An up-conversion and down-conversion dual-mode structured light illumination microscopy system

Abstract

This application discloses an upconversion and downconversion dual-mode structured light illumination microscopy imaging system. The system mainly includes: a first structured light generation module for generating a first structured light, which is used to excite downconversion luminescence; a second structured light generation module for generating a second structured light, which is used to excite upconversion luminescence; a two-dimensional scanning module for scanning the structured light in the focal plane of the microscope objective; and an imaging and detection module for detecting the fluorescent signal excited by the sample to be imaged. This application addresses the problems of limited imaging depth and low resolution in deep imaging in optical microscopy technology. By combining the technical advantages of near-infrared second-zone imaging technology, upconversion luminescence technology, and structured light illumination microscopy, this application can simultaneously achieve deep imaging and high-resolution imaging, while effectively ensuring image quality.

Description

Technical Field

[0001] The present application relates to the field of bio-optics, and in particular to an up-conversion and down-conversion dual-mode structured light illumination microscopy imaging system. Background Art

[0002] In recent years, the development of life sciences has put forward new requirements for biological imaging: a gradual shift from surface imaging to deep imaging. Compared to fluorescence in the visible light band (390-780 nm), fluorescence in the near-infrared region II (1000-1700 nm) experiences relatively weak light scattering in biological tissue and possesses greater tissue penetration. Furthermore, the autofluorescence of endogenous molecules in biological tissue is relatively weak within this band. Therefore, imaging of biological tissue in the near-infrared region II offers advantages such as large imaging depth, high resolution, and low background noise. Therefore, developing near-infrared region II imaging technology and systems and applying them to life science research has significant scientific significance and application value.

[0003] Common fluorescent materials are down-conversion materials, whose characteristic is that they absorb a single short-wavelength photon and emit a long-wavelength photon. Conversely, up-conversion materials are characterized by the simultaneous absorption of multiple long-wavelength photons and the emission of a single short-wavelength photon. Endogenous biological molecules are mostly fluorescently excited by down-conversion luminescence, so labeling biological samples with up-conversion materials can further eliminate background light interference and improve image quality. Furthermore, applying up-conversion and down-conversion materials with both excitation and emission wavelengths in the near-infrared region II to biological imaging can effectively increase imaging depth and facilitate the expansion of near-infrared region II imaging systems.

[0004] However, for far-field imaging, image resolution is inevitably constrained by the optical diffraction limit, which is limited by both the fluorescence emission wavelength and the numerical aperture of the objective lens. Compared to fluorescence in the visible and near-infrared regions, fluorescence in the near-infrared region II has a longer wavelength, resulting in image resolution in the micrometer range for conventional near-infrared region II imaging techniques. Furthermore, as imaging depth increases, light scattering and absorption by biological tissues severely degrades image resolution, hindering the resolution of deep tissue details and hindering in-depth analysis and study of their connections.

[0005] The imaging methods of near-infrared second-region fluorescence imaging technology include bright-field imaging and point scanning imaging. Both of the above imaging methods can obtain image information of biological samples in the near-infrared second-region band and have certain effects, but they also have their own problems and shortcomings. The main manifestations are: the image resolution of the former is not very ideal, and the optical path design and system configuration of the latter are relatively complex and the temporal resolution is limited. Similarly, due to the limitations of the optical diffraction limit, the above two imaging technologies cannot well meet the high-resolution imaging requirements in deep tissues. Summary of the Invention

[0006] The embodiments of the present application provide an up-conversion and down-conversion dual-mode structured light illumination microscopy imaging system to at least solve the problems of limited imaging depth and low resolution in deep imaging in the prior art.

[0007] According to one aspect of the present application, an up-conversion and down-conversion dual-mode structured light illumination microscopy imaging system is provided, comprising: a first structured light generating module for generating a first structured light, the first structured light being used to excite down-conversion luminescence; a second structured light generating module for generating a second structured light, the second structured light being used to excite up-conversion luminescence; a two-dimensional scanning module for realizing scanning of the structured light in the focal plane of the microscope objective; and an imaging and detection module for detecting a fluorescent signal excited by a sample to be imaged.

[0008] Furthermore, the first structured light generating module and the second structured light generating module both include: a spatial light modulator or a microlens array, the spatial light modulator is used to perform phase modulation on light, or the microlens array is used to generate a dot matrix illumination light field.

[0009] Furthermore, the first structured light generating module and the second structured light generating module both include: a laser for emitting excitation light of a specific wavelength; a polarization beam splitter for polarization splitting the excitation light; and the spatial light modulator for phase modulating the light passing through the polarization beam splitter.

[0010] Furthermore, the wavelength of the fluorescence signal excited by the sample to be imaged is within the second near-infrared band.

[0011] Furthermore, the two-dimensional scanning module includes a two-dimensional scanning galvanometer or an acousto-optic deflector.

[0012] Furthermore, the first structured light generating module and the second structured light generating module both include: a beam expander, configured to expand the excitation light before the excitation light enters the polarization beam splitter.

