A high-channel fluorescence radiation differential microscopy imaging method and device

Abstract

The present invention discloses a high-channel fluorescence radiation differential microscopy imaging method and device, comprising a laser, a beam splitter, a fiber mode selection module, a light field adjustment module, a microscopy module, a detection module, and a control unit. The fiber mode selection module is used to time-share select N hollow excitation light spots and N solid excitation light spots that overlap in spatial position. The N excitation light spots, the light field adjustment module, the microscopy module, and the N detection optical fibers included in the detection module correspond one-to-one to form N independent FED microscopy imaging channels. This method can acquire structural information of a sample to be tested in parallel, thereby improving the imaging speed of the FED.

Description

Technical Field

[0001] The present invention relates to super-resolution imaging technology, and in particular to a high-channel Fluorescence Emission Difference (FED) microscopic imaging method and device. Background Art

[0002] Optical microscopes play a crucial role in fields such as life sciences and materials science, enabling intuitive observation of the microscopic structure of samples. However, limited by the optical diffraction limit, the spatial resolution of optical microscopes is generally greater than 1 / 2. To this end, super-resolution techniques such as structured illumination microscopy, stochastic optical reconstruction microscopy, and stimulated emission depletion microscopy have emerged in recent decades. Among them, fluorescence emission difference (FED) microscopy uses digital processing of two images acquired by solid and hollow spot excitation to obtain super-resolution microscopic images. Furthermore, this technique is highly universal and has lower requirements for fluorescent dyes, fluorescent labels, and laser power compared to other super-resolution techniques.

[0003] In the early days, FED microscopy technology first used a Gaussian solid excitation spot to acquire a fluorescence confocal microscopic image of one sample, then used a donut-shaped hollow spot to acquire a fluorescence confocal microscopic image of another sample. Finally, a super-resolution image was reconstructed through digital processing. Because the sample was imaged twice in a time-sharing point scanning mode, the imaging speed was relatively slow, and the quality of FED microscopy was easily affected by factors such as vibration and temperature in the imaging environment.

[0004] In order to improve the imaging speed, an array detector is used to receive the fluorescence signal of the sample. Through signal processing, the signal received in the central area of ​​the array detector corresponds to the excitation signal of the virtual solid spot, while the signal received in the edge area corresponds to the fluorescence signal excited by the virtual hollow spot. This can quickly obtain FED super-resolution images.

[0005] By staggering the incident angles of the solid spot excitation beam and the hollow spot excitation beam, a pair of solid excitation spots and hollow excitation spots with a certain deviation in spatial position can be simultaneously formed in the sample. Two independent detectors are used to receive the fluorescence signals excited by the solid excitation spot and the hollow excitation spot respectively, which can increase the speed of single-channel FED microscopy imaging by 2 times.

[0006] By selectively controlling the polarization state of the incident excitation light using a spatial light modulator (SLM), and using the s-component and p-component of the same excitation light beam to be incident on different spatial regions of the SLM for different modulations, a pair of solid and hollow excitation spots with a certain deviation in spatial position can be simultaneously obtained within the sample, thus achieving single-channel rapid FED microscopic imaging with a more stable optical system and more convenient adjustment.

[0007] However, all of the above methods belong to single-channel FED microscopy, and the improvement of imaging speed is still limited. Summary of the Invention

[0008] In order to overcome the above-mentioned defects of the prior art, the purpose of the present invention is to provide a high-channel fluorescence radiation differential microscopy method and device to form multi-channel parallel FED microscopy and improve the FED microscopy speed.

[0009] To achieve the above-mentioned purpose of the invention, an embodiment provides a high-channel fluorescence radiation differential microscopy imaging device, comprising:

[0010] A laser (1) for providing an excitation light beam (8) having at least one central wavelength;

[0011] A beam splitter (2) is used to split the excitation light beam (8) into excitation sub-beams (9) and (10) and introduce them into two excitation light transmission channels of solid spot excitation and hollow spot excitation of the optical fiber mode selection module (3);

[0012] The optical fiber mode selection module (3) is used for time-sharing selection of two excitation light transmission channels, and the optical fiber array outputs multiple independent solid spot excitation light beams and hollow spot excitation light beams;

[0013] A light field adjustment module (4) is used to adjust the light field of the multi-path light beams output by the optical fiber mode selection module (3) to form a linear excitation light spot array; and is also used to image the multi-path fluorescence signals obtained from the microscopic imaging module (5) to the detection module (6);

[0014] A microscopic imaging module (5) is used to image the linear excitation spot array onto the sample to be tested and collect the fluorescence signal generated by the sample after excitation;

[0015] A detection module (6) is used to receive multiple fluorescence signals in parallel using a detector array and convert them into electrical signals;

[0016] A control unit (7) is used to control the operation of the optical fiber mode selection module (3) and the microscopic imaging module (5), and is also used to collect electrical signals from the detection module (6) and perform super-resolution imaging;

[0017] The optical fiber mode selection module (3) outputs N hollow excitation beams or N solid excitation beams in a time-sharing manner. The N excitation beams, the light field adjustment module (4), the microscopic imaging module (5), and the N detection optical fibers included in the detection module (6) correspond to each other to form N independent FED microscopic imaging channels, so as to obtain the structural information of the sample to be tested in parallel.

