Near-infrared light field three-dimensional imaging system and application

Abstract

The invention discloses a near-infrared light field three-dimensional imaging system and application, and the system is based on single-photon imaging and comprises an illumination light path and a light field imaging light path. The illumination light path generates an illumination light beam of a near-infrared band, and the illumination light beam is projected on a sample by adopting vertical illumination to excite fluorescence of the sample in a Rayleigh range of the illumination light beam; a light spot in the Rayleigh range of the illumination light beam is elliptical; a galvanometer is arranged in the illumination light path, and a tube lens is arranged between the galvanometer and the objective lens, so that an illumination light beam scans in an imaging view field in parallel to a main optical axis of the objective lens; the light field imaging light path is provided with a microlens array, collects fluorescence emitted by the sample, records the fluorescence direction to obtain light field information, and performs light field three-dimensional imaging on the sample. According to the invention, selective illumination is realized by shaping the illumination beam of the near-infrared band, the imaging quality, the imaging depth and the imaging speed of light field three-dimensional imaging are considered in combination with beam scanning, and the method is suitable for real-time three-dimensional imaging of a living body.

Description

Technical Field

[0001] The present invention belongs to the field of microscopic imaging, and more specifically, relates to a three-dimensional near-infrared light field imaging system and its application. Background Art

[0002] Three-dimensional dynamic observation of various physiological activities in vivo, such as brain nerve activities, immune responses, heartbeat, blood flow, etc., plays a crucial role in exploring life and understanding diseases. Physiological activities usually have the characteristics of rapid changes and signals originating from deep tissues. How to see deeply and quickly in living tissues to observe various physiological activities has always been the research direction in the field of biomedical imaging.

[0003] The light field microscopy technology developed in recent years can record the direction and position information of light simultaneously in a single exposure, achieving three-dimensional image information in one exposure and significantly improving the three-dimensional imaging speed. It shows great application potential in dynamically observing rapid physiological activities.

[0004] However, the existing light field imaging technology has high requirements for signal-to-noise ratio. The imaging targets are often limited to thin and transparent biological samples, and the signal detection depth is very limited. When migrated to the in vivo imaging scenario, usually only the signals on the tissue surface can be observed, and it cannot be directly used to observe the activities inside the organism. Currently, when the light field microscopy technology is applied to in vivo imaging, its imaging depth is insufficient to meet the requirements of physiological activity observation.

[0005] Sara Madaan et al. designed a light field microscopy system with single-objective selective illumination, which suppresses the background fluorescence outside the region of interest through light sheet selective illumination, thereby improving the contrast of three-dimensional light field imaging. Sara Madaan et al. provided two selective illumination beam field microscopy systems 1P-ASO-SVIM and 2P-ASO-SVIM with obvious advantages. Among them, 1P-ASO-SVIM, based on single-photon excitation, uses an inclined Gaussian light sheet for illumination and has good imaging speed. However, due to the limitation of the single-photon linear excitation form, the excitation light scattering is obvious, resulting in depth priority for 1P-ASO-SVIM; 2P-ASO-SVIM, based on two-photon excitation, uses an epi-illuminated Gaussian beam combined with near-infrared second-region excitation light to effectively improve the imaging depth. However, limited by the two-photon imaging principle, the imaging speed is slow.

[0006] Currently, for light field microscopy systems, it is still necessary to comprehensively improve the imaging quality, imaging depth, and imaging speed performance to meet the requirements of real-time three-dimensional in vivo imaging. Summary of the Invention

[0007] In view of the above deficiencies or improvement requirements of the prior art, the present invention provides a three-dimensional near-infrared light field imaging system and its application. The purpose is to compress the spot of the incident near-infrared band illumination beam into an ellipse, so that the light energy density of the illumination laser drops rapidly outside the Rayleigh range, increasing the imaging depth while reducing out-of-focus single-photon excitation, ensuring the imaging quality, and maintaining the speed advantage of light field imaging with the transverse scanning of the elliptical spot, taking into account the imaging quality, imaging depth, and imaging speed performance of three-dimensional light field imaging, and being suitable for real-time three-dimensional imaging of living bodies, thereby solving the technical problems of slow imaging speed and insufficient imaging depth in the existing three-dimensional imaging technology of biological samples.

