Angular adjustment apparatus and method for graded index lens for deep brain imaging

Abstract

The application discloses an angle adjusting device and method of a gradient refractive index lens for deep brain imaging, and belongs to the field of biological microscopic imaging. The device comprises, from bottom to top, a first displacement table, a second displacement table, a third displacement table, a second electric angle displacement table, a first electric angle displacement table and a sample bearing plate. The first electric angle displacement table and the second electric angle displacement table are coaxially installed, and the rotation centers thereof coincide. The second electric angle displacement table is fixed on the object table of the third displacement table. The base of the third displacement table is fixed on the object table of the second displacement table, and the base of the second displacement table is fixed on the object table of the first displacement table. The sample bearing plate is arranged above the first electric angle displacement table, and is used for placing an animal sample and coaxially arranging the gradient refractive index lens and the first electric angle displacement table. The application can reduce the angle deviation between the gradient refractive index lens and the central optical axis of a microscope, and improve the imaging quality.

Description

Technical Field

[0001] This invention belongs to the field of biological microscopy imaging, and more specifically, relates to an angle adjustment device and method for a gradient refractive index lens for deep brain imaging. Background Technology

[0002] When imaging the deep brain of animals, a graded-index lens is typically implanted in the animal's head, and a built-in microscope system is used to observe the structural and functional information of the deep brain regions. The built-in microscope system often employs a two-photon fluorescence laser scanning microscope, which has a long penetration depth and low scattering.

[0003] Because graded-index lenses inherently possess aberrations, their aberrations are amplified when used with microscopes employing high numerical apertures. This aberrations are further amplified when both the graded-index lens and the microscope also have high numerical apertures. Since graded-index lenses can only correct a limited number of aberrations, in practical applications, if there is an angular deviation between the central optical axis of the graded-index lens and the central optical axis of the microscope when coupled, severe aberrations will occur, affecting image quality. Furthermore, the resulting tilt of the imaging plane due to this angular deviation will reduce the effectiveness of adaptive optics in correcting system aberrations.

[0004] Due to the centrosymmetry of graded-index lenses, during coupling between a graded-index lens and a microscope, the angular deviation between the two central optical axes is typically adjusted by regulating the lens's pitch and roll angles. In existing technologies, this angular deviation is usually manually adjusted to make the two central optical axes coincide. This manual adjustment method has significant errors and low repeatability; furthermore, the angular deviation between the two central optical axes is a small angular deviation, and manual adjustment often fails to achieve perfect alignment. Summary of the Invention

[0005] In view of the shortcomings of the existing technology and the need for improvement, the present invention provides an angle adjustment device and method for a graded refractive index lens for deep brain imaging, the purpose of which is to reduce the angular deviation between the graded refractive index lens and the central optical axis of the microscope and improve the imaging quality.

[0006] To achieve the above objectives, according to a first aspect of the present invention, an angle adjustment device for a gradient refractive index lens for deep brain imaging is provided, comprising, from bottom to top: a first displacement stage, a second displacement stage, a third displacement stage, a second electrically driven angle displacement stage, a first electrically driven angle displacement stage, and a sample carrier plate.

[0007] Two motorized angular displacement stages are coaxially mounted and their rotation centers coincide; the two motorized angular displacement stages are used to adjust the pitch angle and roll angle during the coupling process between the graded refractive index lens and the microscope; the two motorized angular displacement stages are the first motorized angular displacement stage and the second motorized angular displacement stage.

[0008] The second electric angle displacement stage is fixed to the platform of the third displacement stage; the base of the third displacement stage is fixed to the platform of the second displacement stage, and the base of the second displacement stage is fixed to the platform of the first displacement stage; the three displacement stages are respectively used to reverse compensate for the displacement of the graded refractive index lens in the X-axis, Y-axis and Z-axis caused by the electric angle displacement stage during angle adjustment; the Z-axis is parallel to the central optical axis of the graded refractive index lens; the three displacement stages are the first displacement stage, the second displacement stage and the third displacement stage;

[0009] The sample carrier plate is positioned above the first electric angular displacement stage, and is used to place animal samples so that the gradient refractive index lens of the animal's head is coaxial with the first electric angular displacement stage.

