the ring laser to form a composite laser beam; A scanning galvanometer (209) for deflecting the composite laser beam; A scanning tube mirror (210) for adjusting and focusing the composite laser beam; A scanning lens (211) for adjusting the composite laser beam; a fourth dichroic mirror (212) for reflecting the composite laser beam so as to allow it to enter the objective lens (112); A second lens (213) focuses the fluorescent signal returning along the original path; A motorized pinhole (214) to optimize imaging quality; a fifth dichroic mirror (215) for reflecting the fluorescent signal; The second receiving terminal comprises: a first receiving terminal; A photomultiplier tube (216) collects fluorescent photons, amplifies fluorescent signals, and outputs electrical signals to a computer (118) for data processing and analysis; The imaging structure of the multimodal super-resolution imaging probe for ground state depletion super-resolution imaging includes: a third light source component, a third object carrier component, a third optical path component and a third receiving terminal, wherein: The third light source assembly comprises: a second light source assembly; The third carrier assembly includes: a second loading assembly; The third optical path components include: A second optical path component; The third receiving terminal includes: a second receiving terminal; The second electron multiplying charge coupled device (301) detects the number of photons quickly and with high sensitivity, and transmits the detected photons to a computer (118) for data processing and analysis.

6. The multimodal super-resolution imaging structure of the multimodal super-resolution imaging probe according to claim 5, characterized in that: Also included is an optical anti-drift kit, consisting of: A near-infrared laser (401) generates an anti-drift laser; A sixth dichroic mirror (402) for reflecting the anti-drift laser; A seventh dichroic mirror (403) reflects the anti-drift laser and receives a reflected signal of the anti-drift laser; A third reflecting mirror (404) reflects a reflection signal of the anti-drift laser; The four-quadrant detector (405) receives the reflected signal of the anti-drift laser, processes and analyzes it, and performs drift correction.

7. The multimodal super-resolution imaging method of the multimodal super-resolution imaging structure of the multimodal super-resolution imaging probe according to claim 5, characterized in that: The following steps are involved: S1: Multimodal super-resolution imaging probes are used to label cell samples and perform pre-processing for imaging; S2: Assume that two laser beams are used, a short-wavelength Gaussian laser is split into two laser beams, and a long-wavelength ring laser has weak central light and strong outer light; S3: A short-wavelength laser beam is shaped and expanded for wide-field illumination. The multimodal super-resolution imaging probe generates random scintillation. The random scintillation signal generated by the multimodal super-resolution imaging probe is recorded. Multiple emission signals are collected and the image is reconstructed through the reconstruction algorithm to obtain a super-resolution image, that is, random optical reconstruction super-resolution imaging is realized. S4: A short-wavelength laser beam is shaped and combined with a ring laser to excite the multimodal super-resolution imaging probe to an excited state. The ring laser irradiation produces stimulated radiation and radiationless excitation, and the spontaneous emission fluorescence signal in the center of the remaining ring area is collected. The image is reconstructed through a reconstruction algorithm to obtain a super-resolution image, that is, stimulated emission loss super-resolution imaging is realized; S5: A long-wavelength ring laser irradiates the ground state fluorescent molecules to the excited state, depleting the ground state. A short-wavelength Gaussian laser lags behind the ring laser for a period of time to excite the fluorescent molecules in the central area of ​​the ring laser to produce fluorescence emission. The remaining spontaneous radiation fluorescence signal in the central area of ​​the ring area is collected, and the image is reconstructed through a reconstruction algorithm to obtain a super-resolution image, that is, to achieve ground state depletion super-resolution imaging.

8. A storage medium, characterized in that include: Computer instructions readable by a computer (118) are stored, and the computer instructions are used to enable the computer (118) to execute the operations of collecting images, storing data, and processing and analyzing image data in claim 7.

Images