Confocal light field microscope

Abstract

The invention provides a confocal light field microscope, which comprises an objective lens, a dichroic mirror, a microlens array positioned on a conjugate rear pupil plane of the objective lens, a mask positioned on a rear focal plane of the microlens array, a double-sided scanning galvanometer and a reflector group, the double-sided scanning galvanometer comprises a first reflecting surface and a second reflecting surface which are respectively positioned on the front and back surfaces, the first reflecting surface is used for reflecting the exciting light introduced by the dichroic mirror to an imaging object in front of the objective lens, and the reflecting mirror group reflects the excited exciting light to the second reflecting surface so as to be reflected to the photosensitive element through the second reflecting surface; wherein the angle of the double-sided scanning galvanometer is changed, so that the exciting light scans the imaging object through the first reflecting surface and the excited light enters the photosensitive element through the second reflecting surface. According to the confocal light field microscope provided by the invention, the influence of scanning distortion can be eliminated during scanning through a double-sided scanning mechanism, and a series of problems caused by a synchronization error of two times of scanning are avoided.

Description

Technical Field

[0001] The invention relates to the field of optical imaging, and in particular to a confocal light field microscope. Background Art

[0002] A major goal of neuroscience is to explore the relationship between brain neural network activity and animal behavioral states. Recording a wide range of neural network activity in awake animals can better help us understand the mechanisms by which the animal brain implements various complex functions. However, traditional optical imaging techniques (widefield microscopy, two-photon microscopy) are limited in recording high-throughput neural activity in awake animals. The instantaneous imaging flux is low, and it is difficult to achieve high-speed imaging and large-scale imaging at the same time. Light-field microscopy utilizes a highly parallelized imaging acquisition mechanism. In a single exposure moment of the camera, it can simultaneously capture light signals at different depths, reconstruct the entire three-dimensional volume through a deconvolution algorithm, and achieve rapid volume imaging, thereby significantly improving the imaging flux. Therefore, light-field microscopy has great application potential, and its development will help promote research in the field of neuroscience.

[0003] In the prior art, light field imaging technology is mainly divided into traditional light field microscopy and Fourier light field microscopy according to differences in optical design.

[0004] Please refer to Figure 1A-1B , Figure 1A shows a schematic diagram of the optical path of a conventional light-field microscope. Figure 1B A schematic diagram of the light path of a Fourier light-field microscope is shown.

[0005] like Figure 1A As shown, the conventional light field microscope includes a sample 110, an objective lens 120, a lens 130, a microlens array 140 and a camera 150. By adding a set of microlens arrays 140 on the image plane of the wide field microscope and moving the camera 150 to the focal plane of the microlens array 140, the camera 150 records the array combination of the rear pupil image formed by each microlens. In the rear pupil image formed, the brightness of each pixel represents the superposition of each light passing through the corresponding microlens and the pixel. After imaging, all the light in the light field can be superimposed according to the light position and angle to reconstruct the original three-dimensional body.

[0006] like Figure 1BAs shown, the Fourier light field microscope includes a sample 110, an objective lens 120, a lens 131, a lens 132, a microlens array 140, and a camera 150. The microlens array 140 is placed on the rear pupil plane of the objective lens 120, and the camera 150 is at the focal plane of the microlens array 140. Each microlens collects light from a specific viewing angle of the sample, and then forms an image on the camera 150. Here, the specific angle depends on the position of each microlens on the rear pupil of the objective lens 120. The sub-image formed by each microlens on the camera 150 represents the projection view of the three-dimensional body generated from different angles. Therefore, the algorithm related to projection tomography can reconstruct the three-dimensional body from these projection views.

[0007] The light field imaging technology provided by the prior art has the problems of reconstruction distortion and excessive calculation. Through the new eXtended field-of-view Light Field Microscopy (XLFM), that is, the confocal light field microscope, the wide field illumination used in the traditional light field microscope is changed to sheet illumination. Then, through a specially designed slit, a part of the sample is selectively excited. At the same time, the collected signal is also filtered out of the imaging range by the slit, and the volume imaging is realized by scanning in the vertical direction of the illumination. The confocal light field microscope effectively filters out the background noise without sacrificing the volume imaging speed, greatly improves the sensitivity and resolution, and has the ability of optical sectioning and imaging in thick tissues. However, when the confocal light field microscope is scanned at a high speed (for example, when the scanning speed is greater than 100Hz), the synchronization error of the two scanning galvanometers included in the confocal light field microscope becomes difficult to ignore. This imaging method will cause image distortion and it is difficult to correct it later.