[0010] Furthermore, the sample support plate includes: a fixing plate, two support columns, and a base plate;

[0011] The animal's head is placed between the two pillars, which are fixed to the base plate. The fixing plate is mounted on the two pillars and is connected to the fixing plate on the gradient refractive index lens.

[0012] Furthermore, the stage and the base are fixed together by M6 fixing screws; wherein the stage is the stage of the first displacement stage or the second displacement stage, and the base is the base of the second displacement stage or the third displacement stage.

[0013] According to a second aspect of the present invention, an adjustment method for an angle adjustment device as described in any of the first aspects is provided, comprising:

[0014] S1. Determine the rotation radius, which is the distance between the upper surface of the gradient refractive index lens and the rotation center of the two electric angle displacement stages after the animal sample is placed.

[0015] S2. Set the initial positions of the two electric angular displacement stages and the adjacent area centered on the initial positions;

[0016] S3. Drive the two electric angle displacement stages to adjust the pitch angle and roll angle respectively in the vicinity of the initial position at this time with a determined step accuracy; at the same time, the three displacement stages perform reverse compensation on the displacement of the two electric angle displacement stages according to the rotation radius to obtain the position with the clearest image at this time.

[0017] S4. Using the position where the image is clearest at this moment as the initial position, repeat S3 within the same adjacent area size until the position where the image is clearest is the same in at least two adjacent repetitions; at this moment, the position where the image is clearest is the position where the gradient refractive index lens coincides with the central optical axis of the microscope.

[0018] Further, in S1, the rotation radius is a first rotation radius R1 and a second rotation radius R2; determining the rotation radius includes:

[0019] Measure the first rotation radius R1 and calculate the second rotation radius R2; the first rotation radius R1 is the distance between the upper surface of the gradient refractive index lens and the rotation center of the first electric angular displacement stage after the animal sample is placed; where R2 = R1 + h, h represents the height between the stage of the second electric angular displacement stage and the base of the first electric angular displacement stage.

[0020] In S3, the three displacement stages perform reverse compensation on the displacements of the two electric angular displacement stages according to the rotation radius, including:

[0021] The three displacement stages perform reverse compensation on the displacement of the first electric angle displacement stage based on the first rotation radius R1.

[0022] The three displacement stages perform reverse compensation on the displacement of the second electric angle displacement stage according to the second rotation radius R2.

[0023] Further, in S3, the determined stepping accuracy is the optimal stepping accuracy for the corresponding animal sample, and determining the optimal stepping accuracy includes:

[0024] S31. Initialize the angle adjustment range of the two electric angle displacement stages;

[0025] S32. With the minimum step accuracy, the two electric angle displacement stages are traversed sequentially within the initialized angle adjustment range; at the same time, the three displacement stages are compensated for inverse displacement to obtain a microscopic image at each angle; and the image sharpness evaluation function is used to calculate the evaluation value corresponding to the microscopic image at each angle.

[0026] S33. Determine an interval of the angle corresponding to the image with the largest evaluation value. If the microscopic images within the interval are all in focus, and the interval is the largest interval corresponding to all microscopic images being in focus, then the interval is the optimal step accuracy.

[0027] Furthermore, the corresponding reverse displacement compensation formulas for the three displacement stages based on the first rotation radius R1 or the second rotation radius R2 are as follows:

[0028] Δx=RSin[θ e ]-RSin[θ i ]

[0029] Δy=RSin[θ e ]-RSin[θ i ]

[0030] Δz=RCos[θ e ]-RCos[θ i ]

[0031] Where Δx, Δy, and Δz represent the reverse compensation amounts for the displacement offsets of the two electric angular displacement stages in the X, Y, and Z axes, respectively; R is either R1 or R2. When R is R1, it indicates reverse compensation for the displacement of the first electric angular displacement stage; when R is R2, it indicates reverse compensation for the displacement of the second electric angular displacement stage; θ e θ represents the angle after the last movement of the two electrically driven angular displacement stages. i This indicates the angle of the two electrically driven angular displacement stages before their current movement.