[0013] Furthermore, the first structured light generating module and the second structured light generating module both include: a half-wave plate for adjusting the polarization direction of the incident light, which is arranged between the beam expander and the spatial light modulator, or between the beam expander and the microlens array.

[0014] Furthermore, the first structured light generating module and the second structured light generating module both include: wherein, the light emitted from the spatial light modulator or the microlens array passes through one of the two plano-convex lenses, a spatial filter, and the other plano-convex lens of the two plano-convex lenses in sequence before being incident on the two-dimensional scanning module.

[0015] Furthermore, when the first structured light generating module and / or the second structured light generating module includes multiple lasers, the excitation light emitted by the multiple lasers is emitted along the same optical path through their respective corresponding dichroic mirrors and / or reflective mirrors, wherein the wavelength of the excitation light emitted by each of the multiple lasers is different.

[0016] Furthermore, the imaging and detection module includes: a three-dimensional stage for placing the sample to be imaged and adjusting it in three dimensions; a microscope objective lens for focusing the excitation light on the sample to be imaged; and a near-infrared camera for detecting the fluorescence signal excited by the sample to be imaged.

[0017] Furthermore, the imaging and detection module includes: two plano-convex lenses, which guide the output from the two-dimensional scanning module to the dichroic mirror.

[0018] Furthermore, the imaging and detection module includes: the dichroic mirror, which is used to control the direction of the excitation light and filter out the residual excitation light in the fluorescence signal.

[0019] Furthermore, the imaging and detection module includes: a filter for filtering out stray light in the fluorescence signal.

[0020] In the embodiments of the present application, a first structured light generation module is used to generate a first structured light, which is used to excite down-conversion luminescence; a second structured light generation module is used to generate a second structured light, which is used to excite up-conversion luminescence; a two-dimensional scanning galvanometer is used to scan the structured light in the focal plane of the microscope objective; and an imaging and detection module is used to detect the fluorescence signal excited by the sample. This application solves the problems of limited imaging depth and low resolution in deep imaging in the prior art, and can effectively improve and optimize the imaging depth and image resolution of optical microscopy technology in deep imaging, while ensuring image quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of this application. The exemplary embodiments and descriptions of this application are intended to explain this application and do not constitute an improper limitation on this application. In the accompanying drawings:

[0022] Figure 1 Schematic diagram of the imaging system according to an embodiment of the present application.

[0023] Description of reference numerals:

[0024] Figure 1 Middle: 1-980nm laser, 2-850nm laser, 3-785nm laser, 4-first bandpass filter, 5-second bandpass filter, 6-third bandpass filter, 7-first reflector, 8-first dichroic mirror, 9-second dichroic mirror, 10-first beam expander, 11-first half-wave plate, 12-first polarization beam splitter (e.g., polarization beam splitter cube), 13-first spatial light modulator, 14-second reflector, 15-first plano-convex lens, 16-first spatial filter, 17-second plano-convex lens, 18-1550nm laser, 19-fourth bandpass filter, 20-second beam expander, 21-second half-wave plate, 2 2-second polarization beam splitter (e.g., polarization beam splitter cube), 23-third reflector, 24-fourth reflector, 25-second spatial light modulator, 26-fifth reflector, 27-third plano-convex lens, 28-second spatial filter, 29-fourth plano-convex lens, 30-third dichroic mirror, 31-two-dimensional scanning galvanometer, 32-sixth reflector, 33-fifth plano-convex lens, 34-sixth plano-convex lens, 35-fourth dichroic mirror, 36-microscope objective, 37-three-dimensional high-precision stage, 38-filter, 39-tube lens, 40-near-infrared camera, 41-two-dimensional scanning galvanometer connecting cable, 42-near-infrared camera connecting cable, 43-computer. DETAILED DESCRIPTION

[0025] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0026] In this embodiment, an up-conversion and down-conversion dual-mode structured light illumination microscopy imaging system is provided, including: a first structured light generating module, used to generate a first structured light, the first structured light is used to excite the down-conversion luminescence; a second structured light generating module, used to generate a second structured light, the second structured light is used to excite the up-conversion luminescence; a two-dimensional scanning module, used to realize the scanning of the structured light in the focal plane of the microscope objective lens; and an imaging and detection module, used to detect the fluorescence signal excited by the sample.

[0027] In the system of this embodiment, not only the near-infrared two-zone imaging technology and the structured light illumination microscopy imaging technology are integrated, but also the up-conversion luminescence and the down-conversion luminescence are integrated into the same system. On the one hand, it solves the problem of limited imaging depth and low resolution under deep imaging in the existing imaging technology, and obtains high-resolution and high-quality imaging effects in the deep layer; on the other hand, it also makes the system have a broader application scenario. The generation of structured light can be achieved by a spatial light modulator or a microlens array. The first structured light generation module and the second structured light generation module both include: a spatial light modulator or a microlens array, the spatial light modulator is used to phase modulate light, or the microlens array is used to generate a dot matrix structured light illumination light field. Compared with the microlens array, the spatial light modulator has more advantages: it can flexibly design phase graphic files according to specific needs, generate a preset structured illumination light field (not limited to a dot array), and can be reused.

