Long-focal-depth Bessel beam volume microscopic imaging system

Abstract

The invention provides a long-focal-depth Bessel beam volume microscopic imaging system, and belongs to the technical field of optical microscopic imaging. The system comprises a non-diffraction light beam generation module, a scanning and focusing module, a sample control module, a fluorescence signal detection module and a microscopic imaging processing terminal. According to the invention, laser is used as an excitation light source, the non-diffraction beam generation module generates a long-focal-depth Bessel beam with a non-diffraction distance n times that of a traditional Bessel beam, the transmission distance is controllable, the limitation of an effective numerical aperture can be broken through, and under the condition of a certain transverse resolution, an ultra-large imaging depth of field is provided. Volume imaging is realized by using the characteristic of long focal depth and no diffraction, more axial information stacks are obtained, and the imaging speed and the imaging resolution are relatively high.

Description

Technical Field

[0001] The present invention relates to the field of optical microscopy imaging technology, and in particular to a long-depth-of-field Bessel beam volume microscopy imaging system. Background Art

[0002] Most biological samples are three-dimensional structures. In order to obtain complete three-dimensional structure information, a series of images at different depths usually need to be acquired to image the entire volume. Although point-by-point scanning ensures a unified resolution of the entire sample, it will increase the image acquisition time and thus reduce the frame rate. In free space, the self-healing and non-diffracting characteristics of Bessel beams can provide advantages that Gaussian beams cannot match (see the literature "Extended depth of field microscopy for rapid volumetric two-photon imaging", Optics Express, 2013, 21(8): 10095-10104). First, due to the non-diffracting characteristic, Bessel beams have a larger non-diffracting propagation distance than Gaussian beams; second, due to the beam self-healing property, Bessel beams can achieve a larger imaging depth of field in biological samples (see the literature "Video-rate volumetric functional imaging of the brain at synaptic resolution", Nature Neuroscience, 2017, 20(4): 620-628). At this time, with only one scan, the three-dimensional information of the biological sample can be projected onto a two-dimensional plane, realizing the acquisition of information within the biological volume in a short time, which has been widely applied in high-speed multi-photon fluorescence imaging, multi-photon three-dimensional imaging, large-scale light-sheet fluorescence microscopy, etc. (see the literature "In vivo volumetric imaging of calcium and glutamate activity at synapses with high spatiotemporal resolution", Nature Communication, 2021, 12(1): 6630).

[0003] As described above, in optical volume imaging, the capacity of obtaining three-dimensional biological information mainly depends on the propagation distance of light waves within a biological sample. In other words, a longer non-diffracting distance can provide a greater imaging depth, enabling researchers to observe more structural information in a single imaging. However, current traditional optical volume imaging microscopy techniques are often restricted by the numerical aperture of the objective lens (see the literature "50Hz volumetric functional imaging with continuously adjustable depth of focus", Biomedical Optics Express, 2018, 9(4): 1964-1976). Specifically, the smaller the numerical aperture of the objective lens, the longer the propagation distance of the Bessel beam, which is beneficial for improving the capacity of obtaining biological information; however, the lateral size of the Bessel beam becomes larger, resulting in a decrease in the lateral resolution of the optical system; although increasing the numerical aperture of the objective lens can achieve a higher lateral resolution, it will affect the non-diffracting propagation distance of the beam, thus reducing the information acquisition capacity of the optical system (see the literature "High-throughput volumetric mapping of synaptic transmission", Nature Methods, 2024). How to break through the limitation of the information acquisition capacity of the existing optical volume imaging system based on traditional Bessel beam illumination under the same objective lens focusing condition and achieve fast imaging of high-throughput three-dimensional information is the technical bottleneck of existing optical volume imaging and the key to accurately observing biological mechanisms and activities. Summary of the Invention