[0008] To achieve the above object, according to one aspect of the present invention, there is provided a three-dimensional near-infrared light field imaging system. The system is based on single-photon imaging and includes an illumination optical path and a light field imaging optical path;

[0009] The illumination optical path generates an illumination beam in the near-infrared band, which is projected onto the sample by epi-illumination to excite the fluorescence of the sample within the Rayleigh range of the illumination beam; the spot within the Rayleigh range of the illumination beam is elliptical;

[0010] A galvanometer is provided in the illumination optical path, and a tube lens is provided between the galvanometer and the objective lens to scan the illumination beam parallel to the main optical axis of the objective lens within the imaging field of view;

[0011] The light field imaging optical path has a microlens array, which collects the fluorescence emitted by the sample and records the fluorescence direction to obtain light field information, and performs three-dimensional light field imaging on the sample.

[0012] Preferably, in the three-dimensional near-infrared light field imaging system, the Rayleigh range of the illumination beam is between 50 and 100 μm, the short axis of the elliptical spot within the Rayleigh range of the illumination beam is between 2 and 6 μm, and the long axis is adapted to the field of view width.

[0013] Preferably, in the three-dimensional near-infrared light field imaging system, the energy distribution of the illumination beam conforms to a Gaussian function or a Bessel function.

[0014] Preferably, in the three-dimensional near-infrared light field imaging system, the illumination optical path uses a laser light source in the second near-infrared region, with a wavelength between 800 and 1000 nm and an illumination power range of 50 - 200 mW.

[0015] Preferably, in the three-dimensional near-infrared light field imaging system, the illumination optical path includes a cylindrical lens for shaping the collimated laser spot into an ellipse, and the axis of the cylindrical lens is parallel to the rotation axis of the galvanometer, so that the illumination beam scans transversely along the short axis direction of the elliptical spot.

[0016] Preferably, in the near-infrared light field three-dimensional imaging system, the imaging optical path is a Fourier light field imaging optical path. There is a Fourier lens behind the microlens array, and the excited fluorescence forms a Fourier light field image through the microlens array and the Fourier lens; preferably, the depth of field of the microlens array matches the Rayleigh range of the illumination beam.

[0017] Preferably, in the near-infrared light field three-dimensional imaging system, the microlens array is composed of 9 microlenses, and the effective numerical aperture is between 0.1 and 0.2, generating a lateral resolution of 3 - 6 μm.

[0018] Preferably, in the near-infrared light field three-dimensional imaging system, the imaging optical path includes a wide-field imaging optical path, and the wide-field imaging optical path is confocal with the light field imaging optical path.

[0019] Preferably, in the near-infrared light field three-dimensional imaging system, the illumination optical path and the light field imaging optical path share a microscope objective. The illumination beam is incident on the sample through the microscope objective to excite fluorescence, and the microscope objective collects the excited fluorescence; the microscope objective is provided with a motion mechanism to drive the microscope objective to move along the depth direction.

[0020] According to another aspect of the present invention, an application of the near-infrared light field three-dimensional imaging system is provided, which is applied to in vivo sample imaging, especially brain tissue imaging.

[0021] Generally speaking, compared with the prior art by the above technical solutions conceived by the present invention, the following beneficial effects can be achieved:

[0022] The near-infrared light field three-dimensional imaging system provided by the present invention uses an illumination beam in the infrared band for incident illumination to reduce scattering with the increase of depth; by compressing the light spot into an ellipse, the light energy density within the Rayleigh range is increased, while the light energy density outside the Rayleigh range drops rapidly, thereby reducing out-of-focus excitation and improving the signal-to-noise ratio; combining the above characteristics, on the premise of ensuring the imaging quality, the imaging depth of single-photon imaging is greatly improved, reaching more than 200 μm, and up to 500 μm at most, several times that of the current light field imaging depth. Further, combined with the light spot scanning technology, the imaging speed advantage of light field imaging is maintained, and the performance of imaging quality, imaging depth and imaging speed is integrated, making it a more ideal in vivo sample imaging system, especially for applications such as in vivo observation of brain tissue that require high-quality, fast three-dimensional imaging with depth. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the imaging principle diagram of the near-infrared light field three-dimensional imaging system provided by the present invention;

[0024] Figure 2 is the schematic diagram of the optical path structure of the near-infrared light field three-dimensional imaging system provided in Embodiment 1 of the present invention;

[0025] Figure 3 are the light field image and the reconstructed three-dimensional image provided by Embodiment 2 of the present invention, where Figure 3 A is the light field image collected by the microlens array, Figure 3 B is the reconstructed three-dimensional image;

[0026] Figure 4 is the coupled picture of nerves and blood vessels provided by Embodiment 2 of the present invention, where Figure 4 A is the light field image of the cerebral artery blood vessels of a mouse in a resting state, Figure 4 B is the light field image of the cerebral artery blood vessels of a mouse after tactile stimulation.

