Two-photon imaging device and imaging method

Abstract

This disclosure relates to a two-photon imaging device and method. The two-photon imaging device includes: a fixing component comprising a mounting frame and a carrier, the carrier being mounted on the mounting frame; a window component detachably mounted on the carrier and configured to be fixed at a target imaging position; a position adjustment component mounted on the side of the carrier away from the window component; and a two-photon imaging component mounted on the position adjustment component, configured to image through the window component, and capable of adjusting the imaging position via the position adjustment component. This imaging device can achieve stable image quality.

Description

Technical Field

[0001] This disclosure relates to the field of biological imaging technology, and in particular to a two-photon imaging device and imaging method. Background Technology

[0002] Brain-computer interfaces (BCIs) are used to connect the brain to external devices. They can directly decode the activity of neurons in the brain and translate neural intentions into control commands for external devices, which is an important way to solve this problem.

[0003] Among numerous model organisms, non-human primates (such as rhesus macaques and cynomolgus monkeys) share a high degree of homology with humans in terms of cerebral cortex structure, neural circuit connectivity patterns, and higher cognitive functions (vision, decision-making, learning and memory, and social interaction). They serve as ideal large animal models for studying human brain cognitive mechanisms, brain disease pathology, and the clinical translation of brain-computer interfaces, possessing irreplaceable scientific research value and clinical guiding significance. Two-photon fluorescence microscopy, based on the nonlinear absorption principle of near-infrared femtosecond lasers, excites fluorescence only at the focal point. It naturally possesses unique advantages such as deep penetration, inherent optical slicing, submicron-level spatial resolution, low phototoxicity, and low photobleaching, perfectly matching the deep imaging needs of the thick cortex of primates and becoming the core mainstream technology for dynamic observation of in vivo brain nerves.

[0004] Although desktop two-photon imaging has made significant breakthroughs in primate cognition and disease research, imaging primates in a conscious state remains a challenge. Summary of the Invention

[0005] The embodiments of this disclosure provide a two-photon imaging device and imaging method that can obtain stable imaging quality at the target imaging position.

[0006] The first aspect of this disclosure provides a two-photon imaging apparatus, comprising: The fixing component includes a fixing frame and a support base, with the support base mounted on the fixing frame; The window assembly is detachably mounted on the carrier and is configured to be fixed at the target imaging position; A position adjustment component is mounted on the side of the support away from the window component; and The two-photon imaging component, mounted on the position adjustment component, is configured to image through the window component and the imaging position can be adjusted by the position adjustment component.

[0007] In some embodiments, the mounting bracket includes: Two uprights are spaced apart along a predetermined direction in the horizontal plane; Two mounting brackets, located at the bottom area of ​​the upright, are configured to fix the upright; and Two horizontal bars are connected to the top areas of the two vertical bars respectively; The support base is installed on two horizontal bars at both ends and is located between two vertical bars, while the window assembly is located between the two horizontal bars.

[0008] In some embodiments, the mounting bracket further includes: Two adapters are connected between one end of the crossbar and the upright. The adapters have a first hole and a second hole that are perpendicular to each other. The upright passes through the first hole and the crossbar passes through the second hole. The adapter can be adjusted in height along the upright and / or in lateral position along the crossbar.

[0009] In some embodiments, a first through groove is provided on the side wall of the first hole near the first end of the adapter so that the first end of the adapter forms a first clamp, and the first clamp is connected and locked to the upright rod by fasteners; a second through groove is provided on the side wall of the second hole near the second end of the adapter so that the second end of the adapter forms a second clamp, and the second clamp is locked to the crossbar by fasteners.

[0010] In some embodiments, when the first clamp and mounting base are unlocked, the adapter can be rotatably adjusted relative to the upright; when the second clamp is unlocked, the crossbar can be rotatably adjusted relative to the adapter.

[0011] In some embodiments, the mounting base includes a base plate and a third clamp, the third clamp including a surrounding portion and two engaging portions, the surrounding portion surrounding the outer side wall of the upright and having an opening, and the side of the surrounding portion away from the opening being fixed to the base plate, the two engaging portions being respectively connected to the two ends of the surrounding portion, and the two engaging portions being connected by fasteners to lock the upright.

[0012] In some embodiments, a cut is provided between the third clamp and the substrate, and the third clamp is fixed to the substrate through the portion other than the cut; the inner sidewall of the surrounding portion is provided with a first recess at the inner end of the cut, and / or the outer sidewall of the surrounding portion is provided with a second recess at the inner end of the cut.