[0004] In view of this, the present invention provides a long-depth-of-focus Bessel beam volume microscopy imaging system, which uses a laser as an excitation light source. The non-diffracting beam generation module generates a long-depth-of-focus Bessel beam with a non-diffracting distance n times that of the traditional Bessel beam, capable of breaking through the limitation of the numerical aperture of the objective lens and achieving a greater imaging depth of field under the condition of a certain lateral resolution. Using the long-depth-of-focus non-diffracting beam to achieve volume imaging, it breaks through the limitation of the information acquisition capacity of the existing optical volume imaging system based on traditional Bessel beam illumination, obtains a stack of more axial information, and has a high imaging speed and imaging resolution.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A long-depth-of-focus Bessel beam volume microscopy imaging system, comprising:

[0007] A non-diffracting beam generation module, configured to generate a long-depth-of-focus Bessel beam and incident it on a scanning microscope;

[0008] A scanning and focusing module, which is used to focus the long-depth-of-field Bessel beam generated by the non-diffracting beam generation module onto the sample to achieve high-resolution volume imaging;

[0009] A sample control module, which is used to carry the sample and precisely focus and image the area of interest by controlling the positions of the x, y, and z axes;

[0010] A fluorescence signal detection module, which is used to detect the high-resolution fluorescence signal generated by the sample;

[0011] A microscopic imaging processing terminal, which is used for signal acquisition and synchronous control, and for controlling the galvanometer scanning and the signal synchronization of the photomultiplier tube.

[0012] Preferably, the non-diffracting beam generation module includes:

[0013] A laser, which outputs Gaussian excitation light and has the function of realizing power adjustment;

[0014] A first reflector, which is used to reflect the excitation light into a first doublet lens;

[0015] A first doublet lens, which is used to cooperate with a second doublet lens to form a 4f system to shape and expand the light spot;

[0016] A second reflector, which is used to reflect the shaped excitation light into a phase diffraction device;

[0017] A phase diffraction device, which is used to perform phase modulation on the excitation light to generate a Bessel beam with a long depth of field; the phase modulation mainly modulates the incident beam by processing a phase plate or loading a phase pattern on a spatial light modulator, connects N segments of Bessel beams axially, and finally forms a Bessel beam with a long depth of field. Its phase pattern is expressed as:

[0018]

[0019] where N is the number of Bessel beams; s j is responsible for adjusting the amplitude and phase of the j-th Bessel beam; k = 2π / λ, where λ is the wavelength of the incident light; θ is the objective convergence angle; z j is the axial position of the j-th Bessel beam in the focal region of the illumination objective; φ j is the phase parameter for controlling the j-th Bessel beam. Phase is the phase-taking function.

[0020] T h is a high-pass filter, which forms a Bessel beam through Fourier transform, and its transmittance expression is:

[0021]

[0022] Among them, r0 is the effective light-transmitting aperture radius of the filter, which is used to adjust the axial length of the generated single Bessel beam.

[0023] The third doublet lens is used to cooperate with the fourth doublet lens to form a 4f system, which shrinks the light spot and conjugates it to the galvanometer mirror.

[0024] The aperture stop is used to filter out the unmodulated zero-order light and only allow the first-order light to pass through.

[0025] The third mirror and the fourth mirror are used to reflect the long focal-depth Bessel beam to the galvanometer mirror.

[0026] Preferably, the first doublet lens is used to cooperate with the second doublet lens to form a 4f system, which shapes and expands the light spot.

[0027] Preferably, the third doublet lens is used to cooperate with the fourth doublet lens to form a 4f system, which shrinks the light spot to one-fourth of the original and conjugates it to the galvanometer mirror.

[0028] Preferably, the scanning and focusing module includes a scanning device, a scanning lens, a sleeve lens and an objective lens.

[0029] The excitation light output by the non-diffracting beam generation module sequentially passes through the scanning device, the scanning lens, the sleeve lens and the objective lens and is focused on the sample on the sample carrier.