[0027] In all the drawings, the same reference numerals are used to represent the same elements or structures, where: 1 is a laser, 2 is a pinhole, 3 is a collimator, 4 is a first lens, 5 is a second lens, 6 is a cylindrical lens, 7 is a first achromatic lens, 8 is a scanning mirror, 9 is a second achromatic lens, 10 is a dichroic mirror, 11 is a tube lens, 12 is a microscopic objective lens, 13 is an electric displacement stage, 14 is a doublet lens, 15 is a microlens array, 16 is a Fourier lens, 17 is a camera, and 18 is a host computer. Detailed implementation manners

[0028] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0029] In in-vivo imaging applications, there are three reasons affecting the signal-to-noise ratio of light field microscopy technology: First, regarding the illumination beam: The illumination beam is scattered and absorbed by tissues. Biological tissue components such as hemoglobin have strong absorption of visible light, making the energy of the incident light significantly attenuate with depth. In addition, the sub-micron scale structures existing in biological tissues are comparable to the visible light wavelength, resulting in multiple scattering of light in tissues, making the path of light become random, further causing the energy of the incident light to attenuate with the increase of tissue depth, and unable to meet the high signal-to-noise ratio illumination required for light field imaging. Second, regarding the excitation fluorescence: Even if deep tissues are illuminated, the excited signal fluorescence is relatively weak and cannot penetrate biological tissues again to be effectively received by the camera. And simply relying on increasing the intensity of the illumination beam to increase the fluorescence intensity excited by deep tissues, since light field imaging uses epi-illumination, the out-of-focus fluorescence signals excited by shallow and deeper tissues increase in proportion, and the fluorescence signals excited by deep tissues are submerged in the out-of-focus signals. It is impossible to effectively improve the signal-to-noise ratio while increasing the phototoxicity and photobleaching phenomena.

[0030] For the selective illumination beam field imaging technology, to improve the signal-to-noise ratio, either single-photon excitation is adopted to maintain the imaging speed of the light field imaging, which limits the further increase of the imaging depth, or two-photon nonlinear excitation is adopted to increase the imaging depth, but the imaging speed advantage of the light field imaging technology is lost, which is still not ideal for in vivo biological sample imaging.

[0031] The present invention selects a near-infrared band laser as the illumination beam source to improve the penetration ability of the illumination beam into biological tissues. Based on single-photon linear excitation, the incident light spot is shaped into an ellipse, and the energy of the illumination beam is concentrated within the Rayleigh range of the light sheet, effectively exciting the fluorescence signal. While outside the Rayleigh range, the energy density of the illumination beam rapidly decays, reducing the out-of-focus signals in the shallow and deeper layers in the sample depth direction, thereby improving the signal-to-noise ratio of the fluorescence signal and effectively improving the light field imaging quality of deep tissue samples without sacrificing the increase of light toxicity or the aggravation of photobleaching phenomenon in shallow sample tissues. Combining with the illumination beam scanning technology parallel to the main optical axis, while effectively improving the imaging signal-to-noise ratio using the selective illumination beam field imaging technology, it overcomes the depth limitation of single-photon imaging and takes into account the imaging speed, making it a more suitable in vivo imaging system for biological samples.

[0032] Specifically, the near-infrared light field three-dimensional imaging system provided by the present invention includes an illumination optical path and a light field imaging optical path;

[0033] The illumination optical path generates an illumination beam in the near-infrared band and projects it onto the sample by epi-illumination to excite the fluorescence of the sample within the Rayleigh range of the illumination beam; the light spot within the Rayleigh range of the illumination beam is elliptical; the minor axis of the elliptical light spot within the Rayleigh range of the illumination beam is between 2 - 6 μm, and the major axis is adapted to the field of view width. Near-infrared has good penetration ability into biological tissues, and single-photon excitation still scatters along the depth direction. The present invention compresses the beam energy along the minor axis direction through the elliptical light spot, improving the energy density of the illumination area without increasing the power of the laser light source, while the light energy density rapidly decreases outside the Rayleigh range, thereby reducing out-of-focus excitation and achieving imaging quality not inferior to two-photon excitation.

