Design of multicolor super-resolution imaging probe, imaging module and storage medium

Abstract

The invention discloses a design of a multicolor super-resolution imaging probe, an imaging module and a storage medium. The probe is mainly divided into three parts: a donor, an acceptor and a connecting arm, the donor is irradiated by laser, stimulated absorption is generated, and the donor jumps to a high energy level; the connecting arm is used for transferring excitation energy from the donor to the receptor; and the receptor is used for generating spontaneous radiation and emitting fluorescence. Acceptors with different properties are selected to be connected with the same donor, so that fluorescence with different wavelengths is emitted by excitation with the same wavelength. The irradiation laser intensity is adjusted to be annularly distributed, the center of the laser intensity is weak (close to zero), a donor mainly generates absorption excitation, the outer ring is strong, the donor generates absorption excitation and stimulated radiation loss, the donor rapidly returns to the ground state, and energy transfer is blocked. According to the multi-color super-resolution imaging scheme, multi-color super-resolution imaging is achieved through a single laser beam, the multi-color super-resolution imaging device has the advantages of being simple in structure and easy and convenient to operate, and damage of the laser to cells and photobleaching of the probe are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical microscopes, and more specifically, relates to a design of a multicolor super-resolution imaging probe, a nano-microscope technology that achieves super-resolution imaging effects by exciting and depleting a sample with a single-wavelength laser. Background Art

[0002] Super-resolution imaging is one of the advanced imaging technologies that has significantly promoted the progress of scientific research and application fields in recent years. The limitation of resolution of traditional optical microscopes makes it difficult to meet the needs of high-resolution imaging, especially in high-precision observations in fields such as biomedicine and materials science. With the development of super-resolution imaging technology, scientists are able to break through the classical diffraction limit of optical microscopes and achieve unprecedented detailed observation of the internal structure of samples. Super-resolution imaging techniques, including fluorescence confocal microscopy, stimulated emission depletion microscopy (STED), structured light illumination microscopy, etc., have successfully improved imaging resolution. These techniques achieve higher spatial resolution than traditional optical microscopy through different principles, such as stimulated emission, structured light, or detection of multiple emission wavelengths. Nevertheless, these techniques still face many challenges in multicolor imaging.

[0003] Multicolor imaging, that is, imaging of multiple fluorescent markers simultaneously, is key to understanding complex biological systems and material properties. However, traditional super-resolution imaging methods often encounter signal overlap, low spectral resolution, and complex image processing problems in multicolor imaging. This limits their effectiveness and accuracy in multicolor imaging applications. Multicolor super-resolution imaging technology has a wide range of application potential. In biomedical research, it can be used to observe the spatial distribution of different types of proteins and nucleic acid molecules in cells, revealing interactions and functional changes within cells. In materials science, it can be used to study the structural properties and composition of nanomaterials. In clinical diagnosis, this technology helps to improve the detection accuracy of disease markers and provide more data support in personalized medicine.

[0004] Currently, multicolor super-resolution imaging technology faces several challenges. First, the technology requires high spectral resolution to separate and clearly identify different fluorescence signals. Second, improving the imaging speed is crucial for real-time observation of dynamic processes. In addition, reducing the damage to samples caused by phototoxicity and photobleaching is also an important factor in optimizing imaging quality. Finally, efficient data processing algorithms are also particularly important for extracting accurate multicolor imaging information from large amounts of complex data.

[0005] In the existing technology, multi-color super-resolution imaging requires the use of multiple laser beams to irradiate the sample. There are many problems: First, the requirements for the imaging system are high. Second, the system is complex and involves multiple lasers and conversion systems, which is expensive. Third, the laser beams irradiating the sample are numerous and powerful, which can easily cause phototoxicity and photobleaching to damage the sample. Summary of the invention

[0006] The main purpose of the present invention is to provide a design, imaging module and storage medium for a multi-color super-resolution imaging probe, reduce the number of lasers required for imaging, achieve multi-color super-resolution imaging with a single laser beam, simplify the imaging system, lower the cost, and reduce the damage of the light source to the cells and the bleaching of the probe.

[0007] To achieve the above objectives, the present invention proposes a design scheme for a multi-color super-resolution imaging probe.

[0008] The fluorescent probe is mainly composed of two core parts: donor and acceptor, which are tightly connected by a specially designed linker.