[0028] The first and second structured light generation modules may further include a laser for emitting excitation light and a polarization beam splitter for polarization-splitting the excitation light. In this case, a spatial light modulator is used to phase modulate the light passing through the polarization beam splitter. It should be noted that the wavelength of the fluorescence signal excited by the sample is within the near-infrared region II.

[0029] The two-dimensional scanning module includes a two-dimensional scanning galvanometer or an acousto-optic deflector. In the following embodiments, the two-dimensional scanning galvanometer is used as an example for description.

[0030] In an optional embodiment, at least one of the following modules can be added to the first structured light generating module and the second structured light generating module to improve the performance of the system: for example, a beam expander for expanding the excitation light before the excitation light enters the polarization beam splitter; or a half-wave plate for controlling the polarization direction of the incident light, which can be arranged between the beam expander and the spatial light modulator, or between the beam expander and the microlens array. For another example, a spatial filter and two plano-convex lenses can be added, wherein the light emitted from the spatial light modulator or the microlens array passes through one of the plano-convex lenses, the spatial filter and the other plano-convex lens in sequence before being incident on the two-dimensional scanning galvanometer. There are at least two lasers, and multiple lasers are conducive to expanding the use scenarios of this system. In the case where the first structured light generating module and / or the second structured light generating module include multiple lasers, the excitation light emitted by the multiple lasers is emitted along the same optical path through a dichroic mirror and / or a reflector, wherein the wavelength of the excitation light emitted by each of the multiple lasers is different.

[0031] The module composition of imaging and detection can be determined according to the specific implementation situation. For example, in an optional embodiment, the imaging and detection module may include: a three-dimensional high-precision stage for placing the sample to be imaged; a microscope objective lens for focusing the excitation light on the sample to be imaged; and a near-infrared camera for detecting the fluorescent signal emitted by the sample to be imaged when it is excited.

[0032] In a preferred embodiment, to improve performance, the imaging and detection module must include the following components: a dichroic mirror for controlling the direction of the excitation light and further filtering out residual excitation light from the fluorescence signal; and a filter for filtering out stray light from the fluorescence signal. Alternatively, the imaging and detection module may include a 4f system consisting of two additional plano-convex lenses to sequentially relay the light beams.

[0033] The near-infrared dual-band up-conversion and down-conversion dual-modal structured light illumination microscopy system and imaging method proposed in this embodiment combine the advantages of near-infrared dual-band imaging technology, up-conversion luminescence, and structured light illumination microscopy, enabling simultaneous deep-layer, high-spatial-resolution imaging. This system will play a significant role in research in fields such as biomedicine and life sciences. This embodiment utilizes lasers of different wavelengths in conjunction with two spatial light modulators to generate structured light for down-conversion excitation and structured light for up-conversion excitation, respectively. A two-dimensional scanning galvanometer enables rapid scanning of each structured light across the sample surface. Combined with a high-speed, highly sensitive near-infrared camera, this system enables in vivo, real-time imaging of biological tissue. Furthermore, the spatial light modulator and near-infrared camera employed in this embodiment respond to both visible and near-infrared wavelengths, enabling multi-band imaging of biological samples and obtaining more information about the sample's interior.

[0034] The following describes an imaging system of an optional embodiment, which mainly includes: a structured light generating module A for down-conversion excitation (i.e., a first structured light generating module) A, a structured light generating module B for up-conversion excitation (i.e., a second structured light generating module) B, a dichroic mirror, a two-dimensional scanning galvanometer C, a plane reflector, an imaging and detection module D, data lines 41 / 42 and a computer 43.

[0035] The structured light generating module A for up-conversion excitation and the structured light generating module B for down-conversion excitation can respectively generate structured light for up-conversion excitation and structured light for down-conversion excitation. The structured light generated by the above two modules is combined by a dichroic mirror and incident on the two-dimensional scanning galvanometer C; the reflected light of the two-dimensional scanning galvanometer C is reflected by the reflector and then passes through the two plano-convex lenses 33 / 34 in the imaging and detection module in sequence, and then is reflected by the dichroic mirror 35 and enters the microscope objective 36, and finally focuses on the sample placed on the three-dimensional high-precision translation stage 37 to excite the down-conversion fluorescent probe or up-conversion fluorescent probe marked therein and emit fluorescence outward. The emitted fluorescence signal will return along the original path of the microscope objective 36 and first pass through the long-pass dichroic mirror 35, then pass through a bandpass filter 38 to further filter out the residual excitation light, and then be imaged on the chip of the near-infrared detector through the sleeve tube mirror 39 and detected by it; by controlling the galvanometer C, the scanning of structured light in the XY plane can be achieved; at the same time, the near-infrared detector will collect an original image of the sample at each position scanned by the two-dimensional scanning galvanometer (for example, the camera can collect an original image at each scanning position). After these original images are subsequently reconstructed by the computer 43, a high-resolution image of the sample under test can be obtained.