[0018] In an optional embodiment, the beam splitter (2) selects energy beam splitting or polarization beam splitting according to the polarization characteristics of the excitation light beam. When energy beam splitting is selected, the beam splitter (2) adopts an energy beam splitter, including a beam splitter prism or a beam splitter. When polarization beam splitting is selected, the beam splitter (2) adopts a polarization beam splitter, including a polarization beam splitter prism. When the laser (1) is a pigtail output, the beam splitter (2) adopts a 1*2 optical fiber beam splitter.

[0019] In an optional embodiment, the fiber mode selection module (3) includes fiber mode selection units (11) and (12) corresponding to the two excitation light transmission channels of solid spot excitation and hollow spot excitation, respectively. The fiber mode selection units (11) and (12) each include an acousto-optic modulator (AOM) and a fiber mode selector. The two AOMs are used as optical switches to control the gating timing of solid spot excitation and hollow spot excitation, respectively, so as to achieve high-speed switching of the two excitation light spot modes. The two fiber mode selectors are used to select N solid spot output fibers and N hollow spot output fibers, respectively, and respectively control the time-sharing output of N independent solid spot excitation light beams and N independent hollow spot excitation light beams through the AOM.

[0020] The optical fiber mode selection unit (11) includes an optical fiber mode selector that selects a 1*N single-mode optical fiber splitter, a 1*2 single-mode optical fiber coupler combination with N output ends, a 1*4 single-mode optical fiber coupler combination with N output ends, or a 1*2 and 1*4 single-mode optical fiber coupler combination with N output ends;

[0021] The fiber mode selection unit (12) includes a fiber mode selector that selects a 1*N single-mode fiber splitter and N fiber mode selection couplers, a 1*2 single-mode fiber coupler combination at N output ends and N fiber mode selection couplers, a 1*4 single-mode fiber coupler combination at N output ends and N fiber mode selection couplers, or a 1*2 and 1*4 single-mode fiber coupler combination at N output ends and N fiber mode selection couplers, wherein the fiber mode selection coupler is composed of a single-mode fiber and a few-mode fiber.

[0022] In an optional embodiment, at the end face (21) of the first optical fiber array included in the optical fiber mode selection module (3), an optical fiber clamp is used to fix the end faces of the N solid spot output optical fibers and the N hollow spot output optical fibers of the optical fiber mode selection module (3), so that each solid spot output optical fiber and each hollow spot output optical fiber form a group, corresponding to a time-sharing controllable excitation light point source of an FED microscopic imaging channel.

[0023] In an optional embodiment, the light field adjustment module (4) includes a light beam collimating unit (22), a first converging lens (23), a first lens (25), a dichroic mirror (26), a second lens (27), and a second converging lens (30);

[0024] The beam collimation unit (22) controls the N solid spot excitation beams and N hollow spot excitation beams outputted from the first optical fiber array end face (21) of the optical fiber mode selection module (3) to be collimated and outputted in parallel with each other;

[0025] The first converging lens (23) controls the time-sharing gated N solid spot excitation light beams or N hollow spot excitation light beams to form a first linear excitation light spot array (24) with overlapping spatial positions, wherein the first linear excitation light spot array (24) is composed of the gated N solid excitation light spots or the gated N hollow excitation light spots;

[0026] The first lens (25) and the second lens (27) control the first linear excitation light spot array (24) to be imaged into a second linear excitation light spot array (28), and the second linear excitation light spot array (28) is imaged again through the microscopic imaging module (5) to achieve linear excitation illumination;

[0027] The dichroic mirror (26) is provided between the first lens (25) and the second lens (27) and is used to separate the excitation light beam and the fluorescence signal;

[0028] The second lens (27), the dichroic mirror (26) and the second converging lens (30) sequentially branch and transmit the multi-path fluorescence signals obtained by the microscopic imaging module (5), including collimating the N-path independent fluorescence signals output by the microscopic imaging module (5) through the second lens (27), separating the excitation beam and the fluorescence signal beam through the dichroic mirror (26), and converging the N-path fluorescence signal beams through the second converging lens (30) to enter the detection module (6).

[0029] In an optional embodiment, the microscopic imaging module (5) comprises at least a scanning unit and a microscope objective lens, wherein the microscope objective lens is used to image the second linear excitation light spot array (28) into the sample to be tested to form a third linear excitation light spot array (29), and to receive the fluorescence signal generated by the excitation light field; the scanning unit is connected to the control unit (7) via a data line to realize external communication control, and is used to guide the third linear excitation light spot array (29) to scan and move in two dimensions or three dimensions on the sample according to the scanning command of the control unit (7).

[0030] In an optional embodiment, the beam collimating unit (22) is a microlens array; and the first converging lens (23) is a cylindrical lens.

[0031] In an optional embodiment, the detection module (6) includes a second optical fiber array end face (31), a detection optical fiber array, and a detector array, and the multi-channel fluorescence signals received by the second optical fiber array end face (31) are respectively introduced into the detector array through the detection optical fiber array to be converted into multi-channel electrical signals.