[0030] Preferably, the scanning device includes an XY two-dimensional galvanometer mirror, or a resonant mirror in the X direction and a galvanometer in the Y direction, or a first galvanometer in the X direction and a second galvanometer in the Y direction, or a first acousto-optic deflector in the X direction and a second acousto-optic deflector in the Y direction, or a digital micromirror array.

[0031] Preferably, the sample control module includes a sample holder and an XY two-axis displacement stage. The sample holder is used for fixing cells, slices and samples, and the XY two-axis displacement stage is used for manually or electrically moving the sample to select the region of interest for imaging.

[0032] Preferably, the fluorescence signal detection module includes a backscattered light signal detection module.

[0033] The backscattered light signal detection module includes a dichroic mirror, a lens, a filter and a photomultiplier tube.

[0034] The dichroic mirror is placed between the sleeve lens and the objective lens and is used to reflect the non-linear signal generated by the sample to the lens and transmit the excitation light.

[0035] The lens is placed between the dichroic mirror and the photomultiplier tube and is used to focus the high-resolution fluorescence signal.

[0036] The filter removes the excitation light at once and detects it through the photomultiplier tube.

[0037] Preferably, the information acquisition and synchronization control module includes an acquisition card and an industrial controller;

[0038] The acquisition card is used to control the scanning galvanometer of the scanning and focusing module and the acquisition of voltage signals in the fluorescence signal detection module;

[0039] The industrial controller is used to display the high-resolution image obtained and reconstructed by the acquisition card.

[0040] Preferably, the microscopic imaging processing terminal includes:

[0041] An information acquisition and synchronization control module, which is used to realize the synchronous acquisition of high-resolution fluorescence signals, the synchronous control of the scanning microscope, and the reconstruction and storage of high-resolution volume imaging.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The long focal depth Bessel beam volume microscopic imaging system provided by the present invention uses near-infrared light as the excitation light source, which has small damage to the sample and strong penetration, making the volume imaging technology have great potential in exploring cell interactions and performing in vivo pathological diagnosis. The non-diffracting beam generation module generates a long focal depth Bessel beam with a non-diffracting distance n times that of the traditional Bessel beam, which can break through the limitation of the effective numerical aperture. Under the condition of a certain lateral resolution, it provides a larger imaging depth of field, realizes volume imaging by using the characteristics of long focal depth and non-diffraction, and simultaneously obtains a stack of more axial information, with high imaging speed and imaging resolution.

[0044] Other beneficial effects of the present invention will be described in combination with specific structures in the following specific embodiments. Description of the Drawings

[0045] Figure 1 It is a structural schematic block diagram of implementing a long focal depth Bessel beam volume microscopic imaging system of the present invention;

[0046] Figure 2This is a schematic diagram of the structure of a long-focus depth Bessel beam volume microscopy system according to the present invention. In the figure, 1 is a laser; 2 is a first reflector; 3 is a first doublet lens; 4 is a second doublet lens; 5 is a second reflector; 6 is a phase diffraction element; 7 is a third doublet lens; 8 is a fourth doublet lens; 9 is a third reflector; 10 is an aperture stop; 11 is a fourth reflector; 12 is a scanning device; 13 is a scanning lens; 14 is a tube lens; 15 is a dichroic mirror; 16 is an objective lens; 17 is a sample carrier; 18 is a lens; 19 is a filter; 20 is a photomultiplier tube; 21 is an acquisition card; 22 is an industrial controller; 101 is a non-diffracting beam generation module; 102 is a scanning and focusing module; 103 is a sample control module; 104 is a fluorescence signal detection module; and 105 is an information acquisition and synchronization control module.