[0036] The structured light generation module A for down-conversion excitation used in this embodiment may include three single-mode continuous lasers 1 / 2 / 3 with wavelengths of 785nm, 850nm and 980nm, respectively, bandpass filters 4 / 5 / 6, a plane reflector 7, a dichroic mirror 8 / 9, a first laser beam expander 10, a half-wave plate 11, a polarization beam splitter 12, a spatial light modulator 13, a plano-convex lens 15 / 17, a spatial filter 16 and other components. In the structured light generation module used for down-conversion excitation, the specific type of dichroic mirror is determined according to the direction of the optical path; the half-wave plate and polarization beam splitter used have a transmittance of more than 95% and reliable working performance in the light wavelength range of 700-1100nm; the spatial light modulator used has a good phase modulation effect in the light wavelength range of 700-1100nm; the reflector used has a transmittance of more than 95% in the light wavelength range of 700-1100nm; the plano-convex lenses used are all achromatic cemented lenses, and have a transmittance of more than 95% in the working band of 700-1100nm.

[0037] The structured light generation module B for upconversion excitation used in this embodiment may include components such as a single-mode continuous laser 18 with a wavelength of 1550 nm, a bandpass filter 19, a laser beam expander 20, a half-wave plate 21, a polarization beam splitter 22, plane mirrors 23 / 24, a spatial light modulator 25, plano-convex lenses 27 / 29, and a spatial filter 28. In the structured light generation module for upconversion excitation, the half-wave plate and polarization beam splitter used have a transmittance of over 95% and reliable working performance within the wavelength range of 1100-1650 nm; the spatial light modulator used has a good phase modulation effect within the wavelength range of 1100-1650 nm; the mirrors used have a transmittance of over 95% within the wavelength range of 1100-1650 nm; and the plano-convex lenses used are all plano-convex lenses with a transmittance of over 95% within their operating wavelength range of 1100-1650 nm.

[0038] The imaging and detection module D in this embodiment may include a plano-convex lens 33 / 34 , a dichroic mirror 35 , a microscope objective 36 , a three-dimensional high-precision translation stage 37 , a filter 38 , a sleeve tube mirror 39 and a near-infrared camera 40 .

[0039] The dichroic mirror outside the module in this system is a short-pass dichroic mirror, which cooperates with the plane mirror to realize the beam combination of the up-conversion excitation light path and the down-conversion excitation light path and the subsequent beam guidance; the two-dimensional scanning galvanometer is a high-speed scanning galvanometer to ensure the system's temporal resolution, and the X-axis and Y-axis mirrors of the two-dimensional scanning galvanometer have a reflectivity of more than 95% in the wavelength band of 700-1650nm to ensure the utilization efficiency of the incident light.

[0040] The microscope objective lens used in the imaging and detection module D in this embodiment is a water-immersion objective lens with an operating wavelength range of 380-1500nm. The operating wavelength range of the filter used is selected according to the specific experimental requirements. The three-dimensional high-precision translation stage used has a movement accuracy of no less than 1μm and a travel range of no less than 5mm in the XY plane, and a movement accuracy of no more than 1μm and a travel range of no less than 10mm in the Z direction to ensure accurate focusing and movement of the sample. The near-infrared camera used has a quantum efficiency of more than 80% in the range of 950-1600nm, which can effectively ensure the detection efficiency and signal intensity of the fluorescence signal. In the entire imaging system, the spatial light modulator, two-dimensional scanning galvanometer, near-infrared camera, and three-dimensional high-precision translation stage are all connected to the computer via data cables, and communication and control are achieved through a control program to ensure the system's integration and stability.

[0041] In this embodiment, the down-conversion luminescent structured light illumination microscopy experiment and the up-conversion luminescent structured light illumination microscopy experiment cannot be performed at the same time. In the imaging and detection module D, the dichroic mirror and the filter can be switched according to the specific experiment: for example, when the down-conversion luminescent structured light illumination microscopy experiment is required, the dichroic mirror in the optical path is switched to a long-pass dichroic mirror, and the filter should be a long-wave pass filter or a band-pass filter of the corresponding wavelength band; when the up-conversion luminescent structured light illumination microscopy experiment is required, the dichroic mirror in the optical path is switched to a short-pass dichroic mirror, and the filter should be a short-wave pass filter or a band-pass filter of the corresponding wavelength band.