[0008] In order to overcome the above-mentioned defects of the prior art, there is an urgent need in the art for a confocal light field microscope that can eliminate the influence of scanning distortion during scanning and avoid a series of problems caused by synchronization errors between two scans. Summary of the invention

[0009] A brief summary of one or more aspects is given below to provide a basic understanding of these aspects. This summary is not an exhaustive overview of all conceived aspects, and is neither intended to identify the key or critical elements of all aspects nor to define the scope of any or all aspects. Its only purpose is to give some concepts of one or more aspects in a simplified form as a prelude to a more detailed description that will be given later.

[0010] In order to overcome the above-mentioned defects in the prior art, the present invention provides a confocal light field microscope, which can eliminate the influence of scanning distortion during scanning through a double-sided scanning mechanism and avoid a series of problems caused by the synchronization error of the two scans.

[0011] Specifically, the above-mentioned confocal light field microscope provided according to the first aspect of the present invention includes: an objective lens, a color separation mirror, and a microlens array located at the conjugate rear pupil plane of the objective lens and a mask located at the rear focal plane of the microlens array, characterized in that the confocal light field microscope also includes a double-sided scanning galvanometer and a reflection mirror group, the double-sided scanning galvanometer includes a first reflection surface and a second reflection surface respectively located on the front and back sides, the first reflection surface is used to reflect the excitation light introduced by the color separation mirror to the imaging object in front of the objective lens, and the reflection mirror group reflects the stimulated light to the second reflection surface to reflect it to the photosensitive element through the second reflection surface, wherein the double-sided scanning galvanometer changes the angle so that the excitation light scans the imaging object via the first reflection surface and at the same time the stimulated light scans into the photosensitive element via the second reflection surface.

[0012] Preferably, in one embodiment of the present invention, the reflector group includes a first reflector and a second reflector, the double-sided scanning galvanometer is located at the conjugate rear pupil plane of the objective lens, and a first light path is formed from the objective lens to the first reflective surface of the double-sided scanning galvanometer; a second light path is formed from the first reflective surface of the double-sided scanning galvanometer to the first reflector; a third light path is formed from the second reflective surface of the double-sided scanning galvanometer to the second reflective surface of the double-sided scanning galvanometer, and the second reflector reflects the stimulated light from the first reflector to the second reflective surface of the double-sided scanning galvanometer via the third light path; a fourth light path is formed from the second reflective surface of the double-sided scanning galvanometer to a photosensitive element; the stimulated light emitted by the imaging object after being excited by the excitation light is collected and imaged by the photosensitive element via the first light path, the second light path, the third light path and the fourth light path.

[0013] Preferably, in an embodiment of the present invention, the first light path is perpendicular to the second light path, the first light path is parallel to the third light path, and the third light path is perpendicular to the fourth light path.

[0014] Preferably, in an embodiment of the present invention, the mask plate comprises a plurality of slits, and positions of the plurality of slits correspond one-to-one to positions of a plurality of microlenses on the microlens array.

[0015] Preferably, in one embodiment of the present invention, it further includes a first telecentric lens and a second telecentric lens, the first telecentric lens is located in the third optical path, and the second reflective surface of the double-sided scanning galvanometer is located at the back focal plane of the first telecentric lens; the second telecentric lens is located in the fourth optical path, and the second reflective surface of the double-sided scanning galvanometer is located at the back focal plane of the second telecentric lens.

[0016] Preferably, in an embodiment of the present invention, it further comprises a first relay lens and a second relay lens, wherein the first relay lens and the second relay lens are located in the first optical path.

[0017] Preferably, in an embodiment of the present invention, a third relay lens and a fourth relay lens are further included, the third relay lens is located in the second optical path, and the fourth relay lens is located between the first reflector and the second reflector.

[0018] Preferably, in an embodiment of the present invention, the dichroic mirror is located between the third relay lens and the fourth relay lens.