[0047] Figure 3 Graphs showing the experimental results of connecting three Bessel beams in the axial direction using phase plates with different phase distributions in an embodiment of the present invention; (a) is the phase plate distribution diagram corresponding to the positions of the three Bessel foci in the focusing area at (0, 0, -20), (0, 0, 0) and (0, 0, +20), (b) is the phase plate distribution diagram corresponding to the positions of the three Bessel foci in the focusing area at (0, 0, -15), (0, 0, 0) and (0, 0, +15), (c) is the phase plate distribution diagram corresponding to the positions of the three Bessel foci in the focusing area at (0, 0, -10), (0, 0, 0) and (0, 0, +1 (a) is the phase plate distribution diagram corresponding to the phase plate shown in (0), (b) is the phase plate distribution diagram corresponding to the phase plate shown in (0), (c) is the phase plate distribution diagram corresponding to the positions of the three Bessel focal points in the focusing area at (0, 0, -9.35), (0, 0, 0) and (0, 0, +9.35), (e) is the axial total light intensity distribution of the light beam at the rear focal plane of the objective lens after being modulated by the phase plate shown in (a), (f) is the axial total light intensity distribution of the light beam at the rear focal plane of the objective lens after being modulated by the phase plate shown in (b), (g) is the axial total light intensity distribution of the light beam at the rear focal plane of the objective lens after being modulated by the phase plate shown in (c), and (h) is the axial total light intensity distribution of the light beam at the rear focal plane of the objective lens after being modulated by the phase plate shown in (d).

[0048] Figure 4 : The intensity distribution diagrams of three light beams under the same numerical aperture conditions and the corresponding phase plates in an embodiment of the present invention; wherein, (a) is the phase plate distribution diagram for generating a Gaussian beam, (b) is the phase plate distribution diagram for generating a Bessel beam, and (c) is the phase plate distribution diagram for generating a Bessel beam with a long focal depth; (d) is the lateral total light intensity distribution of the Gaussian beam, (e) is the lateral total light intensity distribution of the Bessel beam, (f) is the lateral total light intensity distribution of the Bessel beam with a long focal depth, and (g) is the axial total light intensity distribution of the Gaussian beam; (h) is the axial total light intensity distribution of the Bessel beam; and (i) is the axial total light intensity distribution of the Bessel beam with a long focal depth. Detailed implementation manners

[0049] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0050] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "upper", "lower", "inner", "outer", "top / bottom end", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as a limitation of the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0051] In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, the terms "installed", "provided with", "sheathed / connected", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0052] As Figure 1-2 shown, a long-depth-of-focus Bessel beam volume microscopy imaging system includes:

[0053] A non-diffracting beam generation module 101 for generating a long-depth-of-focus Bessel beam that is 1.5 to 3 times longer than a traditional Bessel beam and incident on a scanning microscope;

[0054] A scanning and focusing module 102 for focusing the long-depth-of-focus Bessel beam generated by the non-diffracting beam generation module 101 onto a sample to achieve high-resolution volume imaging, where the sample is preferably a sample placed on a sample carrier 17;

[0055] A sample control module 103 for carrying the sample on the sample carrier 17 and accurately focusing and imaging the region of interest by controlling the positions of the x, y, and z axes;

[0056] A fluorescence signal detection module 104 for detecting the high-resolution fluorescence signal generated by the sample on the sample carrier 17;

[0057] A microscopic imaging processing terminal for signal acquisition and synchronous control, and for controlling the signal synchronization of galvanometer scanning and photomultiplier tube 20.

[0058] Among them, the above microscopic imaging processing terminal can be based on mobile terminals such as mobile phones, tablet computers, laptop computers, palmtop computers, personal digital assistants (PDAs), portable media players (PMPs), navigation devices, wearable devices, smart bracelets, etc., and fixed terminals such as digital TVs and desktop computers.

[0059] The long focal depth Bessel beam volume microscopic imaging system provided by the present invention can use the non-diffracting beam generation module 101 to generate a long focal depth Bessel beam. Combining the point-by-point scanning of the galvanometer can achieve high-resolution volume imaging, capture a stack of high-resolution information in the axial direction, so as to quickly obtain the high-resolution structural information of the sample, and has a high imaging speed.

