Miniature two-photon microscopic imaging system based on Bessel beam, imaging method and application

Abstract

The invention provides a miniature two-photon microscopic imaging system based on Bessel beam excitation, an imaging method and application. A miniature conical lens is processed at the output end of a large-mode-field optical fiber to generate a Bessel beam, so that the volume imaging function of a miniature two-photon microscopic system is realized; a dispersion compensation strategy of the large-mode-field photonic crystal fiber is established; a symmetrical scanning light path and a micro objective lens matched with the scanning light path are designed, so that other aberrations are effectively controlled while dispersion is eliminated; by adopting a detection light path design strategy of an objective lens with a high collection aperture and a silicon photomultiplier without a front focusing lens, the structure of a microscopic system is simplified, and meanwhile, the fluorescence collection efficiency is improved; the miniature two-photon microscopic system provided by the invention has the characteristics of high-flux volume imaging, high fluorescence collection efficiency, flexible dispersion adjustment and simple structure, and can effectively meet the nerve imaging requirement of high-flux volume imaging in a free activity state of a living sample.

Description

Technical Field

[0001] This application relates to the field of optical imaging technology, and in particular to a miniature high-throughput two-photon microscope system, imaging method and application based on Bessel beam volume imaging. Background Technology

[0002] Miniature head-mounted multiphoton microscopy systems offer advantages in both deep imaging and optical section imaging, making them an important tool for imaging neural activity in freely moving small animals (mice, rats, etc.). In recent years, with the continuous development of photonic crystal fibers, miniature laser scanning devices, fast-focusing lenses, fluorescence detectors, and miniature objectives, the performance of miniature head-mounted multiphoton microscopy systems has been significantly improved, greatly increasing the number of neurons that can be imaged in freely moving animals.

[0003] However, existing miniature multiphoton microscopy systems have several limitations. First, their imaging throughput is low due to the need for point-by-point scanning. When using high numerical aperture (NA) objectives for high-resolution imaging, it is difficult to record the activity of all neurons within a single volume. Although high-performance tunable lenses can perform axial scans in milliseconds, current techniques can only image a limited number of thin optical sections at a time, and the imaging speed or resolution is correspondingly reduced. Second, the depth of focus in microscopy systems is typically very small, making fluorescence signals susceptible to tissue movement. This problem is particularly severe in imaging freely moving animals, as their head movements are unrestricted. Finally, the size of microscopy systems is typically only a few cubic centimeters, increasing the difficulty of optical design and system assembly; consequently, the system often exhibits aberrations, making it difficult to achieve diffraction-limited imaging effects.

[0004] Compared to the widely used Gaussian beam, tabletop two-photon microscopy systems utilizing Bessel beams have demonstrated several unique advantages in recent years. By scanning an axially extended focal point (increased depth of field) in a two-dimensional plane, Bessel beams enable volumetric imaging, improving imaging speed, increasing information throughput, and reducing data volume. Simultaneously, the extended depth of field makes imaging more resistant to axial motion of brain tissue, which is highly advantageous for monitoring neural circuits in freely moving animals. Furthermore, with the same excitation NA, Bessel beams offer better lateral resolution than Gaussian beam two-photon microscopy systems; and their insensitivity to aberrations such as spherical aberration and coma indicates a potential for simplifying optical system design.

[0005] To address the current problems in the field of microscopy, particularly in imaging the neural activity of freely moving small animals, this invention aims to provide a miniature two-photon microscopy system based on a Bessel beam. This system is the first to introduce a Bessel beam into a head-mounted miniature two-photon microscopy system, generating the beam by fabricating a conical lens at the fiber end face. While using fiber optics to transmit light beams is a conventional technique in optical systems, this application miniaturizes existing large-scale optical systems based on Bessel beams, overcoming the difficulties of optical design and successfully achieving a miniature two-photon microscopy system weighing only 2.6 grams. Based on the characteristics of the Bessel beam, the simplified optical design in this application plays a crucial role in the successful development of the head-mounted microscopy system. Summary of the Invention

[0006] A miniature two-photon microscopic imaging system based on Bessel beam excitation includes: a laser source for two-photon fluorescence excitation; a laser transmission fiber for coupling and transmitting the laser output from the laser; a miniature probe including an excitation optical path and a collection optical path, wherein the excitation optical path converts the laser transmitted by the laser transmission fiber into a Bessel focus and performs two-dimensional scanning and fluorescence excitation, and the collection optical path collects the fluorescence; and a photodetector for detecting the fluorescence signal excited by the two-photon excitation.