[0013] In some embodiments, the crossbar has a flat surface for mounting a support seat, and a set of mounting holes is provided at the position where the support seat connects to the crossbar. The set of mounting holes includes a positioning hole and an arc-shaped adjustment hole. The support seat is mounted to the crossbar by fasteners that pass through the positioning hole and the adjustment hole respectively.

[0014] In some embodiments, the support base is provided with a plurality of fixing hole groups, which are located on the outer periphery of the window assembly, and the position adjustment assembly can be selectively installed in one of the fixing hole groups; and / or The window assembly includes a transparent window panel, a support base with an opening, and multiple fasteners arranged around the opening on the support base to secure the window assembly. The window panel is located within the opening; and / or The support base is equipped with a positioning groove, and the window component is embedded in the positioning groove.

[0015] In some embodiments, the window assembly includes: a base, a locking ring, and a transparent window panel, the window panel being embedded within the locking ring, the base being disc-shaped and having a mounting hole, and the locking ring engaging with the mounting hole via a thread.

[0016] In some embodiments, the base is provided with a plurality of through holes spaced apart around the outer periphery of the mounting hole, the through holes being configured to be mounted at the target imaging position by means of a fastener; and / or The end face of the base facing the two-photon imaging assembly has an annular inner conical surface; and / or The locking ring has an operating part on its end face facing the two-photon imaging assembly. The operating part is configured to receive external operation to unscrew the locking ring.

[0017] In some embodiments, the window assembly includes a transparent window pane, the two-photon imaging assembly includes a lens that images through the window pane, and the imaging area of ​​the lens is smaller than the area of ​​the window pane. The two-photon imaging assembly is configured to obtain local images of different positions of the window pane by moving, so as to stitch them together to form a whole image.

[0018] In some embodiments, the position adjustment component includes: Base; The first connecting seat is fixed to the base; The first slider engages with the first connecting seat and its position is adjustable along the first direction; The second connecting seat is connected to the first slider; The second slider engages with the second connecting seat and is position-adjustable along a second direction, which is perpendicular to the first direction. The third slider cooperates with the second slider and its position is adjustable along a third direction, which is perpendicular to the first and second directions; a two-photon imaging component is installed at the end of the third slider near the base.

[0019] In some embodiments, the position adjustment component further includes: A three-dimensional micro-manipulation component, connected between a first slider and a second connecting seat, is configured to fine-tune the position of the two-photon imaging component along a first direction, a second direction, and a third direction.

[0020] In some embodiments, the two-photon imaging device further includes a clamp assembly mounted on a mounting frame and configured to limit the fiber optic cable of the two-photon imaging assembly.

[0021] A second aspect of this disclosure provides an imaging method based on the two-photon imaging apparatus of the above embodiments, comprising: Fix the window component to the area of ​​the head where imaging is needed; Fix the support base to the window component; Install the mounting bracket onto the support base and fix the mounting bracket in the preset position; The position adjustment component containing the two-photon imaging unit is mounted on the carrier. Imaging is performed through a window component using a two-photon imaging component.

[0022] In some embodiments, the step of imaging through a window component using a two-photon imaging component includes: The position adjustment component allows the lens of the two-photon imaging component to be positioned at different locations within the window component, thereby obtaining local images at different locations. The individual images are stitched together to form the overall image.

[0023] In some embodiments, after mounting the position adjustment assembly on the carrier with the two-photon imaging component, the imaging method further includes: The fiber optic cable of the two-photon imaging component is fixed by a clamp assembly, which is mounted on a mounting bracket.

[0024] In some embodiments, the imaging method further includes: After imaging is complete, remove the position adjustment component and place it on the support base; Remove the support from the mounting bracket; Remove the entire mounting bracket.

[0025] Based on the above technical solution, the two-photon imaging device of this disclosure fixes a window assembly at the target imaging position and provides a support on the mounting frame to install the two-photon imaging component. Since the support and window assembly are fixed, even if the target imaging position moves while the animal is awake, the relative position between the two-photon imaging component and the target imaging position remains fixed, eliminating interference with image quality caused by body movement. This improves the stability and accuracy of imaging. In particular, when the mounting frame is fixed in a preset position, movement of the target imaging position is also restricted, resulting in more stable imaging.