[0060] In the present invention, the Bessel beam generation module in the non-diffracting beam generation module 101 includes a Bessel beam generating device and a lens group (such as the first doublet lens 3 and the second doublet lens 4). The Bessel beam generating device includes, but is not limited to, an axicon, a deformable mirror, an annular slit lens, and a phase diffraction device 6. The lens group is used for collimating and expanding the Bessel beam and matching the parameter requirements of the objective lens 16 for the incident light.

[0061] In the present invention, the non-diffracting beam generation module 101 includes:

[0062] A laser 1 that outputs Gaussian excitation light and has the function of realizing power adjustment. Among them, if near-infrared light is used as the excitation light source, it has little damage to the sample and strong penetration, making the volume imaging technology have great potential in exploring cell interactions and performing in-vivo pathological diagnosis;

[0063] Among them, the Gaussian excitation light is preferably femtosecond-level excitation light or near-infrared light;

[0064] A first mirror 2 for reflecting the excitation light into the first doublet lens 3;

[0065] A first doublet lens 3 for cooperating with the second doublet lens 4 to form a 4f system to shape and expand the light spot;

[0066] A second mirror 5 for reflecting the shaped excitation light into the phase diffraction device 6. Among them, the phase diffraction device 6 can preferably be a spatial light modulator;

[0067] The phase diffraction device 6 is used to perform phase modulation on the excitation light to generate a Bessel beam with a long depth of focus;

[0068] The above-mentioned phase modulation mainly modulates the incident light beam by processing a phase plate or loading a phase pattern on a spatial light modulator, connects N segments of Bessel beams axially, and finally forms a Bessel beam with a long depth of focus. Its phase pattern is expressed as:

[0069]

[0070] where N is the number of Bessel beams. In this embodiment, N = 3. s j is responsible for adjusting the amplitude and phase of the j-th Bessel beam; k = 2π / λ, where λ is the wavelength of the incident light; θ is the objective lens convergence angle; z j is the axial position of the j-th Bessel beam within the focal region of the illumination objective lens; φ j is the phase parameter for controlling the j-th Bessel beam. Phase is the phase function.

[0071] T h is a high-pass filter, which forms a Bessel beam through Fourier transform. Its transmittance expression is:

[0072]

[0073] where r0 is the effective clear aperture radius of the filter, which is used to adjust the axial length of the generated single Bessel beam. For easy understanding, an example is given:

[0074] The incident light is linearly polarized light with a diameter of 8 mm and a wavelength of 1040 nm. Other wavelengths of light beams can be used in specific implementations;

[0075] In this example, the numerical aperture of the objective lens 16 is 0.9, and the number of Bessel foci N = 3. Other values of numerical aperture and number of foci can be used in specific implementations. As Figure 3 shown, Figure 3 (a) shows the phase plate distribution diagrams corresponding to the positions of three Bessel foci in the focusing region being (0, 0, -20), (0, 0, 0), and (0, 0, +20) respectively, Figure 3 (b) shows the phase plate distribution diagrams corresponding to the positions of three Bessel foci in the focusing region being (0, 0, -15), (0, 0, 0), and (0, 0, +15) respectively, Figure 3 (c) shows the phase plate distribution diagrams corresponding to the positions of three Bessel foci in the focusing region being (0, 0, -10), (0, 0, 0), and (0, 0, +10) respectively, Figure 3(d) shows the phase plate distribution maps corresponding to the positions of three Bessel foci in the focusing region being (0, 0, -9.35), (0, 0, 0), and (0, 0, +9.35) respectively. Figure 3 (e, f, g, h) are the total light intensity distribution maps of the outgoing beams in the above four cases respectively. In practical applications, the phase plate can be pixelated and dynamically controlled in real time through a phase-type spatial light modulator or other optical elements to precisely control the positions of the foci of each Bessel beam in the focusing region.