[0032] In an optional embodiment, when the control unit (7) controls the optical fiber mode selection module (3) to work, it sends an instruction to the optical fiber mode selection module (3) to select the excitation light transmission channels for solid spot excitation and hollow spot excitation, thereby controlling the imaging sequence corresponding to the solid spot excitation and the hollow spot excitation;

[0033] When the control unit (7) controls the microscopic imaging module (5) to work, it sends a scanning instruction to the scanning unit of the microscopic imaging module (5) to control the third linear excitation light spot array (29) to perform a two-dimensional / three-dimensional scan on the sample;

[0034] When the control unit (7) collects the electrical signal of the detection module (6) and performs super-resolution imaging, after obtaining the solid spot excitation sample image and the hollow spot excitation sample image obtained based on the electrical signal according to the scanning sequence, the FED technology is used to reconstruct and display the super-resolution microscopic image of the sample.

[0035] To achieve the above-mentioned object of the invention, an embodiment further provides a high-channel fluorescence radiation differential microscopy imaging method, which uses the above-mentioned device and includes the following steps:

[0036] providing an excitation light beam (8) having at least one central wavelength by a laser (1);

[0037] The excitation light beam (8) is split into excitation sub-beams (9) and (10) by a beam splitter (2) and introduced into two excitation light transmission channels of solid spot excitation and hollow spot excitation of the optical fiber mode selection module (3);

[0038] The two excitation light transmission channels are time-sharedly selected by the optical fiber mode selection module (3), and the optical fiber array outputs multiple independent solid spot excitation light beams and hollow spot excitation light beams;

[0039] The optical field adjustment module (4) is used to adjust the optical field of the multi-path light beams outputted by the optical fiber mode selection module (3) to form a linear excitation light spot array; and is also used to image the multi-path fluorescence signals obtained from the microscopic imaging module (5) to the detection module (6);

[0040] Imaging the linear excitation spot array onto the sample to be tested through the microscopic imaging module (5), and collecting the fluorescence signal generated by the sample after excitation;

[0041] The detector array included in the detection module (6) receives multiple fluorescence signals in parallel and converts them into electrical signals;

[0042] The control unit (7) controls the operation of the optical fiber mode selection module (3) and the microscopic imaging module (5), collects the electrical signal of the detection module (6), and performs super-resolution imaging.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1) The fiber mode selection module is used to excite multiple solid and hollow light spots in a time-sharing manner. Combined with the parallel reception of the array detector of the detection module, a multi-channel FED microscopic imaging channel is formed, which can improve the imaging speed of FED.

[0045] 2) Using AOM to select the excitation channel in a high-speed time-sharing manner and optical fiber to select the excitation light mode (solid spot and hollow spot) can make the optical path structure of the device more stable and compact, and reduce the influence of the external environment on FED microscopic imaging.

[0046] 3) The field of view of FED microscopy can be improved by using a fiber clamp and a light field adjustment module to overlap the spatial positions of each set of hollow and solid excitation spots. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0048] Figure 1 1 is a schematic structural diagram of a high-channel fluorescence radiation differential microscopy imaging device provided in an embodiment;

[0049] Figure 2 is a schematic structural diagram of the end face of the excitation fiber array provided in the embodiment;

[0050] Figure 3 is a schematic structural diagram of the end face of the detection optical fiber array provided in an embodiment;

[0051] Figure 4 It is a flow chart of the high-channel fluorescence radiation differential microscopy imaging method provided in the embodiment. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not limit the scope of protection of the present invention.

[0053] like Figure 1As shown, the high-channel FED microscopic imaging device provided in the embodiment includes a laser 1, a beam splitter 2, a fiber mode selection module 3, a light field adjustment module 4, a microscopic imaging module 5, a detection module 6, and a control unit 7. The imaging principle of this device is based on FED microscopic imaging technology. Two confocal fluorescence microscopic images are acquired through solid and hollow spot excitation, and super-resolution fluorescence information is obtained using differential technology. The device's structural arrangement uses the light mode selection module 3 to time-share N hollow excitation beams or N solid excitation beams. The N excitation beams, the light field adjustment module 4, the microscopic imaging module 5, and the N detection fibers included in the detection module 6 correspond to each other, forming N independent FED microscopic imaging channels. This allows for parallel acquisition of structural information of the sample under test, thereby improving FED imaging speed. The key is that the two AOMs contained in the fiber mode selection module 3 quickly and time-share the two excitation light transmission channels, enabling the stable output of N hollow excitation beams and N solid excitation beams. The light field adjustment module 4 ensures that the spatial positions of each set of selected hollow excitation light spots and solid excitation light spots are highly overlapped during transmission. The end face of the excitation fiber contained in the fiber mode selection module 3 is aligned with the end face of the detection fiber contained in the detection module 6 to form the N excitation light output ports and N signal light detection ports of the multi-channel FED imaging channel. Each component is explained in detail below:

[0054] In the embodiment, the laser 1 is used to provide an excitation light beam 8 with at least one central wavelength, that is, to provide a monochromatic or polychromatic excitation light beam, which can make the present invention better applicable to the different optical characteristics of the sample being tested based on the imaging principle, and realize high-throughput FED microscopic imaging of one or more fluorescent dye labels, so that the dynamic information of the sample being tested can be more comprehensively studied or observed. Preferably, the laser 1 can be a single wavelength laser, a wavelength tunable laser or a white light laser. When the output beam of the laser 1 is output to free space, a beam expander composed of a telescope system is required to collimate the output beam into a parallel beam.