[0034] A galvanometer is provided in the illumination optical path, and a tube lens is provided between the galvanometer and the objective lens to scan the illumination beam parallel to the main optical axis of the objective lens within the imaging field of view. The light spot shaping technology of the present invention combined with light spot scanning improves the imaging speed, which is mainly limited by the acquisition rate of the camera, maintaining the speed advantage of light field imaging.

[0035] Such as Figure 1As shown in the figure; the illumination optical path uses a laser light source with a wavelength band in the near-infrared band, with a wavelength between 800 - 1000 nm, preferably 808 nm, and the illumination power range is between 50 - 200 mW, preferably 160 mW. To overcome the problems of light scattering and light absorption of visible light in in-vivo imaging, through extensive research, it is found that near-infrared light (650 - 2500 nm) scatters less in biological tissues, and photons can propagate deeper. Existing research has shown that under the excitation of a near-infrared light source, due to the red-shift characteristic of fluorescence, the emitted fluorescence is in the second near-infrared band, and the penetration depth in biological tissues can reach millimeters or even centimeters, meeting the in-vivo imaging scenario, so it is selected as the illumination light beam source for this patent.

[0036] However, simply increasing the light source intensity will enhance the single-photon imaging scattering with depth, increase the out-of-focus excitation on the surface, and reduce the imaging quality. A cylindrical lens is set in the illumination optical path to shape the collimated laser spot into an ellipse, compress the spot to increase the light energy density, reduce the fluorescence excitation outside the Rayleigh range, and at the same time weaken the degree of photobleaching and reduce the phototoxicity. The Rayleigh range of the illumination light beam is between 50 - 100 μm, the short axis of the elliptical spot within the Rayleigh range of the illumination light beam is between 2 - 6 μm, the long axis is adapted to the field of view width, and with the above-mentioned near-infrared band light source, the light energy density is controlled within the area with a maximum depth of 500 μm in biological tissues, having good fluorescence excitation efficiency.

[0037] In the preferred scheme, the energy distribution of the illumination light beam conforms to the Gaussian function or the Bessel function; among them, the Rayleigh range of the illumination light beam is 69 μm, and experiments prove that it can maximize the elimination of crosstalk within a depth of field of 200 μm, while maintaining the signal intensity in the region of interest. A 25x microscope objective lens is preferably used to compress the short axis of the elliptical spot to 3 μm, which is smaller than the diameter of a single red blood cell in a blood vessel, avoiding the reduction in resolution caused by selective illumination. Select the energy distribution type of the illumination light beam according to the imaging scenario. When the imaging area is relatively fine, select an illumination light beam with an energy distribution conforming to the Gaussian function, which is characterized by being relatively simple to generate and control, being able to focus and scan the sample well, being able to provide a relatively precise focus point, and being suitable for imaging of small structures such as cells and molecules. When imaging deeper tissues, preferably select an illumination light beam with an energy distribution conforming to the Bessel function, which is characterized by having better penetration and anti-scattering capabilities, but being relatively complex to generate and control and having relatively low resolution.

[0038] To overcome the problem of weak deep signals, selective illumination technology is adopted instead of wide-field epi-illumination. By shaping the laser beam into an ellipse and combining a scanning galvanometer, area illumination is achieved, and only the fluorescence signal at the imaging plane in the tissue is excited. Compared with oblique light sheet illumination, the energy is more concentrated, effectively reducing the light excitation and background signals of the upper and lower layers of the sample, greatly improving the imaging quality and the effective imaging depth. Compared with two-photon excitation selective illumination systems, the selective illumination system maintains the imaging speed advantage of light field imaging. In addition, since selective illumination only excites fluorescence in a thin layer region inside the sample, it reduces photobleaching and phototoxicity and minimizes damage to the sample.

[0039] In some embodiments, a cylindrical lens is used for laser shaping. In its illumination optical path, a cylindrical lens is provided to shape the collimated laser spot into an ellipse.

[0040] In a preferred embodiment, the axis of the cylindrical lens is parallel to the rotation axis of the galvanometer, so that the illumination beam scans laterally along the short axis direction of the elliptical spot. The lateral scanning range matches the field of view of the camera in the imaging optical path. During one cycle of the illumination beam scanning, time-integrated exposure imaging is performed to obtain a two-dimensional light field image of the three-dimensional sample within the camera's field of view.