[0076] In this example, the numerical aperture of the objective lens is 0.9, the incident light is linearly polarized light with a diameter of 8 mm and a wavelength of 1040 nm. After being modulated by the phase plate shown in Figure 4 (a, b, c), the beams emitted by the objective lens 16 are a Gaussian beam, a conventional Bessel beam, and a long-depth-of-focus Bessel beam respectively. Figure 4 (d, g) are the total lateral and axial light intensity distribution maps of the Gaussian beam under this condition. Figure 4 (e, h) are the total lateral and axial light intensity distribution maps of the conventional Bessel beam under this condition. Figure 4 (f, i) are the total lateral and axial light intensity distribution maps of the long-depth-of-focus Bessel beam under this condition. Specific implementations can use other values of numerical aperture and beams of other wavelengths.

[0077] The third doublet lens 7 is used to cooperate with the fourth doublet lens 8 to form a 4f system, which shrinks the light spot and conjugates it to the galvanometer. Preferably, it shrinks to one-fourth of the original size. Among them, the galvanometer is preferably the galvanometer of the following scanning device 12.

[0078] The aperture stop 10 is used to filter out the unmodulated zero-order light and only allow the first-order light to pass through.

[0079] The third mirror 9 and the fourth mirror 11 are used to reflect the long-depth-of-focus Bessel beam to the galvanometer. Among them, the galvanometer is preferably the galvanometer of the following scanning device 12.

[0080] In the present invention, the first doublet lens 3 is used to cooperate with the second doublet lens 4 to form a 4f system to shape and expand the light spot.

[0081] In the present invention, the third doublet lens 7 is used to cooperate with the fourth doublet lens 8 to form a 4f system to shrink the light spot to one-fourth of the original size and conjugate it to the galvanometer.

[0082] In the present invention, the scanning and focusing module 102 includes a scanning device 12, a scanning lens 13, a sleeve lens 14, and an objective lens 16.

[0083] The excitation light output by the non-diffracting beam generation module 101 sequentially passes through the scanning device 12, the scanning lens 13, the sleeve lens 14, and the objective lens 16 and is focused on the sample on the sample carrier 17, where the sample carrier 17 can be arranged on a three-dimensional displacement stage.

[0084] In the present invention, the scanning device 12 includes an XY two-dimensional galvanometer mirror, or a resonant mirror in the X direction and a galvanometer in the Y direction, or a first galvanometer in the X direction and a second galvanometer in the Y direction, or a first acousto-optic deflector in the X direction and a second acousto-optic deflector in the Y direction, or a digital micromirror array.

[0085] In the present invention, the sample control module 103 includes a sample holder and an XY two-axis displacement stage. The sample holder is used for fixing cells, sections, and samples, and the XY two-axis displacement stage is used to manually or electrically move the sample to select an area of interest for imaging.

[0086] In the present invention, the fluorescence signal detection module 104 includes a backscattered light signal detection module;

[0087] The backscattered light signal detection module includes a dichroic mirror 15, a lens 18, a filter 19, and a photomultiplier tube 20;

[0088] The dichroic mirror 15 is placed between the sleeve lens 14 and the objective lens 16 and is used to reflect the non-linear signal generated by the sample to the lens 18 and transmit the excitation light;

[0089] The lens 18 is placed between the dichroic mirror 15 and the photomultiplier tube 20 and is used to focus the high-resolution fluorescence signal;

[0090] The filter 19 filters out the excitation light at one time and is detected by the photomultiplier tube 20, and the filter 19 can also select a group of filters 19 according to actual needs.

[0091] In the present invention, the excitation beam generated by the non-diffracting beam generation module 101 excites the sample in the sample control module 103 through the scanning and focusing module 102. The generated fluorescence signal is collected by the objective lens 16 and then reflected by the dichroic mirror 15 into the backscattered fluorescence processing module. The backscattered fluorescence processing module includes a lens 18, a filter 19, and a photomultiplier tube 20. The collected high-resolution fluorescence signal is converted into a voltage signal by the photomultiplier tube 20 and processed by a computer;

[0092] In the present invention, the information acquisition and synchronous control module 105 includes an acquisition card 21 and an industrial controller 22;

[0093] The acquisition card 21 is used to control the scanning galvanometer of the scanning and focusing module 102 and the acquisition of voltage signals in the fluorescence signal detection module 104;

[0094] The industrial controller 22 is used to display the high-resolution images obtained and reconstructed by the acquisition card 21.