[0026] Furthermore, the two-photon imaging module is mounted on the support base via a position adjustment component, allowing for adjustment of its position during imaging. This facilitates flexible adjustment to the target imaging position or acquisition of imaging of different regions. The two-photon imaging module and position adjustment component are mounted on the side of the support base away from the window component, facilitating easy adjustment and preventing interference with body parts during position adjustment. The support base also provides a transitional load, transferring most of the imaging device's weight to the mounting frame, thus distributing the load and preventing weight concentration on the window component. This reduces the risk of the window component becoming loose, resulting in highly stable two-photon imaging images. Attached Figure Description

[0027] The accompanying drawings, which are included to provide a further understanding of this disclosure and form part of this application, illustrate exemplary embodiments of the present disclosure and are used to explain the disclosure, but do not constitute an undue limitation thereof. In the drawings: Figure 1 This is a schematic diagram of the structure of some embodiments of the two-photon imaging device disclosed herein in use.

[0028] Figure 2 This is a schematic diagram of the structure of some embodiments of the window component in the two-photon imaging device of this disclosure.

[0029] Figure 3 This is a schematic diagram of the structure of some embodiments of the position adjustment component in the two-photon imaging device of this disclosure.

[0030] Figure 4 This is a schematic diagram of the structure of some embodiments of the wire clamp assembly in the two-photon imaging device of this disclosure.

[0031] Figure 5 This is a schematic diagram of the structure of some embodiments of the fixed components in the two-photon imaging device of this disclosure.

[0032] Figure 6 for Figure 5 Schematic diagrams of some embodiments of the fixed base.

[0033] Figure 7 This is a schematic diagram of the position adjustment component placed on the support base when not in use.

[0034] Figure 8 This is a schematic diagram of the displacement distribution in the XY plane during two-photon imaging.

[0035] Figure 9 This is a graph showing the displacement of two-photon imaging in the XY plane over time.

[0036] Explanation of reference numerals in the attached figures 1. Window assembly; 11. Window piece; 12. Locking ring; 121. Operating part; 13. Base; 131. Threaded hole; 132. Inner conical surface; 133. Through hole; 14. Fixing component; 2. Position adjustment component; 21. Base; 22. First connecting seat; 23. First slider; 24. Three-dimensional micro-manipulation component; 25. Second connecting seat; 26. Second slider; 27. Limiting component; 28. Third slider; 3. Wire clamp assembly; 31. First part; 311. First fixing hole; 312. Groove; 313. Cable routing groove; 32. Second part; 33. Pull rod; 34. Magnet; 35. Protrusion; 36. Hinge shaft; 4. Fixing component; 41. Third clamp; 411. Surrounding part; 412. Engaging part; 413. First recessed part; 414. Second recessed part; 42. Base plate; 421. Fourth fixing hole; 43. Fastener; 44. Upright post; 441. Third fixing hole; 45. Crossbar; 451. Flat surface; 46. Support; 461. Annular part; 462. Extension part; 463. Positioning hole; 464. Adjustment hole; 465. Second fixing hole; 466. Opening; 48. Adapter; 481. First hole; 482. Second hole; 483. First through groove; 484. Second through groove; 5. Target imaging location; 6. Fiber optic cables; 7. Two-photon imaging assembly; 71. Lens; 72. Main body; 8. Support base; 81. Fixed base; 82. Support column; X, first direction; Y, second direction; Z, third direction. Detailed Implementation

[0037] Various exemplary embodiments of the present disclosure will now be described in detail with reference to the accompanying drawings. The descriptions of the exemplary embodiments are merely illustrative and are in no way intended to limit the present disclosure or its application or use. The present disclosure may be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are provided so that the present disclosure will be thorough and complete, and will fully express the scope of the disclosure to those skilled in the art. It should be noted that, unless specifically stated otherwise, the relative arrangement of components and steps, the composition of materials, numerical expressions, and values ​​set forth in these embodiments should be interpreted as exemplary only and not as limiting.

[0038] The terms "first," "second," and similar words used in this disclosure do not indicate any order, quantity, or importance, but are merely used to distinguish different parts. Words such as "including" or "contains" mean that the element preceding the word encompasses the element listed after it, and do not exclude the possibility of encompassing other elements as well. Terms such as "above," "below," "left," and "right" are used only to indicate relative positional relationships; when the absolute position of the described object changes, this relative positional relationship may also change accordingly.