[0032] Further, measuring the first rotation radius R1 includes:

[0033] S11. After placing the animal sample on the sample support plate, estimate the range of the first rotation radius;

[0034] S12. Under the same first angle displacement stage deflection angle, traverse each estimated first rotation radius; at the same time, the three displacement stages perform reverse displacement compensation according to each estimated first rotation radius to obtain the corresponding microscopic image.

[0035] S13. The sharpest microscopic image is determined using a sharpness evaluation function; the rotation radius corresponding to the sharpest microscopic image is the first rotation radius R1.

[0036] According to a third aspect of the present invention, a system for deep brain imaging of animals is provided, comprising: a microscopic imaging device, a gradient refractive index lens disposed on the head of the animal, and an angle adjustment device as described in any one of the first aspects;

[0037] During the coupling process between the microscope and the graded refractive index lens in the microscopic imaging device, the angle adjustment device is used to adjust the angle between the central optical axis of the microscope and the graded refractive index lens so that the central optical axis of the microscope and the graded refractive index lens coincide.

[0038] In summary, the above-described technical solutions conceived in this invention can achieve the following beneficial effects:

[0039] (1) The angle adjustment device for the graded refractive index lens for deep brain imaging of the present invention adjusts the pitch angle and roll angle of the graded refractive index lens during the coupling process between the lens and the microscope through two electric angle displacement stages. In conjunction with three displacement stages, it performs reverse compensation for the displacement offset of the graded refractive index lens in the X-axis, Y-axis and Z-axis directions caused by the angle adjustment of the two electric angle displacement stages. It can automatically adjust the angle between the graded refractive index lens and the central optical axis of the microscope, thereby reducing the angle deviation between the graded refractive index lens and the central optical axis of the microscope and improving the imaging quality.

[0040] (2) Further, the designed sample carrier plate 3 places the animal head between two pillars and connects the fixed plate on the pillars with the fixed plate on the gradient refractive index lens of the animal head, so that the relative position of the animal head and the gradient refractive index lens remains unchanged, thereby ensuring that the gradient refractive index lens of the animal head is coaxial with the two electric angle displacement stages.

[0041] (3) Based on the angle adjustment device of the present invention, a corresponding adjustment method is also provided. By driving two electric angle displacement stages to automatically adjust the angle in the vicinity of the initial position, and at the same time, the three displacement stages perform corresponding displacement reverse compensation to obtain the position where the image is clearest. Then, taking the position where the image is clearest as the initial position, the above process is repeated in the vicinity of the same size until the position where the image is clearest remains unchanged, so as to realize the coincidence of the gradient refractive index lens and the central optical axis of the microscope. This automatic adjustment device of the present invention can quickly and accurately adjust the angle between the gradient refractive index lens and the central optical axis of the microscope to reduce the aberration of the image and thus improve the image quality. Moreover, the electric angle displacement stages and the three displacement stages have high adjustment accuracy. Through the design method of the present invention, they can cooperate with each other to achieve the adjustment of small angle deviations during automatic angle adjustment.

[0042] (4) Preferably, based on the device of the present invention, a method for finding the optimal stepping accuracy is provided. By using the optimal stepping accuracy to adjust the angle of the two electric angle displacement stages, the speed of finding the position where the graded refractive index lens coincides with the central optical axis of the microscope can be improved.

[0043] (5) Preferably, based on the device of the present invention, a method for measuring the radius of rotation is provided. This method is independent of the depth of the graded refractive index lens buried in the animal brain region, avoids the error caused by the different depths of the graded refractive index lens buried in the animal brain region, improves the accuracy of the radius of rotation measurement, and further reduces the angular deviation between the graded refractive index lens and the central optical axis of the microscope. Attached Figure Description

[0044] Figure 1This is a schematic diagram of an angle adjustment device for a gradient refractive index lens used for deep brain imaging in an embodiment of the present invention.

[0045] Figure 2 This is a schematic diagram of the sample carrier plate structure in an embodiment of the present invention.

[0046] Figure 3 This is a flowchart illustrating the measurement of the rotation radius in an embodiment of the present invention.