[0095] In the present invention, the microscopic imaging processing terminal includes:

[0096] An information acquisition and synchronization control module 105, which is used to realize the synchronous acquisition of high-resolution fluorescence signals, the synchronous control of the scanning microscope, and the reconstruction and storage of high-resolution volume imaging.

[0097] The working principle of the present invention is as follows:

[0098] In the present invention, after the laser 1 in the non-diffracting beam generation module 101 outputs the excitation light, it is incident on the first doublet lens 3 through the first mirror 2, and the light spot is expanded in cooperation with the second doublet lens 4, and then is incident on the phase diffraction device 6 through the second mirror 5; The third doublet lens 7 and the fourth doublet lens 8 form a 4f system, conjugate the light spot emerging from the phase diffraction device 6 to the scanning galvanometer in the scanning device 12, and filter out other order lights through the aperture stop 10, only retaining the first-order light, and use the third mirror 9 and the fourth mirror 11 to straighten the optical path to ensure that the light beam is vertically incident on the scanning galvanometer in the scanning device 12.

[0099] The light beam emerging from the scanning device 12 is then incident on the objective lens 16 through the scanning lens 13 and the sleeve lens 14 to generate a long focal-depth Bessel light on the focal plane. The excited high-resolution fluorescence signal is reflected by the dichroic mirror 15 to the lens 18, and after passing through the filter 19, it is focused into the photomultiplier tube 20.

[0100] The acquisition card 21 controls the scanning galvanometer of the scanning and focusing module 102 and the acquisition of voltage signals in the fluorescence signal detection module 104, and the industrial controller 22 is used to display the high-resolution images obtained and reconstructed by the acquisition card 21.

[0101] The present invention provides a long focal-depth Bessel beam volume microscopic imaging system, including but not limited to non-diffracting beam volume imaging and two-photon excited fluorescence, and the non-diffracting beam includes but not limited to Bessel beam.

[0102] In all embodiments of the present invention, in order to facilitate the transmission of the laser, mirrors can be used to change the direction of the laser.

[0103] The above is only a preferred specific embodiment of the present invention; however, the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, making equivalent substitutions or changes according to the technical solution of the present invention and its improved conceptions, shall be covered by the protection scope of the present invention.

Claims

1. A long-depth-of-field Bessel beam volumetric microscopy imaging system, characterized in that, Comprising: A non-diffracting beam generation module for generating a long depth-of-field Bessel beam and incident on a scanning microscope; A scanning and focusing module for focusing the long depth-of-field Bessel beam generated by the non-diffracting beam generation module onto a sample to achieve high-resolution volume imaging; A sample control module for carrying the sample and accurately focusing and imaging an area of interest by controlling the positions of the x, y, and z axes; A fluorescence signal detection module for detecting the high-resolution fluorescence signal generated by the sample; A microscopic imaging processing terminal for signal acquisition and synchronous control, and for controlling galvanometer scanning and signal synchronization of a photomultiplier tube.