[0009] The main function of the donor part is to receive the excitation light, which determines the excitation wavelength of the probe. When the laser is irradiated to the donor part, the particles in the donor transition from the ground state to the excited state and absorb energy. Subsequently, this energy is transferred to the acceptor part through the connecting arm. The choice of the donor determines the excitation wavelength of the probe, and therefore has a direct impact on the overall performance and application of the probe.

[0010] The linker acts as a connector in the molecular structure, connecting the donor and acceptor parts together and ensuring efficient transfer of energy from the donor to the acceptor.

[0011] The receptor part is responsible for emitting fluorescence and determining the wavelength of fluorescence emitted by the probe. By accurately selecting different receptors, it is possible to generate fluorescence of multiple different wavelengths from the same wavelength of excitation light. This design enables the probe to provide rich fluorescence signals in multi-color super-resolution imaging, greatly improving the resolution and sensitivity of imaging.

[0012] Based on the above theory, a series of probes are designed. The characteristics of the probes are the same donor and different acceptors. Since the donor determines the absorption wavelength and the acceptor determines the emission wavelength, different emission wavelengths can be obtained by the same laser excitation. When this series of probes is applied to the same sample, multiple probes can be excited and depleted by a single laser beam, thereby obtaining super-resolution imaging.

[0013] Preferably, the donor is a short-wavelength organic dye, a metal complex and a rare earth.

[0014] Preferably, the connection mode of the connecting arm is a rigid group connection such as a benzene ring, an alkynyl group, a pyridine ring and a naphthalene ring.

[0015] Preferably, the acceptor is an organic dye with a longer wavelength than that of the donor, such as coumarin, fluorescein, naphthylimide, BODIPY, rhodamine and cyanine dye, an organic complex, or a metal complex such as a rare earth complex.

[0016] Based on the above probe, the present invention proposes a multi-color super-resolution imaging method of the probe, and the process is as follows:

[0017] S1. Use a beam of continuous short-wavelength pulsed laser and pass it through a spatial light modulator or a spiral phase plate to make its light intensity distribution weak in the center and strong in the outer circle.

[0018] S2. The laser intensity of the outer ring position irradiated by the laser beam is high. The sample is irradiated by the laser to produce stimulated absorption, and the particles absorb energy from the low energy level to the high energy level. Due to the high laser intensity, stimulated radiation is emitted at the same time as stimulated absorption, blocking the process of energy transfer from the donor to the acceptor. Therefore, no fluorescence is generated in the ring area.

[0019] S3, at the center, the laser intensity is low, only stimulated absorption occurs, the particle jumps to a high energy level, and then is transferred from the connecting arm to the receptor, where spontaneous radiation occurs, generating fluorescence. The size of the imaging area is controlled, thus generating super-resolution.

[0020] Based on the above method, the present invention proposes a corresponding imaging module, the structure of which is as follows:

[0021] Light source assembly: used to emit short-wavelength pulsed laser with weak center and strong outer circle to the sample.

[0022] Optical path component: connected to the light source component, used to adjust the system optical path and perform a comprehensive scan of the sample.

[0023] Receiving terminal: used to collect fluorescence signals, acquire images, store data and process image data.

[0024] Preferably, the light source assembly includes a single-wavelength picosecond pulse laser, a collimator and a spatial light modulator.

[0025] Preferably, the optical path components include a scanning galvanometer, a scanning lens, an objective lens, a sleeve lens, a dichroic mirror, a reflector, a lens, a stage and a filter.

[0026] Preferably, the receiving terminal includes a multimode optical fiber, a photomultiplier tube and a computer.

[0027] To achieve the above-mentioned purpose, the present invention also proposes a computer-readable storage medium, on which computer instructions are stored, and the computer instructions are used to enable a computer to execute image acquisition, data storage and image data processing.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention selects energy acceptors of different properties to connect with the same energy donor through a rigid group to achieve the same wavelength excitation and different emission wavelength fluorescence. The intensity distribution of the irradiated laser is adjusted. The laser intensity is weak in the center, and the donor mainly undergoes stimulated absorption. The outer circle is strong, and the donor not only absorbs and generates a transition from the machine to the excited state, but also undergoes stimulated radiation to block energy transfer, thereby controlling the imaging range and achieving super-resolution imaging effects. It has the characteristics of simple structure and easy operation, and reduces the damage of the light source to cells and probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A schematic diagram of the structure and absorption and emission spectra of a probe for multi-color super-resolution imaging according to the present invention;

[0031] Figure 2 This is a schematic diagram of laser intensity distribution of the present invention;

[0032] Figure 3 It is a principle diagram of light emission and loss of the probe of the present invention;

[0033] Figure 4 is a schematic diagram of an imaging module of the present invention;

[0034] Figure 5 Schematic diagram of the microscopic imaging device of the present invention;

[0035] Figure 6 It is a schematic diagram of the structure of a computer-readable storage medium embodiment of the present invention.