[0007] The micro two-photon microscopic imaging system based on Bessel beam excitation as described in claim 1 is characterized in that the laser transmission fiber is a large-mode-field photonic crystal fiber, and a micro cone lens is processed on the output end face of the fiber, or the fiber and the micro cone lens are combined to generate a Bessel beam.

[0008] A miniature two-photon microscopic imaging system based on Bessel beam excitation, characterized in that the laser source is a femtosecond laser, preferably the laser has a built-in dispersion compensation module; more preferably the femtosecond laser has a wavelength of 920nm.

[0009] A miniature two-photon microscopic imaging system based on Bessel beam excitation is characterized in that the excitation optical path includes a collimating lens, a scanning galvanometer, a scanning lens, a tube lens, a dichroic mirror, and a miniature objective lens; and the collection optical path includes a miniature objective lens, a dichroic mirror, and a filter.

[0010] A miniature two-photon microscopy imaging system based on Bessel beam excitation is characterized in that, in the excitation optical path, a scanning galvanometer scans the laser; a scanning lens and a tube lens are used to conjugate the beams scanned by the scanning galvanometer at different angles onto the back focal plane of the miniature objective; a dichroic mirror is used to separate the excitation optical path from the collection optical path; and the miniature objective is used to focus the scanning beam into the interior of the sample under test, generating an axially extended Bessel focus on the focal plane to achieve volumetric imaging.

[0011] A miniature two-photon microscopy imaging system based on Bessel beam excitation, characterized in that the photodetector is a silicon photomultiplier tube.

[0012] A miniature two-photon microscopy imaging system based on Bessel beam excitation is characterized in that, in the collection optical path, fluorescence is collected by a miniature objective lens, and after passing through a dichroic mirror and a filter, it is directly irradiated onto the detector for detection without the need for additional lenses for focusing.

[0013] A miniature two-photon microscopic imaging system based on Bessel beam excitation is characterized in that the astigmatism of the scanning lens and tube lens group in the excitation optical path is mutually compensated with the astigmatism of the miniature objective, and the overall astigmatism tends to be zero.

[0014] A microscopic two-photon microscopy imaging method based on Bessel beam excitation is characterized in that the laser emitted by the laser source is transmitted through the transmission optical fiber and the generated Bessel beam enters the micro probe, passing sequentially through a collimating lens, a scanning galvanometer, a scanning lens, a tube lens, a dichroic mirror, and a micro objective lens, forming Bessel focusing at the focal plane of the micro objective lens, thereby exciting the two-photon fluorescence signal of the sample; the fluorescence is collected by the micro objective lens, and after passing through the dichroic mirror and a filter, it is detected by a silicon photomultiplier tube.

[0015] Application of a miniature two-photon microscopy imaging system or method based on Bessel beam excitation in neuronal activity imaging, particularly mouse neuronal activity imaging, and more particularly mouse neuronal calcium ion activity imaging.

[0016] A miniature two-photon microscopy imaging system based on Bessel beam excitation is a head-mounted miniature two-photon microscope device for the study of freely moving mice or rats.

[0017] A miniature multiphoton microscopic imaging system based on Bessel beam excitation includes: a laser source for multiphoton fluorescence excitation; a laser transmission fiber for coupling and transmitting the laser output from the laser; a miniature probe including an excitation optical path and a collection optical path, wherein the excitation optical path converts the laser beam into a Bessel focus and performs two-dimensional scanning and fluorescence excitation, and the collection optical path collects the fluorescence; and a photodetector for detecting the fluorescence signal excited by two photons.