[0055] In the embodiment, the beam splitter 2 is used to split the excitation light beam 8 into excitation sub-beams 9 and 10 and introduce them into the two excitation light transmission channels of the solid spot excitation and hollow spot excitation of the optical fiber mode selection module 3. Among them, for the collimated parallel excitation light beam output by the laser 1, the beam splitter 2 can select energy splitting or polarization splitting according to the polarization characteristics of the excitation light beam. When energy splitting is selected, the beam splitter 2 adopts an energy beam splitter, including a beam splitter prism or a beam splitter. When polarization splitting is selected, the beam splitter 2 adopts a polarization beam splitter, including a polarization beam splitter prism. For the laser 1 using a pigtail to output the excitation light beam, the beam splitter 2 adopts a 1*2 optical fiber beam splitter. Specifically, when the energy beam splitter is used, the energy beam splitter splits the excitation light beam 8 into the excitation sub-beams 9 and 10 according to the energy ratio of 1:1.

[0056] In this embodiment, the fiber mode selection module 3 is used to time-share select two excitation light transmission channels, allowing the fiber array to output multiple independent solid-spot excitation beams and hollow-spot excitation beams. The fiber mode selection module 3 includes fiber mode selection units 11 and 12, corresponding to the two excitation light transmission channels for solid and hollow spot excitation, respectively. Each fiber mode selection unit 11 and 12 includes an acousto-optic modulator (AOM) and a fiber mode selector. Both AOMs function as optical switches, controlling the operating time of the excitation beams entering the two excitation transmission channels. This control, in turn, controls the gating timing for solid and hollow spot excitation, enabling high-speed switching between the two excitation light spot modes. The two fiber mode selectors are used to select N solid-spot output fibers and N hollow-spot output fibers, respectively, and control the time-shared output of the N independent solid-spot excitation beams and N independent hollow-spot excitation beams, respectively, through the AOMs.

[0057] Among them, the fiber mode selector included in the fiber mode selection unit 11 selects a 1*N single-mode fiber splitter, a 1*2 single-mode fiber coupler combination with N output ends, a 1*4 single-mode fiber coupler combination with N output ends, or a 1*2 and 1*4 single-mode fiber coupler combination with N output ends to output N solid spot excitation beams.

[0058] The fiber mode selector included in the fiber mode selection unit 12 selects a 1*N single-mode fiber beam splitter and N fiber mode selective couplers, a 1*2 single-mode fiber coupler combination at N output ends and N fiber mode selective couplers, a 1*4 single-mode fiber coupler combination at N output ends and N fiber mode selective couplers, or a 1*2 and 1*4 single-mode fiber coupler combination at N output ends and N fiber mode selective couplers to output N hollow-core spot excitation beams. The fiber mode selective coupler is composed of a single-mode fiber and a few-mode fiber. The single-mode fiber directly controls the output of a single solid-spot excitation beam, while a single fiber mode selective coupler can control the output of a single hollow-spot excitation beam.

[0059] A fiber coupler is used to split the laser beam into multiple excitation beams of equal intensity based on energy ratio. A 1x2 fiber beam splitter outputs two excitation light transmission channels. A fiber input / output AOM is preferred to control the gating timing for solid-spot excitation and hollow-spot excitation. For the solid-spot excitation mode, a 1xN fiber coupler composed of single-mode fiber is preferred to output N independent solid-spot excitation beams. For the hollow-spot excitation mode, a fiber mode selective coupler composed of single-mode fiber and few-mode fiber is preferred, with N few-mode fibers outputting independent hollow-spot excitation beams.

[0060] In order to simplify the structure of the system optical path and improve the stability of the system, the AOM preferably adopts the optical fiber input and output type. Figure 2As shown, in the system, a fiber clamp is used to secure the end faces of the N solid-spot output fibers and N hollow-spot output fibers of the fiber mode selection module 3. Each solid-spot output fiber and each hollow-spot output fiber form a group, corresponding to a time-controlled excitation light point source for a FED microscopic imaging channel. In this embodiment, a fiber clamp with a low thermal expansion coefficient is preferred.

[0061] Specifically, give examples, such as Figure 1 and Figure 2 As shown, the fiber mode selection unit 11 outputs four independent solid-spot excitation beams 13, 14, 15, and 16, while the fiber mode selection unit 12 outputs four independent donut-shaped hollow-spot excitation beams 17, 18, 19, and 20. A fiber clamp is used to secure the fiber bundles transmitting the four solid-spot excitation beams 13, 14, 15, and 16 and the hollow-spot excitation beams 17, 18, 19, and 20 to the end face 21 of the first fiber array. This allows each pair of fibers outputting the hollow-spot and solid-spot excitation beams to form a group, for example, fibers 13 and 17 form a group. Each group corresponds to the excitation light source of a single FED microscopic imaging channel.

[0062] In the embodiment, the light field adjustment module 4 is used to collimate and adjust the light field of the multi-path light beams output by the fiber mode selection module 3 to form a linearly distributed N parallel excitation spot array, and is also used to image the multi-path fluorescence signals obtained from the microscopic imaging module 5 to the detection module 6. Figure 1 As shown, the light field adjustment module 4 includes a beam collimating unit 22 , a first converging lens 23 , a first lens 25 , a dichroic mirror 26 , a second lens 27 , and a second converging lens 30 .