[0047] Figure 4(a) is an experimental diagram of deep brain imaging of a sample before using the angle adjustment device in the embodiment of the present invention.

[0048] Figure 4(b) is an experimental diagram of deep brain imaging of the same sample using the angle adjustment device in the embodiment of the present invention.

[0049] In all the accompanying drawings, the same reference numerals are used to denote the same elements or structures, wherein:

[0050] 1-Microscope, 2-Graduated refractive index lens, 3-Sample support plate, 4-First motorized angular displacement stage, 5-Second motorized angular displacement stage, 6-Third displacement stage, 7-Second displacement stage, 8-First displacement stage;

[0051] 31-Fixing plate, 32-Support column, 33-Base plate. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0053] In this invention, the terms "first," "second," etc., used in the invention and accompanying drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence.

[0054] like Figure 1 As shown, the angle adjustment device for the graded refractive index lens for deep brain imaging in this embodiment of the invention includes, from bottom to top:

[0055] First displacement stage 8, second displacement stage 7, third displacement stage 6, second electric angle displacement stage 5, first electric angle displacement stage 4 and sample support plate 3;

[0056] The first electric angular displacement stage 4 and the second electric angular displacement stage 5 are coaxially mounted, and the rotation centers of the two electric angular displacement stages coincide.

[0057] The bases of the first displacement stage 8, the second displacement stage 7, and the third displacement stage 6 are fixed to each other with the platform, that is, the base of the second displacement stage 7 is fixed to the platform of the first displacement stage 8, and the base of the third displacement stage 6 is fixed to the platform of the second displacement stage 7; the second electric angle displacement stage 5 is fixed to the platform of the third displacement stage 6.

[0058] The sample carrier plate 3 is positioned above the first electric angular displacement stage 4 to place the animal sample and to make the gradient refractive index lens of the animal head coaxial with the two electric angular displacement stages.

[0059] In this invention, one of the two electrically operated angle-shifting stages, the first electric angle-shifting stage 4 and the second electric angle-shifting stage 5, is used to adjust the pitch angle during the coupling process between the graded-index lens and the microscope, while the other electric angle-shifting stage is used to adjust the roll angle. In this embodiment, the corresponding angles of the two electric angle-shifting stages are adjusted by commands input from a computer, and the three shifting stages below the two electric angle-shifting stages ultimately achieve the alignment of the central optical axis of the graded-index lens with the central optical axis of the microscope.

[0060] One of the three displacement stages is used to reverse the displacement of the graded refractive index lens in the X-axis direction caused by the angle adjustment of the two electric angle displacement stages.

[0061] Another of the three displacement stages is used to reverse the displacement of the graded refractive index lens in the Y-axis direction caused by the angle adjustment of the two electric angle displacement stages.

[0062] The remaining one of the three displacement stages is used to reverse the displacement of the graded refractive index lens in the Z-axis direction caused by the angle adjustment of the two electric angle displacement stages; wherein, the Z-axis direction is parallel to the central optical axis of the graded refractive index lens; the three displacement stages are the first displacement stage 8, the second displacement stage 7 and the third displacement stage 6; the two electric angle displacement stages are the first electric angle displacement stage and the second electric angle displacement stage.

[0063] In this embodiment of the invention, the first displacement stage is used to reverse compensate for the displacement offset of the graded refractive index lens in the X-axis direction caused by the angle adjustment of the two electric angle displacement stages; the second displacement stage is used to reverse compensate for the displacement offset of the graded refractive index lens in the Y-axis direction caused by the angle adjustment of the two electric angle displacement stages; and the third displacement stage is used to reverse compensate for the displacement offset of the graded refractive index lens in the Z-axis direction caused by the angle adjustment of the two electric angle displacement stages.

[0064] In this embodiment of the invention, the bases and platforms of the first displacement stage 8, the second displacement stage 7, and the third displacement stage 6 are fixed to each other using M6 fixing screws; similarly, the platforms of the second electric angle displacement stage 5 and the third displacement stage 6 are also fixed to each other using M6 fixing screws. In other embodiments, other fixing components may be used to fix the bases and platforms, as well as the platforms of the third displacement stage and the second electric angle displacement stage.