[0042] The purpose of switching filters is as follows:

[0043] (1) During up-conversion luminescence, the wavelength of the excitation light is greater than the wavelength of the emitted light (i.e., fluorescence), and the dichroic mirror used is a short-pass dichroic mirror (light with a wavelength less than a specific wavelength is allowed to pass through the dichroic mirror, while light with a wavelength greater than the specific wavelength is not allowed to pass through and is reflected at the dichroic mirror interface. The size of the specific wavelength depends on the parameters of the dichroic mirror used), and then the fluorescence is filtered out;

[0044] (2) When down-converting luminescence, the wavelength of the excitation light is shorter than the wavelength of the emitted light (i.e., fluorescence), and the dichroic mirror used is a long-pass dichroic mirror (light with a wavelength longer than a specific wavelength is allowed to pass through the dichroic mirror, while light with a wavelength shorter than the specific wavelength is not allowed to pass through and is reflected at the dichroic mirror interface. The size of the specific wavelength depends on the parameters of the dichroic mirror used), and then the fluorescence is filtered out;

[0045] (3) Due to process limitations, the dichroic mirror cannot completely filter out the excitation light: the function of the filter is to further filter out the residual excitation light in the fluorescence, and the determination of the filter parameters is determined by the fluorescence wavelength.

[0046] From (1)(2)(3), we can know that since the relationship between fluorescence and excitation light wavelength is different in the up-conversion luminescence and down-conversion luminescence processes, it is necessary to switch the filter module (a combination of a dichroic mirror and a filter) to detect the fluorescence emitted by the sample during the up-conversion luminescence and down-conversion luminescence processes; if the sample is labeled with fluorescent dyes that can simultaneously emit up-conversion luminescence and down-conversion luminescence, up-conversion luminescence and down-conversion luminescence can occur simultaneously, but in the process of detecting fluorescence, it is necessary to switch the filter module (a combination of a dichroic mirror and a filter) to detect the fluorescence in each process.

[0047] The purpose of this embodiment is to achieve deep and high-resolution imaging effects at the same time. Therefore, the integration of upconversion luminescence and downconversion luminescence in one system is to give the system a larger application space (the dye in the sample can be a fluorescent dye that upconverts luminescence and downconversion luminescence), and there will be more choices for the dyes to be used in the sample; in addition, the autofluorescence process in organisms is usually a downconversion luminescence process. If upconversion luminescence technology is selected, the image quality can be further improved.

[0048] Regarding the selection of excitation light, this embodiment uses continuous-wave lasers in the near-infrared band as excitation light (wherein, lasers with wavelengths of 785nm, 850nm, and 980nm are used as excitation light sources for down-conversion luminescence, and a laser with a wavelength of 1550nm is used as an excitation light source for up-conversion luminescence). Each incident light is phase-modulated by a spatial light modulator to generate an illumination light field with a certain spatial distribution. A two-dimensional scanning galvanometer is used to achieve rapid scanning of the illumination light field on the sample surface to excite the near-infrared second-zone up-conversion or down-conversion fluorescent probe in the sample and then emit a near-infrared second-zone fluorescence signal. The fluorescence signal is finally detected by a near-infrared camera. By collecting multiple original images of the sample under test and performing image reconstruction, a high-resolution image of the sample under test at a deep level can be obtained.

[0049] Figure 1 This is a schematic diagram of the imaging system according to an embodiment of the present application. Figure 1 This embodiment will be described.

[0050] refer to Figure 1 As shown in , the system of this embodiment mainly includes: (A) a structured light generation module for down-conversion excitation, (B) a structured light generation module for up-conversion excitation, (C) a two-dimensional scanning galvanometer, (D) an imaging and detection module, a dichroic mirror, a plane reflector, a data cable and a computer, etc.

[0051] In this embodiment, the switching states of the three lasers used for upconversion excitation are determined by the absorption wavelength of the fluorescent probe used in the biological sample being tested. When selecting a laser, a laser with linearly polarized output light should be selected as much as possible to fully utilize the laser energy.

[0052] The determination of the beam size at different positions in this embodiment is mainly based on the following considerations: (1) The liquid crystal windows corresponding to the first spatial light modulator 13 and the second spatial light modulator 25 are both rectangular. In order to obtain a more ideal phase modulation effect, it is necessary to ensure that the beam diameter of the incident light is not less than the short side of each liquid crystal window. Therefore, it is necessary to reasonably select and determine the parameters of the first beam expander and the second beam expander; (2) The structured light used for up-conversion excitation and the structured light used for down-conversion excitation must both be scanned by the two-dimensional scanning galvanometer 31. Therefore, the spatial size of each structured light must match the size of the X-axis and Y-axis mirror surfaces of the two-dimensional scanning galvanometer 31; the incident beam size of the X-axis and Y-axis mirror surfaces of the two-dimensional scanning galvanometer 31 can be controlled by controlling the ratio of the focal lengths of the first plano-convex lens 15 to the second plano-convex lens 17, and the ratio of the focal lengths of the third plano-convex lens 27 to the fourth plano-convex lens 29.