[0063] The beam collimation unit 22 controls the output of the N solid spot excitation beams and the N hollow spot excitation beams from the first optical fiber array end face 21 to be collimated and output in parallel. The first converging lens 23 controls the time-sharing selection of the N solid spot excitation beams or the N hollow spot excitation beams to form a first linear excitation spot array 24 with overlapping spatial positions. The first linear excitation spot array 24 is composed of N selected solid excitation spots or N selected hollow excitation spots. The first lens 25 and the second lens 27 provide appropriate magnification to control the imaging of the first linear excitation spot array 24 into a second linear excitation spot array 28, so that a third linear excitation spot array 29 with minimal interference between adjacent excitation spots can be obtained through the microscopic imaging module 5.

[0064] The dichroic mirror 26 is positioned between the first lens 25 and the second lens 27 to separate the excitation beam from the fluorescence signal. The second lens 27, the dichroic mirror 26, and the second converging lens 30 sequentially split and transmit the multiple fluorescence signals obtained by the microscopic imaging module 5. Specifically, the second lens 27 collimates the N independent fluorescence signals output by the microscopic imaging module 5, the dichroic mirror 26 separates the excitation beam from the fluorescence signal beam, and the second converging lens 30 converges the N fluorescence signal beams into the second fiber array end face 31 of the detection module 6.

[0065] Based on the numerical aperture angle, mode field radius, fiber spacing, and operating wavelength of the hollow excitation light spots and the solid excitation light spots, the beam collimating unit 22 is preferably a microlens array, which can collimate each group of hollow spot excitation light beams and solid spot excitation light beams, and make the output beams of the two light spot modes parallel to each other; the first converging lens 23 is preferably a cylindrical lens, which can converge the above-mentioned each group of collimated hollow spot excitation light beams and solid spot excitation light beams at the same spatial position in the focal plane to form a first linear excitation light spot array 24. Based on the entrance pupil and field of view size of the microscopic imaging system, the first lens 25 and the second lens 27 constitute an imaging system, which images the first linear excitation light spot array 24 into a second linear excitation light spot array 28 of appropriate size and spacing; the second lens 27 and the second converging lens 30 constitute an imaging system, which images the fluorescence signal output by the microscopic imaging module 5 onto the second fiber array end face 31 of the detection module 6.

[0066] In the embodiment, the microscopic imaging module 5 is used to image the second linear excitation spot array 28 onto the sample under test, forming a third linear excitation spot array 29 in which the spacing between adjacent spots is not less than the size of an Airy disk. The third linear excitation spot array 29 generates a multi-point parallel excitation area to excite the sample under test. The sample under test generates a fluorescence signal under excitation, and the fluorescence signal is received and input into the light field adjustment module 4 in an imaging manner.

[0067] The microscopic imaging module 5 comprises at least a scanning unit and a microscope objective lens. The microscope objective lens is used to image the second linear excitation spot array 28 into the sample being measured, forming a third linear excitation spot array 29 with minimal interference between adjacent excitation spots, and to receive the fluorescence signals generated by the excitation light field. The scanning unit is connected to the control unit 7 via a data line for external communication control, and is used to guide the third linear excitation spot array 29 in a two-dimensional or three-dimensional scanning motion across the sample according to scanning commands from the control unit 7.

[0068] In terms of imaging principles, the illumination area of ​​the linear excitation spot array and the input end faces of the linearly arranged detection fiber array are conjugated to each other. Therefore, each excitation spot and its corresponding detection fiber form a confocal imaging mode, i.e., a single FED microscopy channel. By time-sharing N-channel solid spot excitation or N-channel hollow spot excitation, combined with scanning of the linear excitation spot array, sample microscopic images corresponding to solid spot excitation and hollow spot excitation can be rapidly acquired, rapidly reconstructing a super-resolution image of the sample, i.e., multi-channel FED microscopy.

[0069] In the embodiment, the detection module 6 is used to use a detector array to receive multiple fluorescence signals in parallel and convert them into electrical signals. Figure 1 As shown, the detection module 6 includes a second fiber array end face 31, a detection fiber array consisting of detection fibers 32, 33, 34, and 35, and a detector array consisting of detectors 36, 37, 38, and 39. The second fiber array end face 31 receives multiple fluorescence signals and divides them into multiple independent detection channels. The received multiple fluorescence signals are respectively introduced into the detector array through the detection fiber array to be converted into multiple electrical signals. Figure 3 As shown, the detection optical fiber array is arranged linearly.

[0070] Like the first fiber array end face 21, the second fiber array end face 31 is also secured with a fiber clamp. The structure of the fiber clamp is designed based on the numerical aperture, outer diameter, and mode spot radius of the excitation and receiving optical fibers, as well as the imaging resolution and range determined by the light field adjustment module 4 and the microscopic imaging module 5. This ensures that the solid excitation light spot and the hollow excitation light spot within each FED microscopic imaging channel overlap, and that the spacing between the excitation light spots corresponding to adjacent FED microscopic imaging channels within the sample is at least greater than one Airy disk diameter. This allows the fluorescence signal from each FED microscopic imaging channel to be efficiently coupled into the detection fiber array via the microscopic imaging module 5 and the light field adjustment module 4.