[0065] Specifically, such as Figure 2 As shown, the sample carrier plate 3 includes a fixed plate 31, two support columns 32, and a base plate 33; the animal head is placed between the two support columns 32, and the two support columns 32 are respectively fixed on the base plate 33. The fixed plate 31 is set on the two support columns 32, and the fixed plate 31 is connected to the fixed plate on the gradient refractive index lens of the animal head, so that the relative position of the animal head and the gradient refractive index lens remains unchanged, thereby ensuring that the gradient refractive index lens of the animal head is coaxial with the two electric angle displacement stages.

[0066] The method for adjusting the angle of a graded-index lens using the angle adjustment device described in the above embodiments, so that the central optical axis of the graded-index lens coincides with the central optical axis of the microscope, includes:

[0067] S1. Place the animal sample on the sample support plate 3, and determine the distance between the upper surface of the graded refractive index lens after placing the animal sample and the rotation center of the two electric angle displacement stages, i.e., the rotation radius; specifically, the rotation radius includes: a first rotation radius R1 and a second rotation radius R2; determining the rotation radius includes: measuring the first rotation radius R1 and calculating the second rotation radius R2; the first rotation radius R1 is the distance between the upper surface of the graded refractive index lens and the rotation center of the first electric angle displacement stage 4 after placing the animal sample; the second rotation radius R2 = R1 + h, where h represents the height between the stage of the second electric angle displacement stage 5 and the base of the first electric angle displacement stage 4;

[0068] S2. Set the initial positions of the two electric angular displacement stages and the adjacent area centered on these initial positions;

[0069] S3. The motors drive two electric angular displacement stages to adjust the angles (pitch and roll angles) within the vicinity of the initial position at this time using a determined step accuracy. Simultaneously, the three displacement stages perform reverse displacement compensation based on the rotation radius obtained in S1 to determine the position where the microscope image is clearest at this time. Specifically, if the first electric angular displacement stage adjusts the pitch angle during the coupling process between the graded refractive index lens and the microscope using a determined step accuracy, the three displacement stages perform reverse displacement compensation based on the measured first rotation radius R1. Correspondingly, when the second electric angular displacement stage adjusts the roll angle during the coupling process between the graded refractive index lens and the microscope using a determined step accuracy, the three displacement stages perform reverse displacement compensation based on the calculated second rotation radius R2.

[0070] S4. Take the position where the microscope image is clearest at this time as the initial position. Repeat step S3 in a nearby area of ​​the same size until the position where the microscope image is clearest in at least two adjacent repetitions is the same. The position where the microscope image is clearest after this is the position where the central optical axis of the graded refractive index lens coincides with the central optical axis of the microscope.

[0071] Specifically, in S1, measuring the first rotation radius R1 includes:

[0072] S11. After placing the animal sample on the sample support plate 3, estimate the numerical range of the first rotation radius.

[0073] S12. Under the same first angle displacement stage deflection angle, traverse each estimated first rotation radius value, and at the same time, the three displacement stages perform reverse displacement compensation according to each estimated first rotation radius value to obtain the corresponding microscopic imaging results.

[0074] S13. The sharpest image in the microscopic imaging is determined using the sharpness evaluation function; the rotation radius value corresponding to the sharpest image in the microscopic imaging is the required first rotation radius R1, which is the distance between the upper surface of the gradient refractive index lens after the animal sample is placed and the rotation center of the first electric angle displacement stage.

[0075] In this embodiment of the invention, the second electric angle displacement stage is fixed stationary, while the angle of the first electric angle displacement stage is adjusted, such as... Figure 3As shown, starting from the minimum value of the estimated first rotation radius range, the first rotation radius is increased by the same step radius ΔR with each rotation radius value traversal. Specifically, for the current rotation radius, the first electric angle displacement stage and the three displacement stages are all reset to zero. In the current traversal, the first electric angle displacement stage is adjusted by 1°, and the corresponding three displacement stages are subjected to corresponding reverse compensation, and the imaging result is recorded. In the next traversal, the rotation radius is increased by the step radius ΔR, and the first electric angle displacement stage and the three displacement stages are reset to zero again. The first electric angle displacement stage is adjusted by 1°, and the corresponding three displacement stages are subjected to corresponding reverse compensation, and the imaging result is recorded. This process continues until all values ​​of the estimated first rotation radius have been traversed, resulting in a series of imaging results corresponding to the rotation radius. The rotation radius corresponding to the clearest image is the required first rotation radius R1.