[0041] The light field imaging optical path has a microlens array that collects the fluorescence emitted by the sample and records the fluorescence direction to obtain light field information, and performs three-dimensional light field imaging on the sample. The imaging optical path is a Fourier light field imaging optical path. There is a Fourier lens behind the microlens array, and the excited fluorescence forms a Fourier light field image through the microlens array and the Fourier lens. To achieve large-depth-of-field and fast in-vivo imaging, the Fourier light field mode is selected to collect effective signals. Fourier light field imaging decouples the angle and position using frequency domain information, so a three-dimensional image with a large depth of field can be reconstructed from a two-dimensional image through algorithms. For scenes with a large defocus, Fourier light field imaging can capture clearer images than spatial light field imaging. In addition, the Fourier lens behind the microlens array effectively separates the high-frequency information and low-frequency information of the signal. The low-frequency information containing the effective signal can be easily separated from the high-frequency noise, and the final imaging result is less affected by noise interference. In contrast, the ray splitting introduced by the microlens in spatial light field imaging further reduces the signal-to-noise ratio. Therefore, in low-light conditions or high-tissue-scattering imaging, Fourier light field imaging can better maintain the image quality.

[0042] In a preferred embodiment, the depth of field of the microlens array matches the Rayleigh range of the illumination beam, maximizing the imaging efficiency.

[0043] In a preferred embodiment, the effective numerical aperture of the microlens array is 0.14, generating a lateral resolution of approximately 4.27 μm, which can resolve individual red blood cells and matches the application scenario of in-vivo tissue vascular imaging.

[0044] Preferably, the imaging optical path includes a wide-field imaging optical path, which is confocal with the light-field imaging optical path, that is, the focal plane positions are the same. The same camera is used for imaging, and the imaging optical path can be switched by flipping a reflecting mirror. The illumination optical path shares a microscope objective with the light-field imaging optical path. The illumination beam is incident on the sample through the microscope objective to excite fluorescence, and the microscope objective collects the excited fluorescence. The microscope objective is provided with a motion mechanism to drive the microscope objective to move in the depth direction. In the experiment, due to the depth of field of the light-field imaging system being 200 μm, it is difficult to focus the objective lens at the center of the depth of field during actual imaging. Therefore, a parfocal wide-field imaging optical path is built. Since the depth of field of the wide-field imaging optical path is very shallow, during actual imaging, it is only necessary to first focus the wide-field imaging optical path at the depth of interest, and then switch to the light-field imaging optical path to collect light-field images.

[0045] The near-infrared light-field three-dimensional imaging system provided by the present invention is applied to imaging of living samples, with an imaging depth exceeding 200 μm and a maximum imaging depth reaching 500 μm, which is several times that of ordinary light-field imaging systems.

[0046] The near-infrared light-field three-dimensional imaging system provided by the present invention is applied to imaging of living samples, especially brain tissue imaging. By scanning three times along the Z-axis direction, it can provide three-dimensional image information of a living tissue sample with a depth of up to 500 μm, which is about 10 times the imaging depth of ordinary light-field imaging systems. It has the imaging characteristics of ordinary light-field three-dimensional imaging, realizes three-dimensional imaging in a single acquisition, is suitable for studying the real-time dynamic changes of the structure / function of animals in physiological / pathological states, and eliminates imaging time differences.

[0047] The following are examples:

[0048] Example 1

[0049] The near-infrared light-field three-dimensional imaging system provided in this example, as Figure 2 shown, includes an illumination optical path and a light-field imaging optical path;

[0050] The illumination optical path includes three modules: a light source and its adjustment device, an illumination beam generation device, and an illumination beam scanning device:

[0051] Module 1: Light source and its adjustment device

[0052] The light source can be a narrow-band laser or a laser diode. The spot type is preferably a Gaussian beam, and the wavelength of the laser light source is 808 nm. The beam emitted from the laser can pass through a coupler, a collimator, a convex lens, a pinhole, a filter, etc. to achieve adjustment of the light intensity, adjustment of the beam size, shaping, etc.

[0053] Module 2: Illumination beam generation device

[0054] The collimated and expanded laser passes through a cylindrical lens and is compressed into an elliptical light spot. According to the principle of light spot shaping by a cylindrical mirror, the diameter of the incident beam and the focal length of the lens are changed to generate fundamental mode Gaussian light with different characteristics, so that its Rayleigh range matches the reconstruction depth, thereby avoiding the excitation of defocus signals. The short axis of the elliptical light spot within the Rayleigh range of the illumination beam is between 2 - 6 μm, and the long axis is adapted to the field of view width.