[0019] Preferably, in an embodiment of the present invention, the dichroic mirror is located between the first reflection surface of the double-sided scanning galvanometer mirror and the third relay lens.

[0020] Preferably, in an embodiment of the present invention, the excitation light forms a linear light sheet via a cylindrical lens and a beam expander lens group. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above features and advantages of the present invention can be better understood after reading the detailed description of the embodiments of the present disclosure in conjunction with the following drawings. In the drawings, the components are not necessarily drawn to scale, and components with similar related properties or features may have the same or similar reference numerals.

[0022] Figure 1A shows a schematic diagram of the optical path of a conventional light-field microscope;

[0023] Figure 1B A schematic diagram of the optical path of a Fourier light field microscope is shown;

[0024] Figure 2A A light path diagram of a confocal light-field microscope is shown;

[0025] Figure 2B A schematic diagram showing the three-dimensional imaging field of view of a line light sheet in a confocal light field microscope;

[0026] Figure 3 A schematic diagram of a light path of a confocal light field microscope provided according to some embodiments of the present invention is shown; and

[0027] Figure 4 A schematic diagram of filtering out background noise according to some embodiments of the present invention is shown.

[0028] Reference numerals

[0029] 110: Sample;

[0030] 120, 220, 320: objective lens;

[0031] 130, 131, 132: lens;

[0032] 140, 330: microlens array;

[0033] 150, 230: Camera;

[0034] 200, 300: Confocal light field microscope;

[0035] 210, 310: excitation light;

[0036] 211: Linear light sheet;

[0037] 311: cylindrical lens and beam expander lens group;

[0038] 341: first relay lens;

[0039] 342: second relay lens;

[0040] 343: the third relay lens;

[0041] 344: fourth relay lens;

[0042] 350: mask;

[0043] 361: first telecentric lens;

[0044] 362: second telecentric lens;

[0045] 370: Photosensitive element;

[0046] 410: Slit;

[0047] 420: imaging object;

[0048] 421, 422: Imaging;

[0049] D: long pass dichroic mirror;

[0050] D1: dichroic mirror;

[0051] L1, L2, L3, L4: relay lenses;

[0052] G1, G2: Scanning galvanometer;

[0053] G: Double-sided scanning galvanometer;

[0054] M1: first reflector; and

[0055] M2: Second reflector. DETAILED DESCRIPTION

[0056] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. Note that the aspects described below in conjunction with the accompanying drawings and specific embodiments are only exemplary and should not be construed as limiting the scope of protection of the present invention in any way.

[0057] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it 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 a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0058] In addition, the terms "upper", "lower", "left", "right", "top", "bottom", "horizontal" and "vertical" used in the following description should be understood as the directions shown in the paragraph and the related drawings. Such relative terms are only used for the convenience of description and do not mean that the device described therein must be manufactured or operated in a specific direction, and therefore should not be understood as limiting the present invention.

[0059] It is understood that although the terms "first", "second", "third", etc. may be used herein to describe various components, regions, layers and / or parts, these components, regions, layers and / or parts should not be limited by these terms, and these terms are only used to distinguish different components, regions, layers and / or parts. Therefore, the first component, region, layer and / or part discussed below may be referred to as a second component, region, layer and / or part without departing from some embodiments of the present invention.

[0060] Please refer to Figure 2A , Figure 2A A light path diagram of a confocal light-field microscope is shown.

[0061] like Figure 2A As shown, the confocal light field microscope 200 is mainly composed of an excitation light scanning module, a filtering module and a collection light scanning module. The excitation light 210 is introduced by a long-pass dichroic mirror D, and passes through the relay lens L4, the relay lens L3, the scanning galvanometer G1, the relay lens L2, the relay lens L1 and the objective lens 220 to form a linear light sheet perpendicular to the optical axis, which is vertically irradiated on the sample.

[0062] Please refer to Figure 2B , Figure 2B Schematic diagram showing the three-dimensional imaging field of view of a line light sheet in a confocal light-field microscope.