[0053] The generation of structured light for down-conversion excitation and structured light for up-conversion excitation is specifically achieved by loading preset phase pattern files on the first spatial light modulator 13 and the second spatial light modulator 25. In the above generation process, due to the existence of diffraction, the diffracted light beams reflected by the first spatial light modulator 13 and the second spatial light modulator 25 need to be spatially filtered: therefore, it is necessary to respectively set a customized first spatial filter 16 and a second spatial filter 28 at the back focus of the first plano-convex lens 15 (i.e., the front focus of the second achromatic plano-convex lens 17) and the back focus of the third plano-convex lens 27 (i.e., the front focus of the fourth achromatic plano-convex lens 29) to filter out the zero-order diffracted light that is insufficiently diffracted, thereby ensuring the quality of the structured light for down-conversion excitation and the structured light for up-conversion excitation.

[0054] The first and second spatial light modulators 13 and 25 can also be replaced with microlens arrays as needed, thereby generating a point array illumination light field at the focal plane. However, compared to microlens arrays, the first and second spatial light modulators 13 and 25 offer advantages: they can flexibly design phase pattern files based on specific needs, generating pre-defined structured illumination light fields (not limited to point arrays), and are reusable. Furthermore, the system's two-dimensional scanning galvanometer 31 can be replaced with a two-dimensional scanning unit composed of two single-axis acousto-optic deflectors, depending on specific circumstances, to further improve the system's imaging speed.

[0055] The experimental sample is placed on a three-dimensional high-precision stage 37, which can be finely adjusted in the X and Y dimensions to select the target imaging area of ​​interest. In addition, the three-dimensional high-precision stage 37 can also be adjusted in the Z direction for fast and accurate focusing to obtain images of the sample at different depths.

[0056] The near-infrared second-region fluorescent probes marked inside biological samples mainly emit light in upconversion and downconversion modes. Both must have the characteristics of high quantum efficiency, high and stable luminescence intensity, good biocompatibility and no clustering in the body. They can be reasonably selected or customized according to specific imaging scenarios and needs.

[0057] In this embodiment, the first spatial light modulator 13 and the second spatial light modulator 25 are liquid crystal spatial light modulators with model X15213-07 produced by Hamamatsu of Japan and model E19*12-500-1200-HDM8 produced by Meadowlark Optics of the United States, respectively, which can have good light energy utilization efficiency in the 1550nm and 500-1200nm bands, respectively; the near-infrared camera 40 is the NIRvana HS near-infrared camera produced by Princeton Instruments, which has the characteristics of high speed, high sensitivity and low noise, and has a high quantum efficiency in the near-infrared second band.

[0058] The first polarization beam splitter 12 and the second polarization beam splitter 22 can respectively separate the outgoing light with the polarization direction in the horizontal direction, so that the first spatial light modulator 13 and the second spatial light modulator 25 have a phase modulation effect on their respective incident light; by adjusting the angles of the first half-wave plate 11 and the second half-wave plate 21 respectively, the intensity of the incident light of the first polarization beam splitter 12 and the second polarization beam splitter 22 can be adjusted and controlled.

[0059] In addition, the other optical components selected in this system have high transmittance or reflectivity in the near-infrared II band to ensure that the loss of the near-infrared II fluorescence signal during the optical transmission process is as small as possible, thereby maximizing the advantages of the near-infrared II system in imaging depth.

[0060] According to the principles of structured light illumination microscopy, in order to maximize the improvement in the spatial resolution of the reconstructed image, it is necessary to ensure that the spatial frequency of the generated structured illumination light field is as close as possible to the diffraction limit of this imaging system. To this end, the phase pattern file loaded on the spatial light modulator needs to be pre-designed and optimized.

[0061] When the near-infrared second-region up-conversion and down-conversion dual-mode structured light illumination microscopy imaging system of this embodiment performs imaging, the corresponding specific workflow includes the following parts:

[0062] First, the laser beam emitted by 980nm laser 1 passes through first bandpass filter 4 and is reflected by first reflector 7 onto the rear surface (transmission surface) of first dichroic mirror 8. The laser beam emitted by 850nm laser 2 passes through second bandpass filter 5 and is incident on the front surface (reflection surface) of first dichroic mirror 8. After being transmitted through the rear surface of first dichroic mirror 8, the laser beam emitted by 980nm laser 1 merges with the beam emitted by 850nm laser 2 reflected by the first dichroic mirror 8 and is incident on the rear surface (reflection surface) of second dichroic mirror 9, where it is reflected. The laser beam emitted by 785nm laser 3 passes through third bandpass filter 6 and is incident on the front surface (transmission surface) of second dichroic mirror 9, where it is transmitted. It then merges with the beams emitted by 980nm laser and 850nm laser, which were reflected by the rear surface (reflection surface) of second dichroic mirror 9. After combining the three laser beams of different wavelengths, they are collimated and expanded by the first beam expander 10. They then pass through the first half-wave plate 11 and the first polarization beam splitter 12 in sequence before being incident on the first spatial light modulator 13 and phase modulated. The reflected light beam from the first spatial light modulator 13 is incident on the second reflector 14 and reflected. The reflected light from the second reflector 14 passes through the first plano-convex lens 15, the first spatial filter 16, and the second plano-convex lens 17 in sequence before being incident on the rear surface (transmission surface) of the third dichroic mirror 30 and transmitted. Thus, structured light for down-conversion excitation is obtained. The first dichroic mirror 8 is a long-wavelength pass dichroic mirror, while the second dichroic mirror 9 and the third dichroic mirror 30 are short-wavelength pass dichroic mirrors.