[0071] To improve the signal-to-noise ratio, detection fibers 32, 33, 34, and 35 are preferably multimode fibers. When multiple independent detectors are used to receive signals, each detection fiber is directly connected to the end-face fiber interface of the corresponding detector. Detectors 36, 37, 38, and 39 can be selected to have N independent detectors or be area array detectors containing N detection units. When using an area array detector containing N detection units, based on the area of ​​the detection units of the area array detection, the mode radius of the detection fiber, and the numerical aperture, the detection module 6 must at least be equipped with a telecentric optical imaging system. The telecentric optical imaging system controls each signal output by the detection fibers 32, 33, 34, and 35 so that it can be received by the area array detectors 36, 37, 38, and 39 with an appropriate signal-to-noise ratio. The N independent detectors are preferably PMTs or APDs whose N receiver end faces can be directly connected to optical fibers. PMT or APD array detectors have single-photon detection capabilities. Combined with photon counting, fluorescence lifetime imaging of samples can be performed. This can further expand the system's functionality and perform fluorescence lifetime imaging of the sample under FED mode. The area array detector comprising N detection units is preferably an APD array or a multi-pixel photon counter (MPPC). The detection module 6 is connected to the control unit 7 via a data line to realize data acquisition and transmission functions.

[0072] In this embodiment, the control unit 7 includes a computer, a data acquisition card, a scanning control card, a scanner driver circuit, and an AOM driver circuit. It is used to control the operation of the fiber mode selection module 3 and the microscopic imaging module 5, and is also used to collect electrical signals from the detection module 6 and perform super-resolution imaging. Specifically, when controlling the operation of the fiber mode selection module 3, the control unit 7 sends an excitation spot mode switching instruction to the AOM of the fiber mode selection module 3 to select the excitation light transmission channels for solid spot excitation and hollow spot excitation, and controls the imaging sequence corresponding to solid spot excitation and hollow spot excitation. When controlling the operation of the microscopic imaging module 5, the control unit 7 sends a scanning instruction to the scanning unit of the microscopic imaging module 5, controlling the third linear excitation spot array 29 to perform a two-dimensional / three-dimensional scan of the sample. When collecting electrical signals from the detection module 6 and performing super-resolution imaging, the control unit 7 obtains sample microscopic images corresponding to solid spot excitation and hollow spot excitation based on the electrical signals according to the scanning sequence, and then reconstructs and displays the super-resolution microscopic image of the sample using FED technology.

[0073] Using the above-mentioned imaging device, a high-throughput FED microscopic imaging method uses a laser 1 to provide at least one monochromatic illumination beam, which is guided to two transmission channels of an optical fiber mode selection module 3 through a beam splitter 2; the two AOMs of the optical fiber mode selection module 3 time-share the solid spot excitation and hollow spot excitation channels, and output N solid excitation beams and N hollow excitation beams respectively through a combination of an optical fiber coupler and an optical fiber mode selection coupler; the 2N excitation optical fiber end faces of the optical fiber mode selection module 3 are fixed with an optical fiber clamp so that each solid excitation spot output end and each corresponding hollow excitation spot output end form a group; the optical field adjustment module 4 collimates the N solid excitation beams or N hollow excitation beams selected in time-shared manner so that the spatial positions of each group of solid spots and hollow spots overlap to form a linear excitation spot array; The microscopic imaging module 5 images the linear excitation light spot array into the sample and receives the fluorescence signal of the excited area inside the sample; the light field adjustment module 4 collimates, splits and focuses the independent fluorescence signals output by the microscopic imaging module 5 to the input end face of the detection fiber array; N detection optical fibers are directly connected to the separated N detector input end faces, or a telecentric imaging system is used to image the output end face of the detection fiber to the receiving surface of the array detector; the scanning unit of the microscopic imaging module 5 is used to make the third linear excitation light spot array 29 perform two-dimensional or three-dimensional scanning movement in the sample, and multi-channel parallel acquisition of sample image information; the control unit 7 sends a time selection instruction to the AOM and a scanning command to the scanning unit to collect the electrical signal output by the detection module, and reconstruct and display the super-resolution image of the sample according to the FED technology.

[0074] The embodiment also provides a high-channel fluorescence radiation differential microscopy imaging method, which uses Figure 1 The device shown, such as Figure 4 As shown, the following steps are included:

[0075] , providing an excitation beam 8 of at least one central wavelength through a laser 1;

[0076] The excitation light beam 8 is split into the excitation sub-beams 9 and 10 by the beam splitter 2 and introduced into the two excitation light transmission channels of the solid spot excitation and hollow spot excitation of the optical fiber mode selection module 3;

[0077] The two excitation light transmission channels are time-sharedly selected by the optical fiber mode selection module 3, and the optical fiber array outputs multiple independent solid spot excitation beams and hollow spot excitation beams;

[0078] The optical field adjustment module 4 adjusts the optical field of the multi-path light beams output by the optical fiber mode selection module 3 to form a linear excitation spot array; it is also used to image the multi-path fluorescence signals obtained from the microscopic imaging module 5 to the detection module 6;

[0079] The microscopic imaging module 5 images the linear excitation spot array onto the sample to be tested, and collects the fluorescence signal generated by the sample after excitation;

[0080] The detector array included in the detection module 6 receives multiple fluorescence signals in parallel and converts them into electrical signals;

[0081] The control unit 7 controls the operation of the optical fiber mode selection module 3 and the microscopic imaging module 5 to collect the electrical signals of the detection module 6 and perform super-resolution imaging.