[0063] like Figure 2BAs shown, the linear light sheet 211 scans along the y direction. The linear light sheet 211 presents a light cone shape in the z-y plane, and the thickness of the light cone represents the numerical aperture of the excitation light 210. The linear light sheet 211 presents as a line segment in the x direction, and the length of this line segment represents the field of view excited by the linear light sheet 211 in the x direction. Here, the position of the linear light sheet 211 in the scanning direction, that is, the y direction, is precisely controlled by the angle of the scanning galvanometer G1, and the scanned length is the field of view excited in the y direction. Preferably, the field of view excited in the x direction is the same as the field of view excited in the y direction.

[0064] The linear light sheet 211 and the scanning galvanometer G1 together constitute the excitation light scanning module of the confocal light field microscope 200.

[0065] In the confocal light field microscope 200, the stimulated light emitted by the sample after being excited by the excitation light 210 can be fluorescence. The collection light scanning module of the confocal light field microscope 200 is used to collect this fluorescence signal. The fluorescence signal is emitted by the molecules excited by the excitation light 210 of the sample and is usually consistent with the shape of the excitation light 210. Here, the fluorescence signal can be collected by the camera 230 after passing through the objective lens 220, relay lens L1, relay lens L2, scanning galvanometer G1, relay lens L3, relay lens L4, and scanning galvanometer G2.

[0066] The scanning galvanometer G1 changes the angle to change the angle of the excitation light 210 reflected by the scanning galvanometer G1 to the objective lens 220, thereby changing the position of the linear light sheet 211 in the y direction. According to the principle of reversibility of light, the fluorescence signal generated by the excitation of the excitation light 210 will return along the original path to the relay lens L4. Therefore, no matter how the angle of the scanning galvanometer G1 changes, the optical path from the scanning galvanometer G1 to the relay lens L4 remains unchanged. In this way, the fluorescence signal can always present as a line perpendicular to the paper plane at the focal plane of the relay lens L3, thereby realizing descanning.

[0067] Similarly, if the fluorescence signal is directly collected by the camera 230 after passing through the relay lens L4, the collected record will always present as a line shape, the same as that at the focal plane of the relay lens L3. The information in this line is a function of the angle of the scanning galvanometer G1 (i.e., the position of the linear light sheet 211 in the y direction). Therefore, it is necessary to synchronize the deflection angle of the scanning galvanometer G1 through the scanning galvanometer G2, so as to reverse-scan the collected fluorescence signal into the camera 230 in sequence, thereby realizing the area exposure imaging of the camera 230.

[0068] However, in the case of high-speed scanning of the confocal light field microscope 200 (for example, when the scanning speed is greater than 100 Hz), the synchronization error between the scanning galvanometer G1 and the scanning galvanometer G2 will become difficult to ignore, and such an imaging method will cause image distortion, which is also very difficult to correct later.

[0069] To overcome the above-mentioned deficiencies, the present invention provides a confocal light field microscope. Through a double-sided scanning mechanism, it can eliminate the influence of scanning distortion during scanning and avoid a series of problems caused by the synchronization error of two scans.

[0070] Please refer to Figure 3 , Figure 3 which shows a schematic optical path diagram of a confocal light field microscope provided according to some embodiments of the present invention.

[0071] As Figure 3 shown, the confocal light field microscope 300 introduces the excitation light 310 via the dichroic mirror D1. The confocal light field microscope 300 further includes an objective lens 320, a double-sided scanning galvanometer G, a mirror group, a microlens array 330, and a photosensitive element 370. Here, the mirror group includes a first mirror M1 and a second mirror M2, and the double-sided scanning galvanometer G includes a first reflecting surface and a second reflecting surface located on the front and back sides respectively.

[0072] In Figure 3 the shown embodiment, the first reflecting surface of the double-sided scanning galvanometer G can be used to reflect the excitation light 310 introduced by the dichroic mirror D1 to the imaging object in front of the objective lens 320. In addition, the mirror group can also reflect the excited light to the second reflecting surface of the double-sided scanning galvanometer G, so as to be reflected to the photosensitive element 370 through the second reflecting surface. By changing the angle of the double-sided scanning galvanometer G, the excitation light 310 can scan the imaging object via the first reflecting surface of the double-sided scanning galvanometer G, and at the same time, the excited light can scan into the photosensitive element 370 via the second reflecting surface of the double-sided scanning galvanometer G.

[0073] Preferably, the excitation light 310 forms a linear light sheet through a cylindrical lens and an expander lens group 311.