[0018] A design method for a miniature multiphoton microscope based on Bessel beam excitation is characterized in that: the design method controls the overall astigmatism of the microscope to approach zero, and can still obtain diffraction-limited imaging effect in the presence of other aberrations, including spherical aberration, coma, field curvature and distortion.

[0019] This invention is the first to utilize the unique advantages of Bessel beams in a miniature two-photon microscopy system, addressing the problems existing in the aforementioned miniature two-photon microscopy systems. The invention proposes a miniature high-throughput two-photon microscopy system and imaging method based on Bessel beam volumetric imaging.

[0020] In a first aspect, embodiments of the present invention provide a miniature high-throughput two-photon microscopy system based on Bessel beam volume imaging, the miniature two-photon microscopy system comprising:

[0021] The system comprises a femtosecond pulsed laser, a laser transmission fiber, a scanning controller, a miniature probe, and a detector. The miniature probe is a key module of the miniature two-photon microscopy system. The femtosecond pulsed laser is connected to the miniature probe via the laser transmission fiber. Both the detector and the scanning controller are electrically connected to the miniature probe.

[0022] The femtosecond pulsed laser is a femtosecond pulsed laser with dispersion compensation function, used to generate a femtosecond laser with a wavelength of 920nm; and the dispersion of the output pulsed laser can be adjusted to pre-compensate the dispersion caused by the laser in the laser transmission fiber, so that the final pulse width is reduced to an adjustable minimum value for two-photon fluorescence excitation.

[0023] The laser transmission fiber is a large-mode-field photonic crystal fiber, with a miniature conical lens fabricated at the fiber's output end face. This lens couples the laser output from the femtosecond laser, converts it into a Bessel beam, and transmits it to the miniature probe.

[0024] The scanning controller is used to control the scanning mirror in the miniature probe to scan the laser.

[0025] The micro probe includes a fluorescence excitation optical path and a fluorescence collection optical path. The fluorescence excitation optical path converts the light beam output from the optical fiber into a Bessel focus and performs two-dimensional scanning and fluorescence excitation. The fluorescence collection optical path performs high-throughput collection of fluorescence signals and filters laser signals.

[0026] The detector, a silicon photomultiplier tube, is placed at the end of the fluorescence collection optical path of the miniature probe to collect the fluorescence signal in situ and convert it into an electrical signal to generate an image of calcium ion activity in the neurons of the tested sample. The fluorescence is collected by a miniature objective lens, passes through a dichroic mirror and a filter, and is then directly detected by the silicon photomultiplier tube without the need for an additional focusing lens.

[0027] This invention provides a miniature two-photon microscopy imaging system and method based on Bessel beam excitation. By generating a Bessel beam through a miniature conical lens fabricated at the output end of a large-mode-field optical fiber, the volumetric imaging function of the miniature two-photon microscopy system is realized. A dispersion compensation strategy for the large-mode-field photonic crystal fiber is established. A symmetrical scanning optical path and a miniature objective lens used in conjunction with it are designed, ensuring zero astigmatism in the optical system while effectively controlling other aberrations. By employing a high-collecting-aperture objective lens and a detection optical path design strategy using a silicon photomultiplier tube that eliminates the need for a pre-focusing lens, the structure of the microscopy system is simplified while improving fluorescence collection efficiency. The miniature two-photon microscopy system provided by this invention features high-throughput volumetric imaging, high fluorescence collection efficiency, flexible dispersion adjustment, and a simple structure. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. The accompanying drawings are used to provide a further understanding of the embodiments of the present invention, constitute a part of the specification, and are used together with the embodiments of the present invention to explain the present invention, but do not constitute an improper limitation of the present invention. In the accompanying drawings, the same reference numerals generally represent the same components or steps.

[0029] Figure 1 The diagram shown is a schematic diagram of the micro-microsystem structure provided in an embodiment of the present invention.

[0030] Figure 2 The diagram shown is a schematic diagram of the control system of the micro-microsystem provided in an embodiment of the present invention.