[0063] Then, the laser light emitted by the 1550nm laser 18 passes through the fourth bandpass filter 4 in sequence, and is collimated and expanded by the second beam expander 20; then, it passes through the second half-wave plate 21 and the second polarization beam splitter 22 in sequence, and is incident on the third reflector 23 and then reflected on the fourth reflector 24. The reflected light of the fourth reflector 24 is incident on the second spatial light modulator 25 and is reflected after being phase modulated; the reflected light beam of the second spatial light modulator 25 is incident on the fifth reflector 26 and is reflected. The reflected light of the fifth reflector 26 passes through the third plano-convex lens 27, the second spatial filter 28 and the fourth plano-convex lens 29 in sequence, and is incident on the rear surface (reflection surface) of the third dichroic mirror 30: at this point, structured light for down-conversion excitation is obtained.

[0064] Next, the structured light used for down-conversion excitation is reflected by the rear surface (reflective surface) of the third dichroic mirror 30, merges with the structured light used for down-conversion excitation transmitted by the front surface (transmissive surface) of the third dichroic mirror 30, and then enters the X-axis reflector and Y-axis reflector within the two-dimensional scanning galvanometer 31. The reflected light from the two-dimensional scanning galvanometer 31 is incident on the sixth reflector 32 and reflected. After passing through the fifth plano-convex lens 33 and the sixth plano-convex lens 34, it is incident on the fourth dichroic mirror 35 and reflected by it, uniformly filling the entrance pupil of the microscope objective 36. Then, it enters the microscope objective 36 from the entrance pupil and is focused on the sample surface, thereby exciting the fluorescent probe labeled with the near-infrared second region up-conversion or down-conversion luminescence inside the biological sample.

[0065] Finally, the near-infrared second-region fluorescence signal excited in the biological sample returns along its original path through microscope objective 36 and passes through fourth dichroic mirror 35. The excitation light is then further filtered out by filter 38. Finally, tube lens 39 forms an image of the fluorescence signal onto the detection chip of near-infrared camera 40, which then records the sample fluorescence image. By collecting multiple raw images and performing image reconstruction and data processing using a computer program, a high-resolution reconstructed image can be obtained.

[0066] The two-dimensional scanning galvanometer 31 and the near-infrared camera 40 are connected to the computer 43 through the two-dimensional scanning galvanometer connecting line 41 and the near-infrared camera connecting line 42 respectively. The synchronization and control of the two are realized by the data acquisition card built into the computer 43; in addition, the first spatial light modulator 13, the second spatial light modulator 25 and the three-dimensional high-precision stage 37 are separately controlled and adjusted by the software installed in the computer 43.

[0067] Compared with the existing related technologies, this embodiment has the following advantages:

[0068] (1) This embodiment can use a detector and spatial light modulator with a wide response band, so that it can respond to fluorescence signals in the visible light band and the near-infrared band. Therefore, this embodiment can image fluorescence signals of different bands emitted by the same sample, and through multi-band imaging, more information inside the sample can be obtained;

[0069] (2) In this embodiment, a two-dimensional scanning galvanometer is combined with a spatial light modulator to achieve rapid scanning of the structured illumination light field on the sample surface, which can effectively reduce the adverse effect of the low frame rate of the spatial light modulator on the system's temporal resolution, and enable the system to have a faster imaging speed: Therefore, this embodiment has a higher temporal resolution and can be applied to in vivo, real-time, and rapid imaging of biological samples;

[0070] (3) This embodiment combines near-infrared second-zone imaging, upconversion luminescence and other technical means to effectively reduce tissue absorption and scattering of light and suppress the impact of background noise on image quality: Therefore, this embodiment can achieve a deeper imaging depth (not less than 1mm), which is helpful for exploring and studying more life phenomena;

[0071] (4) Compared with the common near-infrared two-zone imaging system based on wide-field imaging mode, this embodiment proposes an imaging method that combines near-infrared two-zone imaging technology with structured light illumination microscopy technology, which can give full play to the advantages of both imaging technologies; in particular, the introduction of structured light illumination technology can improve the resolution of the final image compared to the wide-field imaging method: therefore, this embodiment can achieve higher resolution imaging effects on samples at deeper imaging depths;

[0072] (5) The imaging system proposed in this embodiment can be combined with a variety of imaging technologies (Bessel beam, polarization microscopy, etc.) without complex modification and adjustment to carry out research in different technical fields, and has strong compatibility and flexibility.