[0055] The fundamental mode laser beam is scanned horizontally by a galvanometer mirror to form a three-dimensional illumination area, which is focused by a microscope objective lens to form an illumination beam. The amplitude and frequency of the scanning galvanometer mirror can be driven and controlled by a signal generator to achieve fast volume illumination of different imaging scenarios. Compared with traditional epi-illumination, only the part of the sample illuminated by the Rayleigh range of the elliptical beam will be excited. Therefore, selective illumination can eliminate out-of-focus light pollution and improve the signal-to-background ratio of the imaging system.

[0056] Module 3: Illumination beam scanning device

[0057] To further achieve scanning illumination at different depths, a motion control system is designed to precisely control the movement of the objective lens along the depth direction. The components of the motion control system are a brushless DC servo controller and a supporting electric displacement stage. The microscope objective lens is installed on the electric displacement stage and can achieve axial displacement, and the minimum displacement interval is the axial resolution of the system. The entire system is controlled and triggered by LabVIEW to achieve the coordination between the objective lens displacement and the signal detection end.

[0058] In this embodiment, the light source is a narrow-band laser. The wavelength of the laser light source is preferably 808 nm, the power range is 0 - 200 mW, the spot type is an elliptical Gaussian beam, the Rayleigh range is 69 μm, the short axis of the elliptical spot is 3 μm, and the long axis is adapted to the field of view width. The beam emitted from the laser 1 is collimated and corrected through the pinhole 2 and the collimator 3. The laser after collimation and beam expansion is adjusted in spot diameter by the 4f system composed of the first lens 4 and the second lens 5, and then is compressed into an ellipse by the cylindrical lens 6. Then, the ellipse is coupled to the rear focal plane of the microscope objective through the first achromatic lens 7 and the second achromatic lens 9 of the achromatic lens group. A scanning mirror 8 is placed between these two lenses to scan the elliptical laser beam in the y direction to form a uniform volume illumination beam. After passing through the dichroic mirror 10, the tube lens 11 and the microscope objective 12, it irradiates into the sample volume. The tube lens 11 scans the illumination beam parallel to the main optical axis of the objective within the imaging field of view. The amplitude and frequency of the scanning galvanometer can be driven and controlled by a signal generator to achieve volume illumination of different regions of interest. To quickly illuminate the light field images at different depths, a motion control system is designed to precisely control the movement of the objective along the depth direction. The components of the motion control system are a brushless DC servo controller and a supporting electric displacement stage 13. The microscope objective is mounted on the electric displacement stage, which can achieve an axial displacement of 25 mm, and the minimum displacement interval is 0.2 μm. Through the control trigger of LabVIEW, the coordination between the objective displacement and the signal detection end is achieved.

[0059] Signal detection and acquisition: The imaging optical path consists of a light field imaging optical path and a wide-field imaging optical path. Since the light field imaging has a large depth of field, during actual imaging, the wide-field imaging optical path needs to be focused on the depth of interest first, and then switched to the light field imaging optical path for light field image acquisition. By switching the states of two flip mirrors, the two imaging modalities can be flexibly switched. Considering the volume resolution and depth of field comprehensively, a microlens array suitable for the near-infrared wavelength is designed and processed, and the Fourier light field imaging modality is preferably used to meet the large depth-of-field requirements of in vivo imaging. After the sample is focused, a signal detection device such as a CCD, EMCCD, CMOS, or sCMOS can be used to collect the near-infrared fluorescence signal, and the ImageJ software is used to process the obtained image data in real time to provide a visualization effect.

[0060] The imaging optical path consists of a light field imaging optical path and a wide-field imaging optical path, and the wide-field imaging optical path is confocal with the light field imaging optical path. The wide-field imaging optical path is used for accurate focusing, and then the optical path is switched to the light field imaging optical path for light field image acquisition. The fluorescence excited by the illumination beam is collected by the objective, passed through the tube lens and the doublet lens 14 and relayed to the surface of the microlens array 15 to form a light field signal, realizing the dimensionality reduction modulation of the three-dimensional fluorescence signal. After being transformed by the Fourier lens 16, it is collected by the near-infrared camera 17, and the data processing is executed by the host computer 18.

[0061] The depth of field of the Fourier light field matches the Rayleigh range of the illumination beam, maximizing the imaging efficiency.