[0082] The devices and methods provided in the above embodiments utilize time-sharing gating of solid and hollow excitation spot arrays for parallel excitation, combined with synchronous and parallel reception by a detector array, to achieve high-channel-count FED microscopy imaging. An AOM is used to time-sharingly gate the solid and hollow excitation beam channels, generating solid and hollow excitation beams via fiber mode selection. A fiber clamp is used to secure the spatial positions of the excitation and detection fiber arrays, and a detector array is used to receive the fluorescence signal array generated by the excitation array. This enables time-sharing excitation of the solid and hollow excitation spot arrays and parallel detection of the array fluorescence signals, forming multiple FED microscopy channels. The device utilizes fiber arrays for transmission at both the excitation and detection ends, stabilizing the optical path structure while allowing the solid and hollow excitation spots in each FED microscopy channel to co-path, thereby improving imaging accuracy and speed and facilitating the observation or measurement of dynamic cellular processes.

[0083] The specific implementation methods described above provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above is only the most preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, supplements and equivalent substitutions made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A high-channel fluorescence radiation differential microscopy imaging device, characterized in that: include: A laser (1) for providing an excitation light beam (8) having at least one central wavelength; A beam splitter (2) is used to split the excitation light beam (8) into excitation sub-beams (9) and (10) and introduce them into two excitation light transmission channels of solid spot excitation and hollow spot excitation of the optical fiber mode selection module (3); The optical fiber mode selection module (3) is used for time-sharing selection of two excitation light transmission channels, and the optical fiber array outputs multiple independent solid spot excitation light beams and hollow spot excitation light beams; A light field adjustment module (4) is used to adjust the light field of the multi-path light beams output by the optical fiber mode selection module (3) to form a linear excitation light spot array; and is also used to image the multi-path fluorescence signals obtained from the microscopic imaging module (5) to the detection module (6); A microscopic imaging module (5) is used to image the linear excitation spot array onto the sample to be tested and collect the fluorescence signal generated by the sample after excitation; A detection module (6) is used to receive multiple fluorescence signals in parallel using a detector array and convert them into electrical signals; A control unit (7) is used to control the operation of the optical fiber mode selection module (3) and the microscopic imaging module (5), and is also used to collect electrical signals from the detection module (6) and perform super-resolution imaging; N hollow excitation beams or N solid excitation beams are output in a time-sharing manner through the optical fiber mode selection module (3). The N excitation beams, the light field adjustment module (4), the microscopic imaging module (5) and the N detection optical fibers included in the detection module (6) correspond to each other to form N independent fluorescence radiation differential microscopic imaging channels, so as to obtain the structural information of the sample to be tested in parallel.

2. The high-channel fluorescence radiation differential microscopy imaging device according to claim 1, characterized in that: The beam splitter (2) selects energy beam splitting or polarization beam splitting according to the polarization characteristics of the excitation light beam. When energy beam splitting is selected, the beam splitter (2) adopts an energy beam splitter, including a beam splitter prism or a beam splitter. When polarization beam splitting is selected, the beam splitter (2) adopts a polarization beam splitter, including a polarization beam splitter prism. When the laser (1) is a pigtail output, the beam splitter (2) adopts a 1*2 optical fiber beam splitter.

3. The high-channel fluorescence radiation differential microscopy imaging device according to claim 1, characterized in that: The fiber mode selection module (3) includes fiber mode selection units (11) and (12) corresponding to the two excitation light transmission channels of solid spot excitation and hollow spot excitation, respectively. The fiber mode selection units (11) and (12) each include an acousto-optic modulator (AOM) and a fiber mode selector. The two AOMs are used as optical switches to control the gating timing of solid spot excitation and hollow spot excitation, respectively, so as to achieve high-speed switching of the two excitation light spot modes. The two fiber mode selectors are used to select N solid spot output optical fibers and N hollow spot output optical fibers, respectively, and respectively control the time-sharing output of N independent solid spot excitation light beams and N independent hollow spot excitation light beams through the AOM. The optical fiber mode selection unit (11) includes an optical fiber mode selector that selects a 1*N single-mode optical fiber splitter, a 1*2 single-mode optical fiber coupler combination with N output ends, a 1*4 single-mode optical fiber coupler combination with N output ends, or a 1*2 and 1*4 single-mode optical fiber coupler combination with N output ends; The fiber mode selection unit (12) includes a fiber mode selector that selects a 1*N single-mode fiber splitter and N fiber mode selection couplers, a 1*2 single-mode fiber coupler combination at N output ends and N fiber mode selection couplers, a 1*4 single-mode fiber coupler combination at N output ends and N fiber mode selection couplers, or a 1*2 and 1*4 single-mode fiber coupler combination at N output ends and N fiber mode selection couplers, wherein the fiber mode selection coupler is composed of a single-mode fiber and a few-mode fiber.