[0074] The double-sided scanning galvanometer G is located at the conjugate rear pupil plane of the objective lens 320. The first optical path is formed from the objective lens 320 to the first reflecting surface of the double-sided scanning galvanometer G. Preferably, a first relay lens 341 and a second relay lens 342 can be provided on the first optical path to extend the first optical path. In this preferred embodiment, the first reflecting surface of the double-sided scanning galvanometer G should be located at the rear focal plane of the second relay lens 342.

[0075] The second optical path is formed from the first reflecting surface of the double-sided scanning galvanometer G to the first mirror M1. Preferably, the first optical path is perpendicular to the second optical path.

[0076] The second mirror M2 is used to receive the excited light reflected from the first mirror M1. In Figure 3 the shown embodiment, the second mirror M2 can directly receive the excited light reflected from the first mirror M1.

[0077] As shown Figure 3 in FIG., the confocal light field microscope 300 may further include a third relay lens 343 and a fourth relay lens 344. The third relay lens 343 is located in the second optical path, and the fourth relay lens 344 is located between the first mirror M1 and the second mirror M2.

[0078] In Figure 3 the embodiment shown, the dichroic mirror D1 is located between the third relay lens 343 and the fourth relay lens 344. Figure 3 The position of the dichroic mirror D1 shown is the most ideal position of the dichroic mirror D1. The dichroic mirror D1 can be used to form a linear light sheet at the focal plane between the fourth relay lens 344 and the first mirror M1. The smaller the required aperture of the reflective surface of the dichroic mirror D1, the smaller the impact on imaging. Therefore, the dichroic mirror D1 should not be too close to the third relay lens 343 or the fourth relay lens 344.

[0079] Furthermore, no optical element should be placed at any focal plane in the optical path to avoid the imaging interference of dust on the optical element on the excited light signal. Therefore, the dichroic mirror D1 should not be placed at the focal plane between the fourth relay lens 344 and the first mirror M1 to avoid the imaging of dust on the dichroic mirror D1 from interfering with the excited light signal generated by the excited imaging object.

[0080] Optionally, the dichroic mirror D1 can also be located between the first reflective surface of the two-sided scanning galvanometer G and the third relay lens 343. Here, a lens with the same focal length as the third relay lens 343 needs to be added before the excitation light 310. And, since the light spot is larger when the dichroic mirror D1 is located between the first reflective surface of the two-sided scanning galvanometer G and the third relay lens 343, the size of the dichroic mirror D1 needs to be specially customized.

[0081] The dichroic mirror D1 can be a long-pass dichroic mirror to reflect the excitation light 310 with a shorter wavelength and pass the excited light with a longer wavelength.

[0082] In addition, the first mirror M1 is preferably placed between the first reflective surface of the two-sided scanning galvanometer G and the fourth relay lens 344 and away from the focal plane between the third relay lens 343 and the fourth relay lens 344 to ensure that the optical path is not blocked by the first mirror M1.

[0083] In Figure 3 the embodiment shown, the excitation light 310 is reflected by the dichroic mirror D1 to the first mirror M1, then through the second optical path to the first reflective surface of the two-sided scanning galvanometer G, and is reflected to the objective lens 320 through the first optical path by the first reflective surface of the two-sided scanning galvanometer G, vertically irradiating the imaging object and illuminating the x-z plane at one time. By changing the angle of the two-sided scanning galvanometer G, the excitation light 310 scans the imaging object.

[0084] After the imaging object is excited by the excitation light 310, it emits stimulated light. The stimulated light is reflected by the first reflecting surface of the double-sided scanning galvanometer G into the second optical path. In Figure 3 the illustrated embodiment, the stimulated light reaches the second mirror M2 via the first mirror M1 and the dichroic mirror D1, while the excitation light 310 is reflected by the dichroic mirror D1 and cannot reach the second mirror M2.

[0085] From the second mirror M2 to the second reflecting surface of the double-sided scanning galvanometer G forms a third optical path. The second mirror M2 reflects the stimulated light from the first mirror M1 to the second reflecting surface of the double-sided scanning galvanometer G via the third optical path. Preferably, the first optical path is parallel to the third optical path.