[0039] In this disclosure, when a specific device is described as being located between a first device and a second device, an intermediary device may or may not be present between the specific device and the first or second device. When a specific device is described as being connected to other devices, the specific device may be directly connected to the other devices without an intermediary device, or it may be not directly connected to the other devices but have an intermediary device.

[0040] All terms used in this disclosure (including technical or scientific terms) have the same meaning as understood by one of ordinary skill in the art to which this disclosure pertains, unless otherwise specifically defined. It should also be understood that terms defined in a general dictionary, such as a dictionary, should be interpreted as having a meaning consistent with their meaning in the context of the relevant art, and not as having an idealized or highly formalized meaning, unless expressly defined herein.

[0041] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, they should be considered part of the specification.

[0042] Based on the embodiments disclosed above, in the absence of explicit denial or conflict, the technical features of one embodiment may be advantageously combined with one or more other embodiments.

[0043] Currently, when imaging the heads of primates using two-photon imaging devices, their movement is typically restricted, and the head is imaged using a separate two-photon imaging device. However, due to the large size of primates and the restricted animal movement, experiments suffer from inherent defects such as stress responses, behavioral distortions, and low ecological validity, making it difficult to obtain stable image quality. Furthermore, the curved shape of the brain, the imaging region, also presents a significant challenge for imaging devices. Therefore, achieving single-cell resolution, long-term, and stable neural observations in freely moving primates has become a core direction urgently needing breakthroughs in current brain science and brain-inspired research.

[0044] like Figures 1 to 7 As shown, this disclosure provides a two-photon imaging device, hereinafter referred to as the "imaging device", which in some embodiments includes: The fixing component 4 includes a fixing frame and a support base 46, with the support base 46 mounted on the fixing frame; Window assembly 1 is detachably mounted on the support 46, and window assembly 1 is configured to be fixed at the target imaging position 5; Position adjustment component 2 is installed on the side of the support 46 away from window component 1; and The two-photon imaging component 7 is mounted on the position adjustment component 2, is configured to image through the window component 1, and can adjust the imaging position through the position adjustment component 2.

[0045] For example, two-photon imaging devices can be used on animals, such as non-human primates, including monkeys such as macaques or cynomolgus monkeys. Such two-photon imaging devices can be head-mounted and fixed to the target imaging position 5, for example, fixed to the animal's head to image the brain, or they can be fixed to other body parts of the animal.

[0046] A window assembly 1 is installed at the target imaging position 5. For example, the window assembly 1 is fixed to the head of the animal. Specifically, the window assembly 1 is embedded in the opening of the imaging brain region and fixed to the target imaging position 5 by a fastener 14. For example, the fastener 14 can be a bone nail or the like.

[0047] The fixing component 4 includes a mounting frame and a support 46, the mounting frame being configured to be fixed in a preset position. For example, when imaging non-human primates, the mounting frame can be fixed to a monkey chair. The fixing component 4 can be designed as a head-mounted device, the support 46 is mounted on the mounting frame, and the support 46 is fixedly connected to the window component 1 and detachably mounted. The two-photon imaging component 7 is mounted on the side of the support 46 away from the window component 1 via the position adjustment component 2. The two-photon imaging component 7 includes a main body 72 and a lens 71, the lens 71 being mounted on the main body 72 and used for imaging through the window component 1.

[0048] This embodiment fixes the window assembly 1 at the target imaging position 5 and provides a support 46 on the mounting frame to install the two-photon imaging assembly 7. Because the support 46 is fixed to the window assembly 1, even if the animal moves at the target imaging position 5 while awake, the relative position between the two-photon imaging assembly 7 and the target imaging position 5 remains fixed, eliminating interference with image quality caused by body movement, thereby improving imaging stability and accuracy. In particular, when the mounting frame is fixed in a preset position, the movement of the target imaging position 5 is also restricted, resulting in more stable imaging.

[0049] Furthermore, the two-photon imaging component 7 is mounted on the support 46 via the position adjustment component 2, allowing for adjustment of its position during imaging. This facilitates flexible adjustment to the target imaging position or the acquisition of imaging in different areas. The two-photon imaging component 7 and the position adjustment component 2 are mounted on the side of the support 46 away from the window component 1, making adjustment convenient and preventing interference with body parts during position adjustment. The support 46 also provides a transitional load, transferring most of the imaging device's weight to the mounting frame, thus distributing the load and preventing weight concentration on the window component 1. This reduces the risk of the window component 1 becoming loose, resulting in highly stable two-photon imaging images.