[0031] Figure 3 The diagram shown is a schematic diagram of the output end structure of a large-mode-field photonic crystal fiber provided in an embodiment of the present invention.

[0032] Figure 4 The figure shows the dispersion compensation results provided by an embodiment of the present invention.

[0033] Figure 5a The diagram shown is a schematic diagram of the excitation optical path of the micro-microscopic system provided in an embodiment of the present invention.

[0034] Figure 5b The figure shows the distribution of the Seid aberration coefficients of the excitation optical system of the micro-micro system provided in an embodiment of the present invention.

[0035] Figure 6a The figure shows the variation of the root mean square wavefront aberration of the excitation optical system provided by the micro-micro system in the embodiment of the present invention with the field of view.

[0036] Figure 6b The image shows a comparison of the focusing effects of a Gaussian beam and a Bessel beam used in the micro-microscopic system provided in this embodiment of the invention.

[0037] Figure 7 The figure shows the resolution test results of the micro-microscopic system provided in the embodiment of the present invention.

[0038] Figure 8 The image shown is a comparison of mouse brain slice images observed by the miniature microscopy system provided in the embodiments of the present invention and the desktop two-photon microscopy system.

[0039] Figure 9a The image shows neuronal activity in the anterior cingulate cortex of a freely moving mouse, observed using the micromicroscopic system provided in this embodiment of the invention.

[0040] Figure 9b The image shows neuronal activity in the anterior cingulate cortex of a head-fixed mouse brain region, as observed using a desktop two-photon microscope system. Detailed Implementation

[0041] To make the objectives, features, and advantages of the embodiments of the present invention clearer, the technical approach of the embodiments of the present invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0042] Figure 1 The diagram shows a schematic of a microscopic system provided in an embodiment of the present invention. The microscopic system provided in this embodiment includes a femtosecond laser (not shown in the figure), a large-mode-field photonic crystal fiber 01 with a conical lens fabricated at the output end, a collimating mirror 02, a micro-electro-mechanical system (MEMS) scanning mirror 03, a scanning lens 04, a tube lens 05, a micro objective 07, a dichroic mirror 08, a filter 09, and a silicon photomultiplier tube 10 (only its effective detection surface is shown in the figure). The excitation optical path includes the collimating mirror 02, the MEMS scanning mirror 03, the scanning lens 04, the tube lens 05, the micro objective 07, and the dichroic mirror 08; the collection optical path includes the micro objective 07, the dichroic mirror 08, and the filter 09.

[0043] Furthermore, the working principle of the excitation optical path of the micro-microscopy system provided in this embodiment of the invention is described. The femtosecond pulsed laser is used to generate 920nm laser light. The generated laser light is coupled into a large-mode-field photonic crystal fiber 01 by a lens, and at the fiber output end, it is converted into a ring beam by a conical lens. After passing through a collimating lens 02, it is reflected by a MEMS scanning mirror 03 and enters a scanning lens 04 and a tube lens 05. Then, it is reflected by a dichroic mirror 08 and enters a micro-objective lens 07. The Bessel beam is finally focused on the sample by the micro-objective lens 07, generating an axially extended focal point at the focal plane 06, thus achieving volumetric imaging.

[0044] Furthermore, the working principle of the light collection path of the microscopic system provided in this embodiment of the invention is described. The fluorescence signal excited by the femtosecond laser is collected by the micro-objective 07, and after passing through the dichroic mirror 08 and the filter 09, it enters the effective detection region of the silicon photomultiplier tube 10. The silicon photomultiplier tube 10 converts the received fluorescence signal into an electrical signal, which is used to generate an image of the neural activity of the sample under test. The dichroic mirror 08 and the filter 09 are used to filter out the transmitted light beam belonging to the fluorescence band.