[0076] Specifically, in S3, the determined stepping accuracy is the optimal stepping accuracy for the corresponding animal sample; the process of finding the optimal stepping accuracy includes:

[0077] S31. Initialize the angle adjustment range of the two electric angle displacement stages; In this embodiment of the invention, the angle adjustment range of the two electric angle displacement stages is initialized according to the estimated deviation between the central optical axis of the gradient refractive index lens and the central optical axis of the microscope.

[0078] S32. With the minimum step accuracy, traverse the two electric angle displacement stages sequentially within the initialized angle adjustment range. That is, for each angle within the initialized angle adjustment range, adjust the angle of the two electric angle displacement stages accordingly. At the same time, the three displacement stages perform corresponding reverse displacement compensation based on the rotation radius obtained in S1 to obtain the microscopic imaging image at each angle. Use the image sharpness evaluation function to obtain the evaluation value corresponding to the microscopic imaging image at each angle.

[0079] S33. Determine an interval of the angle corresponding to the image with the largest evaluation value. Within this interval, if all the microscopic images corresponding to the angles are in the focal plane (i.e., in focus), and this interval is the largest interval corresponding to all the microscopic images being in focus, then this interval is the best stepping accuracy for the animal sample.

[0080] Specifically, the formula for reverse displacement compensation based on the rotation radius of the three displacement stages is as follows:

[0081] Δx=RSin[θ e ]-RSin[θ i ]

[0082] Δy=RSin[θ e ]-RSin[θ i ]

[0083] Δz=RCos[θ e ]-RCos[θ i ]

[0084] Where Δx represents the reverse compensation amount for the displacement of the graded refractive index lens in the X-axis direction caused by the angle adjustment of the two electric angular displacement stages; Δy represents the reverse compensation amount for the displacement of the graded refractive index lens in the Y-axis direction caused by the angle adjustment of the two electric angular displacement stages; Δz represents the reverse compensation amount for the displacement of the graded refractive index lens in the Z-axis direction caused by the angle adjustment of the two electric angular displacement stages; R takes either R1 or R2. When R is R1, it indicates reverse compensation for the displacement of the graded refractive index lens caused by the angle adjustment of the first electric angular displacement stage; when R is R2, it indicates reverse compensation for the displacement of the graded refractive index lens caused by the angle adjustment of the second electric angular displacement stage; θ e θ represents the angle after the last movement of the two electrically driven angular displacement stages. i This indicates the angle of the two electrically driven angular displacement stages before their current movement.

[0085] Specifically, in S3, a sharpness evaluation function is used to determine the position where the microscope image is sharpest at this time.

[0086] Specifically, the imaging end for performing the above-mentioned microscope imaging includes a camera (SCMOS) or a photomultiplier tube (PMT), etc.

[0087] The angle adjustment device described in the above embodiments is applied to a two-photon fluorescence laser scanning microscope to image the deep brain region of mice. In other embodiments, it can also be used for brain implantation imaging in other animals such as zebrafish. In this embodiment of the invention, when no sample is placed, the stage of the first motorized angle displacement stage is 65mm away from its rotation center, the stage size is 90×90mm, the height is 35mm, and the angle adjustment range is ±8°; the stage of the second motorized angle displacement stage is 100mm away from its rotation center, the stage size is 90×90mm, the height is 35mm, and the angle adjustment range is ±8°; the rotation centers of the two motorized angle displacement stages coincide; the specific shape of the sample carrier plate 3 can vary depending on the self-built system. In this embodiment of the invention, the carrier plate is 30mm high, 100mm long, and 75mm wide. Of the three displacement stages, the first stage has a platform size of 90×90mm, a stroke of 100mm, and a height of 45mm; the second stage has a platform size of 90×90mm, a stroke of 100mm, and a height of 45mm; and the third stage has a platform size of 90×90mm, a stroke of 20mm, and a height of 82–102mm. After placing the mouse, as shown in Figures 4(a) and 4(b), it can be seen that before the device was adjusted, the image of a certain brain region of the mouse brain was low in brightness, and the cells in the region of interest were shifted. After adjusting the device using the method in this embodiment of the invention, the same brain region of the mouse brain was imaged again, and the image brightness was significantly enhanced, and the target cells also appeared in the region of interest.