2. The long depth-of-field Bessel beam volume microscopic imaging system according to claim 1, wherein, The non-diffracting beam generation module includes: A laser that outputs Gaussian excitation light and has the function of power adjustment; A first mirror for reflecting the excitation light into a first doublet lens; A first doublet lens for cooperating with a second doublet lens to form a 4f system to shape and expand the light spot; A second mirror for reflecting the shaped excitation light into a phase diffraction device; A phase diffraction device for phase modulating the excitation light to generate a Bessel beam with a long depth of field; the phase modulation is mainly carried out by processing a phase plate or loading a phase pattern on a spatial light modulator to modulate the incident beam, connecting N segments of Bessel beams axially, and finally forming a Bessel beam with a long depth of field, and its phase pattern is expressed as: where N is the number of Bessel beams; s j is responsible for adjusting the amplitude and phase of the j-th Bessel beam; k = 2π / λ, where λ is the wavelength of the incident light; θ is the objective lens convergence angle; z j is the axial position of the j-th Bessel beam within the focal region of the illumination objective lens; φ j is the phase parameter for controlling the j-th Bessel beam. Phase is the phase function. T h is a high-pass filter. After Fourier transformation, it forms a Bessel beam, and its transmittance is expressed as: Where r0 is the effective clear aperture radius of the filter for adjusting the axial length of a single generated Bessel beam. A third doublet lens for cooperating with a fourth doublet lens to form a 4f system to contract the light spot and conjugate it to a galvanometer; An aperture stop for filtering out the unmodulated zero-order light and only allowing the first-order light to pass through; A third mirror and a fourth mirror for reflecting the long depth-of-field Bessel beam onto a galvanometer.

3. The volumetric microscopic imaging system for a long depth-of-field Bessel beam according to claim 2, characterized in that, The long-depth-of-focus Bessel beam is generated by precisely controlling the number N of Bessel beams and the axial position z of each Bessel beam within the focal region of the illumination objective lens. j Generated.

4. A long-depth-of-field Bessel beam volume microscopy imaging system according to claim 2, characterized in that, The non-diffracting propagation distance of the long depth-of-field Bessel beam depends on the number N of Bessel beams.

5. A long-depth-of-field Bessel beam volumetric microscopy imaging system according to claim 1, wherein The scanning and focusing module includes a scanning device, a scanning lens, a sleeve lens, and an objective lens; The excitation light output by the non-diffracting beam generation module sequentially passes through the scanning device, the scanning lens, the sleeve lens, and the objective lens and is focused on the sample on a sample carrier.

6. The volumetric microscopic imaging system of a long depth-of-field Bessel beam according to claim 7, characterized in that, The scanning device includes an XY two-dimensional galvanometer mirror, or a resonant mirror in the X direction and a galvanometer in the Y direction, or a first galvanometer in the X direction and a second galvanometer in the Y direction, or a first acousto-optic deflector in the X direction and a second acousto-optic deflector in the Y direction, or a digital micromirror array.

7. A long-depth-of-field Bessel beam volume microscopy imaging system according to claim 1, characterized in that The sample control module includes a sample holder and an XY two-axis displacement stage. The sample holder is used for cell, section, and sample fixation, and the XY two-axis displacement stage is used for manually or electrically moving the sample to select an area of interest for imaging.

8. A long-depth-of-field Bessel beam volume microscopy imaging system according to claim 1, characterized in that, The fluorescence signal detection module includes a backscattered light signal detection module; The backscattered light signal detection module includes a dichroic mirror, a lens, a filter, and a photomultiplier tube; The dichroic mirror is placed between the sleeve lens and the objective lens for reflecting the non-linear signal generated by the sample to the lens and transmitting the excitation light; The lens is placed between the dichroic mirror and the photomultiplier tube for focusing high-resolution fluorescence signals; The filter removes the excitation light at one time and is detected by the photomultiplier tube.

9. The volumetric microscopic imaging system of a long-depth-of-focus Bessel beam according to claim 1, wherein The information acquisition and synchronization control module includes an acquisition card and an industrial controller; The acquisition card is used to control the scanning galvanometer of the scanning and focusing module and the acquisition of voltage signals in the fluorescence signal detection module; The industrial controller is used to display the high-resolution images obtained and reconstructed by the acquisition card.

10. A long-depth-of-field Bessel beam volume microscopy imaging system according to any one of claims 1-9, characterized in that, The microscopic imaging processing terminal includes: An information acquisition and synchronization control module for realizing the synchronous acquisition of high-resolution fluorescence signals, the synchronous control of the scanning microscope, and the reconstruction and storage of high-resolution volume imaging.

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