[0045] Figure 2 The diagram shown is a schematic of the control system for the micromicroscope system provided in an embodiment of the present invention. The host computer and the micromicroscope system exchange control and information via two electrical signals and one laser signal. Figure 2 From top to bottom, the first electrical signal is the scanning control command sent by the host computer to the scanning mirror controller. After receiving the command, the controller drives the MEMS scanning mirror 03 to perform two-dimensional scanning imaging. The first optical signal is the femtosecond pulse laser signal output by the femtosecond pulse laser controlled by the host computer. This signal is transmitted to the micro-microsystem via fiber optic cable 01 to excite and generate a two-photon fluorescence signal. The second electrical signal is the fluorescence signal detected by the silicon photomultiplier tube 10. The silicon photomultiplier tube 10 converts the detected two-photon fluorescence signal into an electrical signal. After being amplified by the silicon photomultiplier tube drive circuit, the signal is transmitted to the host computer to generate an image of the calcium ion activity of neurons in the sample under test.

[0046] Figure 3 The diagram shows a schematic of the output end face structure of a large-mode-field photonic crystal fiber provided in an embodiment of the present invention. In this embodiment, the front end of the large-mode-field photonic crystal fiber 01 consists of a short collapsed region and a conical lens fabricated within the collapsed region. The length of the collapsed region is approximately 1 mm, and the cone angle of the fiber front end is approximately 120 degrees. Through this structure, the light beam propagating in the photonic crystal fiber can be converted into a Bessel beam with volumetric imaging capabilities, making the micro-microscopic system structure in this embodiment of the present invention more compact.

[0047] It should be noted that the embodiments of the present invention only provide example parameters and related structural descriptions for processing a conical lens at the output end of a photonic crystal fiber, and are not specific limitations on the large-mode-field photonic crystal fiber 01 with a conical lens processed at the output end; as long as it can satisfy the function of converting laser into a Bessel beam, the large-mode-field photonic crystal fiber 01 with a conical lens processed at the output end can be replaced with any other type of fiber, or it can be replaced with any fiber and any optical element (such as a conical lens) that has the function of forming a Bessel beam.

[0048] In this embodiment of the invention, a femtosecond pulsed laser with dispersion compensation (Toptica, FemtoFiber ultra 920, dispersion compensation range -40000 to +1000 fs) is used. 2 This invention can accurately and flexibly compensate for the dispersion caused by large-mode-field photonic crystal fibers of different lengths during laser transmission, bringing the final pulse width to an adjustable minimum value before output. In femtosecond laser transmission, two factors cause pulse broadening: material dispersion and nonlinear effects. The dispersion compensation function of the laser in this embodiment can compensate for the pulse broadening caused by fiber material dispersion. Figure 4 The figure shows the pulse width of the output laser under different transmission powers. It can be seen that as the laser transmission power in the fiber increases, the nonlinear effect of the laser also increases, resulting in a slight increase in the final output pulse width. However, in general, the pulse broadening caused by the nonlinear effect is not severe. For a laser pulse with a pulse width of 100 fs, a maximum average power of 100 mW, and a center wavelength of 920 nm, after transmission through a large-mode-field photonic crystal fiber 01 (NKT Photonics, LMA-12) of approximately 1 m in length, the pulse width can still be maintained in a relatively narrow range. Most existing miniature two-photon microscopy systems use hollow photonic crystal fiber (NKT Photonics, HC-920) for laser transmission, requiring the use of glass pillars of a certain length for dispersion compensation. Different fiber lengths require different glass pillar lengths, and the implementation of dispersion compensation is quite cumbersome, making it unsuitable for users without an optical background.

[0049] It should be noted that the embodiments of the present invention only provide a pulse compensation strategy and are not specific limitations on the laser model, parameters, and fiber type and model. Any femtosecond pulse laser with dispersion compensation function combined with an optical fiber can achieve pulse compensation.