[0036] In the figure: 1. Single wavelength picosecond pulse laser; 2. Collimator; 3. Spatial light modulator; 4. Dichroic mirror; 5. Scanning galvanometer; 6. Scanning lens; 7. Sleeve lens; 8. Objective lens; 9. Stage; 10. Lens; 11. Filter; 12. Multimode optical fiber; 13. Photomultiplier tube; 14. Computer; 15. Cage cube; 16. Cage assembly support rod; 17. Extension rod; 18. Optical breadboard; 19. Vibration isolation rubber base; 20. Cage plate. DETAILED DESCRIPTION

[0037] 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.

[0038] To this end, the present invention proposes a method for realizing multi-color super-resolution imaging using a single-beam loss laser.

[0039] Reference Figure 1 In an embodiment of the present invention, the probe for multicolor super-resolution imaging consists of three parts: a donor, an acceptor and a connecting arm. A short-wavelength organic dye is selected as an energy donor, and the donor is excited by a short-wavelength pulsed laser. The acceptor selects two metal complexes with similar properties, and uses rigid groups of benzene rings, naphthalene rings and alkynyl groups as connecting arms to connect to the donor respectively, to obtain a probe that emits yellow and red multicolor fluorescence. Then, probes with different emission wavelengths are used to dye and mark the same sample.

[0040] Reference Figure 2 and Figure 3 . Emitted by the optical system Figure 2 The laser has a light intensity distribution as shown. When the laser is used to illuminate the sample, the principle of probe light emission and loss is as follows Figure 3 As shown, the laser intensity is high at the outer ring position, and the sample is stimulated to absorb when irradiated by the laser, and the fluorescent dye absorbs energy from the low energy level to the high energy level. Due to the high laser intensity, the stimulated radiation loss process occurs in the excited state, and the energy transfer from the donor to the acceptor is blocked in the ring area. Therefore, no fluorescence is generated in the ring area. At the center position, the laser intensity is low, and only the excited state of the absorption transition occurs, and no stimulated radiation phenomenon is generated. The excitation energy is only transferred to the acceptor through the connecting arm, and the excitation energy acceptor undergoes spontaneous radiation, generating fluorescence. The size of the imaging area is controlled, thereby producing a super-resolution phenomenon.

[0041] Reference Figure 4 Figure 5 . The light source assembly includes a single-wavelength picosecond pulse laser 1, a collimator 2, and a spatial light modulator 3. The laser is emitted by the laser, and after being modulated by the collimator and the spatial light modulator, it forms a laser beam with weak intensity in the center and high intensity in the outer circle, and enters the optical path assembly. The optical path assembly includes a dichroic mirror 4, a scanning galvanometer 5, a scanning lens 6, a sleeve lens 7, an objective lens 8, a stage 9, a lens 10, and a filter 11. The incident laser is reflected by the dichroic mirror to the scanning galvanometer. The scanning galvanometer is used to deflect the laser beam. The scanning lens and the sleeve lens collimate and expand the laser beam to a size that fills the entrance pupil of the microscope objective. Finally, the objective lens focuses the laser on the stained sample on the stage. The fluorescent signal emitted by the sample after being irradiated is then received by the receiving terminal through the optical path assembly. The receiving terminal includes a multimode optical fiber 12, a photomultiplier tube or an avalanche photodiode 13, and a computer 14. The fluorescence signal is transmitted to the photomultiplier tube by multimode optical fiber, amplified by the photomultiplier tube, converted into an electrical signal, and transmitted to the computer for processing. The system is built on an optical platform and connected with a cage structure.

[0042] Reference Figure 6The present invention provides a storage medium that can be used to store computer program instructions to implement software functions. The computer readable storage medium stores computer instructions, which are used to enable the computer to execute image acquisition, data storage and image data processing.

[0043] The processing results show that the present invention selects receptors of different properties to connect with the same donor, realizes excitation at the same wavelength, and emits fluorescence of different wavelengths. The intensity distribution of the irradiated laser is adjusted, the laser intensity is weak in the center, the donor mainly undergoes stimulated absorption, and the outer circle is strong, the donor undergoes both stimulated absorption and stimulated radiation, blocking energy transfer, thereby controlling the imaging range and achieving super-resolution imaging effect. It has the characteristics of simple structure and easy operation, and reduces the damage of the light source to cells and probes.