4. The high-channel fluorescence radiation differential microscopy imaging device according to claim 3, characterized in that: At the end face (21) of the first optical fiber array included in the optical fiber mode selection module (3), an optical fiber clamp is used to fix the end faces of the N solid spot output optical fibers and the N hollow spot output optical fibers of the optical fiber mode selection module (3), so that each solid spot output optical fiber and each hollow spot output optical fiber form a group corresponding to a time-sharing controllable excitation light point source of a fluorescence radiation differential microscopy imaging channel.

5. The high-channel fluorescence radiation differential microscopy imaging device according to claim 1, characterized in that: The light field adjustment module (4) comprises a light beam collimating unit (22), a first converging lens (23), a first lens (25), a dichroic mirror (26), a second lens (27), and a second converging lens (30); The beam collimation unit (22) controls the N solid spot excitation beams and N hollow spot excitation beams outputted from the first optical fiber array end face (21) of the optical fiber mode selection module (3) to be collimated and outputted in parallel with each other; The first converging lens (23) controls the time-sharing gated N solid spot excitation light beams or N hollow spot excitation light beams to form a first linear excitation light spot array (24) with overlapping spatial positions, wherein the first linear excitation light spot array (24) is composed of the gated N solid excitation light spots or the gated N hollow excitation light spots; The first lens (25) and the second lens (27) control the first linear excitation light spot array (24) to be imaged into a second linear excitation light spot array (28), and the second linear excitation light spot array (28) is imaged again through the microscopic imaging module (5) to achieve linear excitation illumination; The dichroic mirror (26) is provided between the first lens (25) and the second lens (27) and is used to separate the excitation light beam and the fluorescence signal; The second lens (27), the dichroic mirror (26) and the second converging lens (30) sequentially branch and transmit the multi-path fluorescence signals obtained by the microscopic imaging module (5), including collimating the N-path independent fluorescence signals output by the microscopic imaging module (5) through the second lens (27), separating the excitation beam and the fluorescence signal beam through the dichroic mirror (26), and converging the N-path fluorescence signal beams through the second converging lens (30) to enter the detection module (6).

6. The high-channel fluorescence radiation differential microscopy imaging device according to claim 1, characterized in that: The microscopic imaging module (5) comprises at least a scanning unit and a microscope objective lens, wherein the microscope objective lens is used to image the second linear excitation light spot array (28) into the sample to be tested to form a third linear excitation light spot array (29), and to receive the fluorescence signal generated by the excitation light field; the scanning unit is connected to the control unit (7) via a data line to realize external communication control, and is used to guide the third linear excitation light spot array (29) to scan and move in two dimensions or three dimensions on the sample according to the scanning command of the control unit (7).

7. The high-channel fluorescence radiation differential microscopy imaging device according to claim 5, characterized in that: The light beam collimating unit (22) is a microlens array; the first converging lens (23) is a cylindrical lens.

8. The high-channel fluorescence radiation differential microscopy imaging device according to claim 1, characterized in that: The detection module (6) comprises a second optical fiber array end face (31), a detection optical fiber array, and a detector array. The multi-channel fluorescence signals received by the second optical fiber array end face (31) are respectively introduced into the detector array through the detection optical fiber array to be converted into multi-channel electrical signals.

9. The high-channel fluorescence radiation differential microscopy imaging device according to claim 1, characterized in that: When the control unit (7) controls the optical fiber mode selection module (3) to work, it sends instructions to the optical fiber mode selection module (3) to select the excitation light transmission channels for solid spot excitation and hollow spot excitation, and controls the imaging sequence corresponding to the solid spot excitation and the hollow spot excitation; When the control unit (7) controls the microscopic imaging module (5) to work, it sends a scanning instruction to the scanning unit of the microscopic imaging module (5) to control the third linear excitation light spot array (29) to perform a two-dimensional / three-dimensional scan on the sample; When the control unit (7) collects the electrical signal of the detection module (6) and performs super-resolution imaging, after obtaining the solid spot excitation sample image and the hollow spot excitation sample image obtained based on the electrical signal according to the scanning sequence, the super-resolution microscopic image of the sample is reconstructed and displayed using the fluorescence radiation difference technology.

10. A high-channel fluorescence radiation differential microscopy imaging method, characterized in that: The method uses the device according to any one of claims 1 to 9, comprising the following steps: providing an excitation light beam (8) having at least one central wavelength by a laser (1); The excitation light beam (8) is split into excitation sub-beams (9) and (10) by a beam splitter (2) and introduced into two excitation light transmission channels of solid spot excitation and hollow spot excitation of the optical fiber mode selection module (3); The two excitation light transmission channels are time-sharedly selected by the optical fiber mode selection module (3), and the optical fiber array outputs multiple independent solid spot excitation light beams and hollow spot excitation light beams; The optical field adjustment module (4) is used to adjust the optical field of the multi-path light beams outputted by the optical fiber mode selection module (3) to form a linear excitation light spot array; and is also used to image the multi-path fluorescence signals obtained from the microscopic imaging module (5) to the detection module (6); Imaging the linear excitation spot array onto the sample to be tested through the microscopic imaging module (5), and collecting the fluorescence signal generated by the sample after excitation; The detector array included in the detection module (6) receives multiple fluorescence signals in parallel and converts them into electrical signals; The control unit (7) controls the operation of the optical fiber mode selection module (3) and the microscopic imaging module (5), collects the electrical signal of the detection module (6), and performs super-resolution imaging.

Images