[0073] In addition, compared with commercial near-infrared two-zone imaging systems on the market, this embodiment has a deeper imaging depth, weaker biological tissue damage, and higher temporal and spatial resolution, and is flexible in operation and has stable performance.

[0074] The aforementioned near-infrared two-zone imaging system and imaging method achieve deep, high-resolution, and high-quality imaging, enabling better research in fields such as life sciences. These embodiments combine the advantages of near-infrared two-zone imaging technology, upconversion luminescence, and structured illumination microscopy, effectively achieving deep, high-spatial-resolution imaging of samples such as biological tissue.

[0075] It should be noted that the steps shown in the flowcharts of the accompanying drawings can be executed in a computer system such as a set of computer-executable instructions, and that, although a logical order is shown in the flowcharts, in some cases, the steps shown or described can be executed in an order different from that shown here.

[0076] In this embodiment, an electronic device is provided, including a memory and a processor. The memory stores a computer program, and the processor is configured to run the computer program to execute the method in the above embodiment.

[0077] The above program can be executed in a processor or stored in a memory (or computer-readable medium), which includes permanent and non-permanent, removable and non-removable media that can implement information storage by any method or technology. Information can be computer-readable instructions, data structures, program modules, or other data. Examples of computer storage media include, but are not limited to, phase change memory (PRAM), static random access memory (SRAM), dynamic random access memory (DRAM), other types of random access memory (RAM), read-only memory (ROM), electrically erasable programmable read-only memory (EEPROM), flash memory or other memory technology, compact disc read-only memory (CD-ROM), digital versatile disc (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other non-transmission media that can be used to store information that can be accessed by a computing device. As defined herein, computer-readable media does not include transitory computer-readable media such as modulated data signals and carrier waves.

[0078] These computer programs can also be loaded onto a computer or other programmable data processing device so that a series of operational steps are executed on the computer or other programmable device to produce a computer-implemented process, thereby providing instructions for executing on the computer or other programmable device to implement the process. Figure 1 a process or multiple processes and / or boxes Figure 1 The steps of the functions specified in one or more blocks can be implemented by different modules corresponding to different steps. The electronic device can also include a device or system composed of a software module, and the modules in the device or system correspond to the steps in the above embodiments.

[0079] The above are merely embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. An up-conversion and down-conversion dual-mode structured light illumination microscopy imaging system, characterized in that: include: A first structured light generating module, configured to generate a first structured light, wherein the first structured light is used for exciting down-conversion luminescence; A second structured light generating module, configured to generate a second structured light, wherein the second structured light is used to excite up-conversion luminescence; A two-dimensional scanning module, used to scan structured light in the focal plane of the microscope objective lens; Imaging and detection module, detecting the fluorescent signal excited by the sample to be imaged; The first structured light generating module and the second structured light generating module both include: A spatial light modulator or a microlens array, wherein the spatial light modulator is used to perform phase modulation on light, or the microlens array is used to generate a dot matrix illumination light field; Laser, used to emit excitation light of a specific wavelength; a polarization beam splitter, configured to perform polarization beam splitting on the excitation light; The spatial light modulator is used to perform phase modulation on the light passing through the polarization beam splitter; a beam expander, configured to expand the excitation light before the excitation light enters the polarization beam splitter; A half-wave plate, used for adjusting the polarization direction of the incident light, disposed between the beam expander and the spatial light modulator, or between the beam expander and the microlens array; A spatial filter and two plano-convex lenses, wherein the light emitted from the spatial light modulator or the microlens array passes through one of the two plano-convex lenses, the spatial filter and the other plano-convex lens of the two plano-convex lenses in sequence before being incident on the two-dimensional scanning module.

2. The system according to claim 1, wherein: The wavelength of the fluorescence signal excited by the sample to be imaged is within the second near-infrared band.

3. The system according to claim 1, wherein: The two-dimensional scanning module includes a two-dimensional scanning galvanometer or an acousto-optic deflector.

4. The system according to claim 1, wherein the first structured light generating module and / or the second structured light generating module comprises a plurality of lasers, and the excitation light emitted by the plurality of lasers is emitted along the same optical path through the corresponding dichroic mirrors and / or reflective mirrors, wherein: The wavelength of the excitation light emitted by each of the plurality of lasers is different.

5. The system according to any one of claims 1 to 4, characterized in that The imaging and detection module includes: A three-dimensional stage, used for placing the sample to be imaged and performing adjustments in three dimensions; A microscope objective lens focuses the excitation light on the sample to be imaged to stimulate the sample to emit a fluorescent signal outward; A near-infrared camera is used to detect the fluorescent signal excited by the sample to be imaged.

6. The system according to claim 5, characterized in that The imaging and detection module includes at least one of the following: two plano-convex lenses, directing the output light from the two-dimensional scanning module to the dichroic mirror; The dichroic mirror is used to control the direction of the excitation light and filter out the residual excitation light in the fluorescence signal; The filter is used to filter out stray light in the fluorescent signal.

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