[0086] The microlens array 330 is located between the second mirror M2 and the second reflecting surface of the double-sided scanning galvanometer G. The second mirror M2 is located in front of the microlens array 330. The microlens array 330 is located on the conjugate rear pupil plane of the objective lens 320.

[0087] As Figure 3 shown, the confocal light field microscope 300 may include a mask 350, and the mask 350 is located on the rear focal plane of the microlens array 330. Through the mask 350, a denoising method for filtering background noise applicable to light field imaging is realized.

[0088] Please refer to Figure 4 , Figure 4 which shows a schematic diagram of a method for filtering background noise provided according to some embodiments of the present invention.

[0089] As Figure 4 shown, after the stimulated light passes through the microlens array 330, each microlens will form a line perpendicular to the paper surface at the position of the mask 350, as Figure 4 shown by the slit 410 of the mask 350 in. The shape of the slit 410 is fixed regardless of the scanning angle of the double-sided scanning galvanometer G. Therefore, the stimulated light can be physically filtered by the mask 350 with a fixed shape.

[0090] In Figure 3 the illustrated embodiment, the effective focal depth range of the computational imaging can be calculated to design a suitable slit width for each microlens, and the slits are arranged on the mask 350 according to the spatial position of the microlens array 330, and then the mask 350 is placed on the rear focal plane of the microlens array 330.

[0091] As Figure 4As shown, the mask 350 includes a plurality of slits 410, and the positions of the plurality of slits 410 correspond one-to-one with the positions of the plurality of microlenses on the microlens array 330. The center of each slit 410 is the center of the corresponding microlens. Each slit 410 has the same length, and the length of the slit 410 represents the length of the imaging field of view. The widths of the slits 410 are different, and the width of the slit 410 represents the thickness of the same imaging field of view. Each slit 410 can exactly allow the stimulated light generated by the imaging object with the target thickness to pass through, and the background noise exceeding the target thickness will be filtered out by the slit 410. The widths of the slits 410 are different because the positions of the different microlenses in the array are different.

[0092] The stimulated light emitted from the focal plane of the imaging object 420 can form a converging image after passing through the microlens array 330, while the stimulated light emitted from a position far from the focal plane can only form a blurred and magnified image, as shown by the blurred imaging 421 in Figure 4 . Therefore, placing the mask 350 with the slits 410 behind the microlens array 330 can allow the stimulated light within the effective focal depth range to pass through while blocking the background noise generated in the out-of-focus area, thus solving the problem of excessive background noise faced by the prior art when imaging a relatively thick imaging object.

[0093] Please continue to refer to Figure 4 , the sheet-shaped excitation light 310 sweeps across the entire imaging object 420 in the y direction. Due to the principle of reversibility of light, the stimulated light excited by the light sheets of the excitation light 310 at different scanning positions can all pass through the slits 410 of the fixed mask 350, and then the double-sided scanning galvanometer G reflects the stimulated light in sequence to the corresponding positions of the photosensitive element 370 (for example, a camera) to form a clear imaging 422.

[0094] As shown in Figure 3 , a fourth optical path is formed from the second reflecting surface of the double-sided scanning galvanometer G to the photosensitive element 370. Preferably, the third optical path is perpendicular to the fourth optical path.

[0095] In addition, the confocal light field microscope 300 may further include a first telecentric lens 361 and a second telecentric lens 362. The first telecentric lens 361 is located in the third optical path, and the second reflecting surface of the double-sided scanning galvanometer G is located at the rear focal plane of the first telecentric lens 361. The second telecentric lens 362 is located in the fourth optical path, and the second reflecting surface of the double-sided scanning galvanometer G is located at the rear focal plane of the second telecentric lens 362. The stimulated light signal is conjugated to the photosensitive element 370 through the first telecentric lens 361 and the second telecentric lens 362. The stimulated light signal is easily affected by the angle after being filtered by the mask 350. By using the first telecentric lens 361 and the second telecentric lens 362, the aberration can be effectively reduced.