[0088] The present invention provides an angle adjustment device for a graded refractive index lens for deep brain imaging. This device uses two motorized angle displacement stages to adjust the pitch and roll angles during the coupling process between the graded refractive index lens and the microscope. Three displacement stages are used to compensate for the displacement of the graded refractive index lens in the X, Y, and Z axes caused by the angle adjustment. This allows for automatic adjustment of the angle between the graded refractive index lens and the central optical axis of the microscope, thereby reducing the angular deviation between the lens and the microscope and improving image quality.

[0089] The angle adjustment method of the present invention can quickly and accurately adjust the angle between the graded refractive index lens and the central optical axis of the microscope, so that the graded refractive index lens coincides with the central optical axis of the microscope, thereby reducing the aberration of the system and improving the image resolution, signal-to-noise ratio and tomography capability of the system imaging.

[0090] The present invention also provides a system for deep brain imaging of animals, including a microscopic imaging device, a graded refractive index lens disposed on the animal's head, and an angle adjustment device in the above embodiments;

[0091] In the process of coupling the microscope and the graded refractive index lens in the microscopic imaging device, the angle adjustment device in the above embodiment is used to adjust the angle between the optical axis of the microscope and the graded refractive index lens so that the optical axis of the microscope and the graded refractive index lens coincide.

[0092] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An angle adjustment device for a graded refractive index lens used for deep brain imaging, characterized in that, From bottom to top, it includes: a first displacement stage (8), a second displacement stage (7), a third displacement stage (6), a second electric angle displacement stage (5), a first electric angle displacement stage (4), and a sample support plate (3); Two electric angle displacement stages are coaxially mounted and their rotation centers coincide; the two electric angle displacement stages are used to adjust the pitch angle and roll angle during the coupling process between the graded refractive index lens and the microscope; the two electric angle displacement stages are the first electric angle displacement stage (4) and the second electric angle displacement stage (5). The second electric angle displacement stage (5) is fixed on the platform of the third displacement stage (6); the base of the third displacement stage (6) is fixed on the platform of the second displacement stage (7), and the base of the second displacement stage (7) is fixed on the platform of the first displacement stage (8); the three displacement stages are respectively used to reverse the displacement offset of the graded refractive index lens in the X-axis, Y-axis and Z-axis caused by the electric angle displacement stage during angle adjustment; the Z-axis is parallel to the direction of the central optical axis of the graded refractive index lens; the three displacement stages are the first displacement stage (8), the second displacement stage (7) and the third displacement stage (6); The sample carrier plate (3) is positioned above the first electric angle displacement stage (4) for placing animal samples and ensuring that the gradient refractive index lens of the animal's head is coaxial with the first electric angle displacement stage (4).

2. The angle adjustment device according to claim 1, characterized in that, The sample support plate (3) includes: a fixing plate (31), two support columns (32) and a base plate (33); The animal head is placed between the two pillars (32), which are fixed on the base plate (33). The fixing plate (31) is set on the two pillars (32) and is connected to the fixing plate on the gradient refractive index lens.

3. The angle adjustment device according to claim 1 or 2, characterized in that, The stage and the base are fixed together by M6 fixing screws; wherein the stage is the stage of the first displacement stage (8) or the second displacement stage (7), and the base is the base of the second displacement stage (7) or the third displacement stage (6).