[0044] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0045] 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 design scheme for a multicolor super-resolution imaging probe, characterized in that: The fluorescent probe is mainly composed of two core parts: an energy donor and an energy acceptor, which are tightly bound by a connecting arm. Donor: It has two absorption peaks and one emission peak. When the donor is excited at a short wavelength, it produces corresponding fluorescence emission. When it is excited at a long absorption wavelength, there is no fluorescence emission. The donor absorbs laser energy and determines the absorption wavelength of the probe. Linker arm: transfers the donor's stimulated absorption transition to a higher energy level to the acceptor. Receptor: emits fluorescence and determines the wavelength of fluorescence emission emitted by the probe.

2. The design of a multicolor super-resolution imaging probe according to claim 1, characterized in that: The donor has two absorption peaks and one fluorescence emission peak. Short-wavelength excitation produces fluorescence, while long-wavelength excitation has no fluorescence emission. When the donor is irradiated by laser, the donor absorbs energy and transitions from the ground state to the excited state, and transfers the excitation energy to the acceptor part through the connecting arm. The choice of the donor determines the absorption and excitation wavelengths of the probe.

3. The design scheme of a multi-color super-resolution imaging probe according to claim 1, characterized in that: The linker acts as a linker in the molecular structure, connecting the donor and acceptor parts together to ensure efficient energy transfer from the donor to the acceptor. Its length and properties affect the efficiency of energy transfer and the overall performance of the probe.

4. The design of a multi-color super-resolution imaging probe according to claim 1, characterized in that: The receptor receives the energy of the excited state of the donor, transitions to the excited state, and then spontaneously radiates to produce fluorescence. The receptor determines the wavelength of the fluorescence emitted by the probe. By selecting receptors with different properties, it is possible to generate fluorescence of multiple different wavelengths by laser excitation with the same wavelength.

5. A multi-color super-resolution imaging method of a probe, based on the design of a multi-color super-resolution imaging probe according to claims 1-4, characterized in that: The following steps are involved: S1. Assume a short wavelength ring laser. S2, the laser beam has weak light in the center and strong light in the outer circle, and the probe is illuminated by the laser; S3. The intensity of the laser in the central area is low, and stimulated absorption mainly occurs. The laser in the annular area is high, and stimulated absorption simultaneously causes stimulated radiation loss, blocking the energy transfer process. The size of the imaging area is limited, thereby achieving super-resolution imaging effects.

6. An imaging module of a multicolor super-resolution imaging probe, based on the design scheme and imaging method of the probe according to claims 1-5, characterized in that: include: Light source assembly, optical path assembly, receiving terminal.

7. The imaging module of a multi-color super-resolution imaging probe according to claim 6, characterized in that: The light source assembly comprises: Single-wavelength picosecond pulse laser: generates pulsed excitation light; Collimator: collimates the laser beam; Spatial light modulator: converts the laser light into a form in which the center is weak and the outer ring is strong. The laser light is emitted by a pulse laser and passes through a lens and a spatial light modulator to form the ring laser as described in claim 5.

8. The imaging module of a multi-color super-resolution imaging probe according to claim 6, characterized in that: The optical path component comprises: Scanning galvanometer: used to synchronously scan the excitation light to achieve area array imaging of the sample; Scan lens: used to collect the laser beam. Objective lens: used to focus the laser onto the focal plane and collect the fluorescence signal reflected from the sample. Tube lens: Combined with the objective lens to form a microscopic imaging system. Dichroic mirror: used to transmit excitation light and reflect fluorescence signals. Lens: Focuses the parallel laser beam. Stage: used to place and fix samples. Reflector: changes the direction of laser transmission. Filter: used to remove stray light other than fluorescence and improve the image signal-to-noise ratio.

9. The imaging module of a multi-color super-resolution imaging probe according to claim 6, characterized in that: The receiving terminal comprises: Multimode optical fiber: used to transmit signals to the photomultiplier tube detector, and its core serves as the detection aperture of the system. Photomultiplier tube or avalanche photodiode: used to collect fluorescent photons, amplify the fluorescent signal, and output electrical signals to the computer. Computer: used to control the software to acquire images, store data and process image data.

10. A computer-readable storage medium, based on the probe design and imaging system according to claims 1-9, characterized in that: The computer-readable storage medium stores computer instructions, which are used to enable the computer to execute image acquisition, data storage and image data processing.

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