[0050] Figure 5a The diagram shows the excitation optical path of the micro-microscopic system provided in an embodiment of the present invention. A is the aperture stop, 04 is the scanning lens, 05 is the tube lens, 07 is the micro-objective lens, and B is the focal plane. Table 1 shows the connection with... Figure 5a The corresponding optical simulation parameters. Figure 5bfor Figure 5a The Seid aberration coefficients in the excitation optical system are shown in Table 1. In this embodiment of the invention, the scanning lens 04 and the tube lens group 05 are arranged in a symmetrical 4f system consisting of four identical plano-convex lenses. This symmetrical system configuration can effectively eliminate coma, distortion, and lateral chromatic aberration. Its astigmatism is used to compensate for the astigmatism in the miniature objective lens 07, making the astigmatism in the entire excitation optical system zero. Figure 6a To explore the relationship between the root mean square wavefront error and the field of view in an optical system, it can be seen that a focusing effect close to the diffraction limit can be obtained when the working wavelength is 920-940nm. Figure 6b This paper presents a simulated focusing effect of a microscopic system using Gaussian and Bessel beams for excitation, as described in an embodiment of the present invention. It can be seen that Gaussian beam focusing is easily affected by residual coma and spherical aberration, resulting in blurred focus; while Bessel beams can achieve diffraction-limited focusing.

[0051] It should be noted that the embodiments of the present invention only provide a configuration of scanning lens 04 and tube lens 05, and are not a specific limitation on the structure of scanning lens and tube lens. Those skilled in the art can select scanning lens and tube lens according to actual application conditions, as long as they can achieve the function of scanning lens and tube lens group in the embodiments of the present invention.

[0052]

[0053] Table 1. Simulation parameters of the excitation optical path of the miniature two-photon microscopy system according to the embodiments of this disclosure (wavelength 920-940nm; aperture diameter 1mm; 0.3NA)

[0054] In this embodiment of the invention, the miniature objective 07 consists of only two plano-convex lenses, featuring small size and light weight. By cooperating with the aforementioned scanning lens 04 and tube lens 05, astigmatism can be eliminated. Further, the working distance of the miniature objective 07 in this embodiment is 1.07 mm, the overall height of the objective is 2.5 mm, and the physical aperture of the optical lens near the focal end is 2.5 mm. The design excitation NA of the miniature objective 07 is 0.3, and the collection NA is 0.95. Further, the silicon photomultiplier tube 10 in this embodiment is directly placed behind the miniature objective 07 (without an additional focusing lens) to collect the two-photon fluorescence signal in situ and convert it into an electrical signal. Based on the detection optical path design strategy of combining the high-collection-aperture miniature objective 07 with the silicon photomultiplier tube 10, the miniature microscopy system in this embodiment has higher fluorescence detection efficiency; and compared with the method of collecting fluorescence signals using fiber bundles, the silicon photomultiplier tube 10 only requires a coaxial cable connection, greatly reducing the constraint of the cable on the free movement of the live sample.

[0055] It should be noted that the embodiments of the present invention only provide a configuration of the miniature objective 07, and are not a specific limitation on the miniature objective 07. Those skilled in the art can select the miniature objective according to the actual application, as long as it can achieve the function of the miniature objective in the embodiments of the present invention.

[0056] Figure 7 The image shows the actual optical resolution of the micro-microscopy system provided in this embodiment of the invention, measured using 500 nm fluorescent microspheres. In this embodiment, the actual excitation NA is 0.26, and the measured lateral resolution at the center field of view of the micro-microscopy system is 0.98 ± 0.01 μm, and the axial resolution is 83.84 ± 2.87 μm (n = 10). Compared with the imaging parameters of a Gaussian beam at the same NA (theoretical minimum lateral resolution 1.33 μm, theoretical minimum axial resolution 22.46 μm), using a Bessel beam can achieve better lateral resolution while extending the depth of field, thus realizing high-throughput, high-resolution volumetric imaging.