4. A method for adjusting the angle adjusting device as described in any one of claims 1-3, characterized in that, include: S1. Determine the rotation radius, which is the distance between the upper surface of the gradient refractive index lens and the rotation center of the two electric angle displacement stages after the animal sample is placed. S2. Set the initial positions of the two electric angular displacement stages and the adjacent area centered on the initial positions; S3. Drive the two electric angle displacement stages to adjust the pitch angle and roll angle respectively in the vicinity of the initial position at this time with a determined step accuracy; at the same time, the three displacement stages perform reverse compensation on the displacement of the two electric angle displacement stages according to the rotation radius to obtain the position with the clearest image at this time. S4. Using the position where the image is clearest at this moment as the initial position, repeat S3 within the same adjacent area size until the position where the image is clearest is the same in at least two adjacent repetitions; at this moment, the position where the image is clearest is the position where the gradient refractive index lens coincides with the central optical axis of the microscope.

5. The adjustment method according to claim 4, characterized in that, In S1, the rotation radius is a first rotation radius R1 and a second rotation radius R2; determining the rotation radius includes: Measure the first rotation radius R1 and calculate the second rotation radius R2; the first rotation radius R1 is the distance between the upper surface of the gradient refractive index lens and the rotation center of the first electric angle displacement stage (4) after the animal sample is placed; where R2 = R1 + h, h represents the height between the stage of the second electric angle displacement stage (5) and the base of the first electric angle displacement stage (4); In S3, the three displacement stages perform reverse compensation on the displacements of the two electric angular displacement stages according to the rotation radius, including: The three displacement stages perform reverse compensation on the displacement of the first electric angle displacement stage (4) according to the first rotation radius R1; The three displacement stages perform reverse compensation on the displacement of the second electric angle displacement stage (5) according to the second rotation radius R2.

6. The method according to claim 4 or 5, characterized in that, In S3, the determined stepping accuracy is the optimal stepping accuracy for the corresponding animal sample, and determining the optimal stepping accuracy includes: S31. Initialize the angle adjustment range of the two electric angle displacement stages; S32. With the minimum step accuracy, the two electric angle displacement stages are traversed sequentially within the initialized angle adjustment range; at the same time, the three displacement stages are compensated for inverse displacement to obtain a microscopic image at each angle; and the image sharpness evaluation function is used to calculate the evaluation value corresponding to the microscopic image at each angle. S33. Determine an interval of the angle corresponding to the image with the largest evaluation value. If the microscopic images within the interval are all in focus, and the interval is the largest interval corresponding to all microscopic images being in focus, then the interval is the optimal step accuracy.

7. The method according to claim 6, characterized in that, The formulas for the three displacement stages to perform corresponding reverse displacement compensation based on the first rotation radius R1 or the second rotation radius R2 are as follows: Δx=RSin[θ e ]-RSin[θ i ] Δy=RSin[θ e ]-RSin[θ i ] Δz=RCos[θ e ]-RCos[θ i ] Wherein, Δx, Δy, and Δz represent the reverse compensation amounts for the displacement offsets of the two electric angular displacement stages in the X, Y, and Z axes, respectively; R is either R1 or R2. When R is R1, it indicates reverse compensation for the displacement of the first electric angular displacement stage (4); when R is R2, it indicates reverse compensation for the displacement of the second electric angular displacement stage (5); θ e θ represents the angle after the last movement of the two electrically driven angular displacement stages. i This indicates the angle of the two electrically driven angular displacement stages before their current movement.

8. The method according to claim 5, characterized in that, Measuring the first rotation radius R1 includes: S11. After placing the animal sample on the sample support plate (3), estimate the range of the first rotation radius; S12. Under the same first angle displacement stage deflection angle, traverse each estimated first rotation radius; at the same time, the three displacement stages perform reverse displacement compensation according to each estimated first rotation radius to obtain the corresponding microscopic image. S13. The sharpest microscopic image is determined using a sharpness evaluation function; the rotation radius corresponding to the sharpest microscopic image is the first rotation radius R1.

9. A system for deep brain imaging in animals, characterized in that, include: Microscopic imaging device, a graded refractive index lens disposed on the head of an animal, and the angle adjustment device as described in any one of claims 1-3; During the coupling process between the microscope and the graded refractive index lens in the microscopic imaging device, the angle adjustment device is used to adjust the angle between the central optical axis of the microscope and the graded refractive index lens so that the central optical axis of the microscope and the graded refractive index lens coincide.

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