[0062] The excited fluorescence passes back through the microlens array and the Fourier lens to form a Fourier light field image, which is recorded by the area array detector as f. Mathematically, there is a relationship f = Hg, where the vector f represents the light field image, the vector g is the three-dimensional image to be reconstructed, and H is the matrix generated by forward modeling of the imaging process. The coefficients of H largely depend on the point spread function of the light field microscope. The analytical model of a point source in the image plane of the microlens array in space can be calculated using the scalar Debye theory, and then the light propagation process from the microlens array to the sensor can be calculated according to the Fresnel propagation theory. The final matrix is the point spread function H of the light field. With the help of Matlab software, the point spread function of the designed optical path is simulated using the wave optics model. The simulation results are three-dimensionally deconvolved with f recorded by the area array detector to obtain the three-dimensional image.

[0063] The near-infrared light field three-dimensional imaging system provided in this embodiment has an imaging field of view of 550*550 μm, a lateral resolution of about 4.27 μm, a maximum imaging depth of 500 μm, and a maximum imaging rate of up to 20 Hz. This system can capture the dynamic changes of tissues in real time. For in vivo imaging, especially for neuronal activities, blood flow changes, or pathological changes, etc., it provides detailed information on the dynamic changes. At the same time, it can reveal the relationship between structural changes and functional activities, which helps in the in-depth understanding of disease mechanisms and the exploration of treatment methods.

[0064] Example 2: In vivo imaging of blood vessels and nerves in the cerebral cortex of mice

[0065] In this example, mice were used as experimental subjects, and the near-infrared light field three-dimensional imaging system provided in Example 1 was used for imaging to conduct an in vivo imaging experiment of blood vessels in the cerebral cortex and analyze the three-dimensional imaging results.

[0066] 8-week-old mice were used, anesthetized with isoflurane during the operation, and fixed on a stereotactic device after being fully sedated. A craniotomy was performed using a cranial drill, and a cover glass was covered after removing the cranial bone piece for observation. A near-infrared probe was injected into the mice to label the blood vessels in the brain. After focusing in the wide-field optical path, it was switched to the light field optical path for exposure imaging. The exposure time was set to 200 ms, and the three-dimensional imaging volume was set to a cylinder with a depth of 200 μm and a diameter of 550 μm. The sample was excited by the illumination beam, and 9 light field images were formed after passing through the microlens array, as shown in Figure 3 A, and relayed to the photosensitive surface of the imaging camera by a pair of lenses. For details, see Figure 3 . Image reconstruction was achieved using a three-dimensional reconstruction algorithm, and a three-dimensional image with a large depth of field and high resolution was obtained. For details, see Figure 3 B.

[0067] Using a near-infrared light field microscopy device, we restored the out-of-focus and blurred structures that were lost under visible light wide-field microscopy or light field microscopy. The high spatial resolution enabled us to observe brain blood vessels at the micron scale, and the large depth of field allowed us to observe three-dimensional structures of 200 μm in a single exposure, with the total imaging depth of field reaching 500 μm.

[0068] The near-infrared light field three-dimensional imaging system provided in Example 1 has the ability to resist scattering and a large imaging depth of field, allowing for the observation of finer three-dimensional structural changes.

[0069] Functional hyperemia of mouse cerebral blood vessels is a homeostatic response. The local increase in blood flow caused by neuronal activity is one of several basic processes to ensure that the brain obtains sufficient glucose and oxygen under different physiological conditions. Functional hyperemia is mediated by neurovascular coupling, which is a signaling mechanism that links an increase in neuronal activity to the dilation of nearby blood vessels. Studies have shown that after the mouse whiskers are stimulated, the cerebral cortex will release glutamate and the vasoactive product prostaglandin to induce the dilation of neurovessels. We observed this neurovascular coupling phenomenon through a cranial window. Figure 4 A is the light field image of the first frame, which is the light field image of the mouse in a resting state. Figure 4 B is the light field image after touching the whiskers of an awake mouse. It can be observed that the diameter of the arterial blood vessels in the corresponding brain region increases and the blood flow increases, which is in line with the physiological phenomenon description of relevant studies.