[0057] Figure 8 The image shown is a comparison of mouse cerebral cortex slices (approximately 80 μm thick, Thy1-GFP) observed by the miniature microscopy system provided in this embodiment of the invention and a benchtop two-photon microscopy system. Figure 8 The left image is the full field-of-view image and corresponding local detail area image obtained based on the embodiment of the present invention (excitation NA 0.26). The upper image has a field of view of 400×400μm. 2 Imaging frame rate 9Hz, average image of 100 frames in the same area; lower image: field of view 60×60μm 2 The imaging frame rate is 9Hz, and the average image of the same area is 100 frames. Figure 8 The image on the right shows the same region obtained using a commercial tabletop two-photon microscope system (excitation NA 0.3). Top image: field of view 400 × 400 μm. 2 The imaging frame rate was 1.9 Hz / layer, the z-axis scan step size was 3 μm, and the total imaging time was approximately 15 seconds. The lower image had a field of view of 60 × 60 μm, an imaging frame rate of 1.9 Hz / layer, a z-axis scan step size of 3 μm, and averaged images were acquired every 10 frames for the same number of layers, resulting in a total imaging time of approximately 176 seconds. Figure 8 As can be seen, compared with commercially available tabletop two-photon microscopy systems based on traditional Gaussian beam excitation, under similar excitation NA conditions, the micro-microscopy system provided in this embodiment of the invention can provide clearer detail information in neural imaging. Figure 8(As indicated by the middle arrow). Furthermore, regarding resolution, the embodiments of this invention, based on Bessel beam imaging, can provide higher lateral resolution (the embodiments of this invention have an NA of 0.26, resulting in a lateral resolution of 0.98 μm; commercial tabletop two-photon microscopy systems with an NA of 0.3 have a measured lateral resolution of approximately 1.34 μm). Regarding imaging speed, the embodiments of this invention utilize a Bessel beam, achieving volumetric imaging to a certain depth (approximately 80 μm) with a single scan. In contrast, commercial tabletop two-photon microscopy systems based on Gaussian beams require multiple layer-by-layer scans to achieve volumetric imaging. These tests demonstrate that the miniature microscopy system provided by the embodiments of this invention has higher resolution, greater imaging throughput, and superior imaging performance compared to tabletop two-photon microscopy systems equipped with similar NA objectives.

[0058] Figure 9a The image shows neuronal activity (GCaMP7f) in the anterior cingulate cortex (ACC) region of a freely moving mouse, observed using the micromicroscopic system provided in this embodiment of the invention. Figure 9a The top left image shows the standard deviation projection of a 5-minute observation of neuronal activity; the top right image shows 1015 neurons identified by the software; and the bottom image shows the activity curves of 8 representative neurons. For comparison, we used a desktop two-photon microscope (excitation NA 0.3) to image neurons in the same region while mice were awake and their heads were fixed. The imaging results are shown below. Figure 9b As shown. In Figure 9b In the middle, we respectively... Figure 9a The upper, middle, and lower layers of the imaging region (corresponding to depths of 145, 185, and 226 micrometers, respectively) were recorded. The figures show the average projected images and the corresponding cell counts for a 2-minute recording time. It can be seen that in a tabletop two-photon microscopy system with similar excitation NA, imaging on a single focal plane can only track approximately 200 cells; however, the micromicroscopy system provided in this embodiment of the invention can track over 1000 cells without sacrificing imaging speed, further verifying the advantage of the micromicroscopy system provided in this embodiment of the invention in terms of imaging throughput.

[0059] The design method and imaging system of the two-photon microscopy system based on Bessel beam excitation described in this application can also be applied to other nonlinear optical microscopy imaging, including but not limited to second harmonic and three-photon microscopy imaging. Its optical design concept is consistent with the two-photon microscopy imaging system claimed, requiring no inventive effort from those skilled in the art.

[0060] It should be noted that the embodiments of the present invention do not limit the type of the sample being tested, such as mice or other types of animals; and there are no restrictions on the observation area and the content of the observation (such as brain nerves, spinal nerves, etc. are all acceptable).

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications or equivalent substitutions made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A miniature two-photon microscopy imaging system based on Bessel beam excitation, comprising: A laser source, wherein the laser source is used for two-photon fluorescence excitation; A laser transmission fiber is used to couple and transmit the laser output from the laser. A miniature probe includes an excitation optical path and a collection optical path. The excitation optical path converts the laser transmitted by the laser transmission fiber into a Bessel focus and performs two-dimensional scanning and fluorescence excitation. The collection optical path collects the fluorescence. A photodetector is used to detect fluorescence signals excited by two photons.