[0096] So far, Figure 3 The optical path design in the illustrated embodiment follows the principle of minimizing the number of optical elements and is the most optimal design solution. Figure 3 The total length of the optical path from the first reflecting surface of the double-sided scanning galvanometer G to the second reflecting surface of the double-sided scanning galvanometer G shown is fixed, and the expression is:

[0097] 2*(F 1 +F 2 +F 3 )+F 4 ;

[0098] F 1 is the focal length of the third relay lens 343, F 2 is the focal length of the fourth relay lens 344, F 3 is the focal length of the first telecentric lens 361, F 4 is the focal length of the microlens array 330.

[0099] Figure 3 The lengths of the three sides of the optical path from the first reflecting surface of the double-sided scanning galvanometer G to the second reflecting surface of the double-sided scanning galvanometer G shown satisfy the Pythagorean theorem.

[0100] In the optical path from the first reflecting surface of the double-sided scanning galvanometer G to the second reflecting surface of the double-sided scanning galvanometer G, the distance between the third relay lens 343 and the first mirror M1 and the distance between the second mirror M2 and the microlens array 330 can be adjusted. In addition, N pairs of 4F lens systems can be added to the optical path. In this way, the total length of such an optical path can become longer. New mirrors can also be added to the optical path from the first reflecting surface of the double-sided scanning galvanometer G to the second reflecting surface of the double-sided scanning galvanometer G. In this way, the optical path is no longer limited to a triangular shape.

[0101] In Figure 3 the illustrated embodiment, the objective lens 320 and the first relay lens 341, the first relay lens 341 and the second relay lens 342, the second relay lens 342 and the third relay lens 343, the third relay lens 343 and the fourth relay lens 344, the microlens array 330 and the first telecentric lens 361, and the first telecentric lens 361 and the second telecentric lens 362 are all 4F systems. Preferably, the microlens array 330 is located on the rear focal plane of the fourth relay lens 344, and the photosensitive element 370 is located on the rear focal plane of the second telecentric lens 362. The stimulated light emitted after the imaging object is stimulated by the excitation light 310 is collected and imaged by the photosensitive element 370 via the first optical path, the second optical path, the third optical path, and the fourth optical path.

[0102] Please refer to Figure 3 and Figure 4When the excitation light sheet of the excitation light 310 sweeps across the entire three-dimensional imaging object 420, the shooting of one frame of image can be completed. Although the exposure of the imaging object 420 is no longer instantaneous, considering that the swinging speed of the double-sided scanning galvanometer G is extremely fast (up to several kilohertz), which is much greater than the single-frame exposure speed of the photosensitive element 370, introducing the confocal excitation and detection strategy will not affect the imaging speed of the confocal light field microscope 300.

[0103] Replace the pair of scanning galvanometers G1 and G2 shown with a single high-speed double-sided scanning galvanometer G Figure 2A to reduce the influence of scanning distortion caused by the synchronization error of the galvanometers G1 and G2. The strategy of folding the optical path enables the optical path of the excited light to be scanned synchronously by the front and back sides of the double-sided scanning galvanometer G respectively. After the excited light is reflected back by the first reflecting surface of the double-sided scanning galvanometer G, the signal is reflected to the second reflecting surface of the double-sided scanning galvanometer G through the first reflecting mirror M1, the second reflecting mirror M2, the third relay lens 343, and the fourth relay lens 344. Considering that the speed of light is extremely fast and the entire optical path does not exceed 2 meters, the time difference of the excited light passing through the first reflecting surface and the second reflecting surface of the double-sided scanning galvanometer G can be stably controlled within 1 nanosecond, which is much higher than the scanning galvanometer synchronization generated by signal triggering in terms of time accuracy, avoiding the noise of the excited light caused by the scanning of the scanning galvanometer. Therefore, no matter how fast the scanning speed of the double-sided scanning galvanometer G is, a series of problems caused by synchronization errors will not occur.