[0070] Use transgenic mice expressing calcium indicators (such as GCaMP6), or transfer calcium indicators into the target area of the mouse brain through viral vectors, and use a head fixation device to reduce the impact of mouse movement on imaging quality during the experiment. Image for a period of time before the stimulation to record the baseline activity level of neurons. Subsequently, apply appropriate stimuli (such as visual, auditory, olfactory, etc.) to the mouse, and record the changes in neuronal calcium signals before and after the stimulation. Use a light field microscope to collect three-dimensional calcium imaging data to capture the rapid response of neurons under stimulation. Use a three-dimensional reconstruction algorithm to achieve image reconstruction, and analyze the dynamic changes of calcium signals of each neuron through the three-dimensionally reconstructed image. Machine learning or automated software can be combined to locate neurons and track their activities.

[0071] Example 3 uses a Bessel illumination beam to achieve in vivo imaging of blood vessels in the mouse cerebral cortex.

[0072] The near-infrared light field three-dimensional imaging system provided in this example is different from that in Example 1 in that: a phase plate is used to generate a Bessel illumination beam. After being focused by a scanning galvanometer and a microscope objective, a Bessel illumination beam is formed for epi-illumination. The specific imaging process is the same as that in Example 2. Using the Bessel illumination beam for in vivo imaging of the mouse cerebral cortex can provide high-depth penetration, strong anti-scattering ability, and low imaging distortion, and is also suitable for in vivo brain imaging.

[0073] Those skilled in the art can easily understand that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A near-infrared light field three-dimensional imaging system, characterized in that: The system is based on single-photon imaging, including an illumination optical path and a light field imaging optical path; The illumination light path generates an illumination light beam in the near-infrared band, which is projected onto the sample using epi-illumination to excite the fluorescence of the sample within the Rayleigh range of the illumination light beam; the light spot within the Rayleigh range of the illumination light beam is elliptical; A galvanometer is provided in the illumination light path, and a tube lens is provided between the galvanometer and the objective lens, so that the illumination light beam is scanned parallel to the principal optical axis of the objective lens in the imaging field of view; The light field imaging optical path has a microlens array, which collects the fluorescence emitted by the sample and records the direction of the fluorescence to obtain light field information, and performs light field three-dimensional imaging of the sample.

2. The near-infrared light field three-dimensional imaging system according to claim 1, characterized in that: The Rayleigh range of the illumination light beam is between 50 and 100 μm, the short axis of the elliptical light spot within the Rayleigh range of the illumination light beam is between 2 and 6 μm, and the long axis is adapted to the width of the field of view.

3. The near-infrared light field three-dimensional imaging system according to claim 1, characterized in that: The energy distribution of the illumination light beam conforms to a Gaussian function or a Bessel function.

4. The near-infrared light field three-dimensional imaging system according to claim 1, characterized in that: The illumination light path adopts a laser light source with a wavelength in the near-infrared region II, a wavelength between 800-1000nm, and an illumination power range of 50200mW.

5. The near-infrared light field three-dimensional imaging system according to claim 1, characterized in that: The illumination light path includes a cylindrical lens for shaping the collimated laser spot into an ellipse. The axial direction of the cylindrical lens is parallel to the rotation axis of the galvanometer, so that the illumination light beam is horizontally scanned along the short axis direction of the ellipse spot.

6. The near-infrared light field three-dimensional imaging system according to claim 1, characterized in that: The imaging optical path is a Fourier light field imaging optical path, a Fourier lens is provided at the rear side of the microlens array, and the excited fluorescence forms a Fourier light field image through the microlens array and the Fourier lens; preferably, the depth of field of the microlens array matches the Rayleigh range of the illumination light beam.

7. The near-infrared light field three-dimensional imaging system according to claim 5, characterized in that: The microlens array consists of 9 microlenses with an effective numerical aperture between 0.1 and 0.2, resulting in a lateral resolution of 3 to 6 μm.

8. The near-infrared light field three-dimensional imaging system according to claim 1, characterized in that: The imaging optical path comprises a wide-field imaging optical path, and the wide-field imaging optical path is confocal with the light-field imaging optical path.

9. The near-infrared light field three-dimensional imaging system according to claim 1, characterized in that: The illumination light path and the light field imaging light path share a microscope objective lens, the illumination light beam is incident on the sample through the microscope objective lens to excite fluorescence, and the microscope objective lens collects the excited fluorescence; the microscope objective lens is provided with a motion mechanism to drive the microscope objective lens to move along the depth direction.

10. Application of the near-infrared light field three-dimensional imaging system according to any one of claims 1 to 9, which is applied to imaging of living samples, especially brain tissue imaging.

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