2. The miniature two-photon microscopic imaging system based on Bessel beam excitation as described in claim 1, characterized in that... The laser transmission fiber is a large-mode-field photonic crystal fiber, and a microconical lens is processed on the output end face of the fiber, or the fiber and the microconical lens are combined to generate a Bessel beam.

3. The miniature two-photon microscopic imaging system based on Bessel beam excitation according to any one of claims 1-2, characterized in that... The laser source is a femtosecond laser, preferably with a built-in dispersion compensation module; more preferably, the wavelength of the femtosecond laser is 920nm.

4. The miniature two-photon microscopic imaging system based on Bessel beam excitation according to any one of claims 1-3, characterized in that... The excitation optical path includes a collimating lens, a scanning galvanometer, a scanning lens, a tube lens, a dichroic mirror, and a miniature objective lens; the collection optical path includes a miniature objective lens, a dichroic mirror, and a filter.

5. The miniature two-photon microscopic imaging system based on Bessel beam excitation according to any one of claims 1-4, characterized in that... In the excitation optical path, the scanning galvanometer scans the laser; the scanning lens and the tube lens are used to conjugate the beams scanned by the scanning galvanometer at different angles onto the back focal plane of the miniature objective; the dichroic mirror is used to separate the excitation optical path from the collection optical path; the miniature objective is used to focus the scanning beam onto the interior of the sample being tested, generating an axially extended Bezier focus on the focal plane to achieve volumetric imaging.

6. The miniature two-photon microscopic imaging system based on Bessel beam excitation according to any one of claims 1-5, characterized in that... The photodetector is a silicon photomultiplier tube.

7. The miniature two-photon microscopic imaging system based on Bessel beam excitation according to any one of claims 1-6, characterized in that... In the optical collection path, fluorescence is collected by a miniature objective lens, and after passing through a dichroic mirror and a filter, it is directly irradiated onto the detector for detection without the need for an additional lens for focusing.

8. The miniature two-photon microscopic imaging system based on Bessel beam excitation according to any one of claims 1-7, characterized in that... The astigmatism of the scanning lens and tube lens group in the excitation optical path compensates for the astigmatism of the miniature objective lens, and the overall astigmatism approaches zero.

9. A method for miniature two-photon microscopy imaging based on Bessel beam excitation, characterized in that... The laser emitted by the laser source is transmitted through the transmission fiber and the generated Bessel beam enters the miniature probe. It passes sequentially through the collimating lens, scanning galvanometer, scanning lens, tube lens, dichroic mirror and miniature objective lens. Bessel focusing is formed at the focal plane of the miniature objective lens, which excites the two-photon fluorescence signal of the sample. The fluorescence is collected by the miniature objective lens, and after passing through the dichroic mirror and filter, it is detected by the silicon photomultiplier tube.

10. The application of the miniature two-photon microscopy imaging system or method based on Bessel beam excitation as described in any one of claims 1-9 in neuronal activity imaging, particularly mouse neuronal activity imaging, and more particularly mouse neuronal calcium ion activity imaging.

11. The miniature two-photon microscopy imaging system based on Bessel beam excitation according to any one of claims 1-8 is a head-mounted miniature two-photon microscopy device for the study of freely moving mice or rats.

12. A miniature multiphoton microscopy imaging system based on Bessel beam excitation, comprising: A laser source, wherein the laser source is used for multiphoton fluorescence excitation; A laser transmission fiber is used to couple and transmit the laser output from the laser. A miniature probe includes an excitation optical path and a collection optical path. The excitation optical path converts a laser beam into a Bessel focus and performs two-dimensional scanning and fluorescence excitation. The collection optical path collects the fluorescence. A photodetector is used to detect the fluorescence signal excited by two photons.

13. A design method for a miniature multiphoton microscope based on Bessel beam excitation, characterized in that: The design method controls the overall astigmatism of the microscope to approach zero, and can still achieve diffraction-limited imaging effects even when other aberrations exist, including spherical aberration, coma, field curvature, and distortion.

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