[0104] In summary, the system design of the confocal light field microscope provided by the present invention is simple. While achieving the advantages of large field of view and high signal-to-noise ratio in awake animal brain imaging, through the double-sided scanning method of the double-sided scanning galvanometer, the imaging speed of the confocal light field microscope is pushed to the limit, making it the one with the largest imaging range in the current single-cell resolution in vivo high-speed imaging technology. The confocal light field microscope provided by the present invention can be applied to the observation of various rapid dynamic changes in living animals. Taking high-speed voltage imaging as an example, the confocal light field microscope can record the voltage signals of nearly a thousand neurons in the entire three-dimensional field of view at a speed greater than 400 Hz within the range of 800 microns in diameter and 160 microns in thickness of the mouse cerebral cortex, which is the voltage imaging microscope with the highest imaging throughput currently. The high-throughput information acquisition ability of the confocal light field microscope provided by the present invention provides a new tool for studying the functions of neural networks. Moreover, the application scope of the confocal light field microscope provided by the present invention is not limited to the field of voltage imaging. All scenarios that require high-speed imaging in transparent or semi-transparent biological tissues (such as blood cell imaging, organoid dynamic imaging, etc.) can be realized using this confocal light field microscope.

[0105] The foregoing description of the disclosure is provided to enable any person skilled in the art to make or use the disclosure. Various modifications to the disclosure will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other variations without departing from the spirit or scope of the disclosure. Thus, the disclosure is not intended to be limited to the examples and designs described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A confocal light field microscope, comprising: an objective lens, a dichroic mirror, a microlens array located on the conjugate rear pupil plane of the objective lens, and a mask plate located on the rear focal plane of the microlens array, characterized in that the confocal light field microscope further comprises a two-sided scanning galvanometer and a mirror group, the two-sided scanning galvanometer includes a first reflecting surface and a second reflecting surface located on the front and back sides respectively, the first reflecting surface is used to reflect the excitation light introduced by the dichroic mirror to the imaging object in front of the objective lens, and the mirror group reflects the excited light to the second reflecting surface to be reflected by the second reflecting surface to the photosensitive element, wherein the two-sided scanning galvanometer changes the angle so that the excitation light scans the imaging object via the first reflecting surface and at the same time the excited light scans into the photosensitive element via the second reflecting surface.

2. The confocal light field microscope according to claim 1, characterized in that the mirror group includes a first mirror and a second mirror, the two-sided scanning galvanometer is located on the conjugate rear pupil plane of the objective lens, and the first optical path is formed from the objective lens to the first reflecting surface of the two-sided scanning galvanometer; the second optical path is formed from the first reflecting surface of the two-sided scanning galvanometer to the first mirror; the third optical path is formed from the second mirror to the second reflecting surface of the two-sided scanning galvanometer, and the second mirror reflects the excited light from the first mirror to the second reflecting surface of the two-sided scanning galvanometer via the third optical path; the fourth optical path is formed from the second reflecting surface of the two-sided scanning galvanometer to the photosensitive element; the excited light emitted by the imaging object after being excited by the excitation light is collected and imaged by the photosensitive element via the first optical path, the second optical path, the third optical path and the fourth optical path.

3. The confocal light field microscope according to claim 2, characterized in that the first optical path is perpendicular to the second optical path, the first optical path is parallel to the third optical path, and the third optical path is perpendicular to the fourth optical path.

4. The confocal light field microscope according to claim 1, characterized in that the mask plate includes a plurality of slits, and the positions of the plurality of slits correspond one-to-one to the positions of the plurality of microlenses on the microlens array.

5. The confocal light field microscope according to claim 2, characterized in that it further comprises a first telecentric lens and a second telecentric lens, the first telecentric lens is located in the third optical path, and the second reflecting surface of the two-sided scanning galvanometer is located on the rear focal plane of the first telecentric lens; the second telecentric lens is located in the fourth optical path, and the second reflecting surface of the two-sided scanning galvanometer is located on the rear focal plane of the second telecentric lens.

6. The confocal light field microscope according to claim 2, characterized in that it further comprises a first relay lens and a second relay lens, and the first relay lens and the second relay lens are located in the first optical path.

7. The confocal light field microscope according to claim 2, characterized in that It further includes a third relay lens and a fourth relay lens. The third relay lens is located in the second optical path, and the fourth relay lens is located between the first reflector and the second reflector.

8. The confocal light field microscope according to claim 7, wherein, the dichroic mirror is located between the third relay lens and the fourth relay lens.

9. The confocal light field microscope according to claim 7, wherein, the dichroic mirror is located between the first reflecting surface of the double-sided scanning galvanometer and the third relay lens.

10. The confocal light field microscope according to claim 1, wherein, the excitation light forms a linear light sheet via a cylindrical lens and a beam expander lens group.

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