[0050] In some embodiments, the window assembly 1 includes a transparent window 11, through which the lens 71 of the two-photon imaging assembly 7 images, and the imaging area of ​​the lens 71 is smaller than the area of ​​the window 11. The two-photon imaging assembly 7 is configured to obtain local images of different positions of the window 11 by moving, so as to stitch together an overall image.

[0051] This embodiment can reduce the size of the lens 71, which is conducive to the lightweighting of the imaging device, thereby reducing the weight on the animal's head, reducing the stress response during the experiment, and obtaining more stable imaging quality. On the other hand, the small field of view lens has stronger fluorescence signal and higher imaging resolution in single-frame imaging. The position adjustment component 2 drives the lens 71 to scan and stitch images in sections, which can fully cover the entire observation area of ​​the window 11 while taking into account high-precision imaging.

[0052] In some embodiments, such as Figure 5 As shown, the mounting bracket includes: Two uprights 44 are spaced apart along a predetermined direction in the horizontal plane; Two mounting brackets, located at the bottom area of ​​the upright 44, are configured to fix the upright 44; and Two horizontal bars 45 are connected to the areas near the top of two vertical bars 44 respectively; The bearing seat 46 is installed at both ends on two horizontal bars 45 and is located between two vertical bars 44, and the window assembly 1 is located between the two horizontal bars 45.

[0053] Specifically, two horizontal bars 45 are spaced apart to avoid obstructing the window assembly 1, and the support base 46 is detachably installed at both ends of the horizontal direction onto the inner ends of the two horizontal bars 45. For example, the support base 46 may have a plate-like structure, including an annular portion 461 and two extensions 462, with the two extensions 462 respectively connected to both ends of the annular portion 461. The window assembly 1 is installed on one side of the annular portion 461, and the two extensions 462 are respectively installed at the inner ends of the two horizontal bars 45. Thus, the two uprights 44, the two horizontal bars 45, and the support base 46 form a headband-style support frame.

[0054] This embodiment uses two uprights 44 and a crossbar 45 to form a stable mounting frame. The mounting base at the bottom of the uprights 44 can lock and fix the entire mounting frame, for example, by assembling the mounting frame onto a monkey chair, thereby stably supporting the weight of the two-photon imaging assembly 7 and the position adjustment assembly 2. Moreover, the support base 46 is installed between the two uprights 44, for example, in the middle area between the two uprights 44. After the support base 46 is fixed to the window assembly 1, the animal can be positioned between the two uprights 44.

[0055] In addition, by setting two crossbars 45 at intervals, space can be left for the installation of window assembly 1. When it is necessary to install the imaging device, the support 46 can be fixed to window assembly 1 first, and then the uprights 44 and crossbars 45 can be installed side by side. There is no need to install the heavy fixing component 4 as a whole on window assembly 1, which can reduce the assembly difficulty.

[0056] In some embodiments, such as Figure 5As shown, the fixing frame also includes: two adapters 48, which are connected between one end of the crossbar 45 and the upright 44. The adapters 48 are provided with a first hole 481 and a second hole 482 that are perpendicular to each other. The upright 44 passes through the first hole 481 and the crossbar 45 passes through the second hole 482. The adapters 48 are adjustable in height along the upright 44 and / or adjustable in lateral position along the crossbar 45.

[0057] The uprights 44, the crossbars 45, and the adapters 48 can all be cylindrical. An adapter 48 is provided between one end of each upright 44 and the crossbar 45. The adapter 48 has a first hole 481 and a second hole 482 that are perpendicular to each other and are spaced apart along its length. The first hole 481 can be set vertically, and the second hole 482 can be set horizontally.

[0058] This embodiment connects the uprights 44 and the crossbar 45 using an adapter 48, which facilitates more stable assembly and easier connection, and eliminates the need to process and install interfaces on the uprights 44 and the crossbar 45. Moreover, it enables more stable operation and flexible adjustment when adjusting the height of the crossbar 45, the horizontal distance between the two uprights 44, and the lateral position of the support 46 relative to the uprights 44, thus adapting to different experimental needs.

[0059] In some embodiments, a first through groove 483 is provided on the side wall of the first hole 481 near the first end of the adapter 48, so that the first end of the adapter 48 forms a first clamp, and the first clamp is connected to and locks the upright 44 through a fastener 43; a second through groove 484 is provided on the side wall of the second hole 482 near the second end of the adapter 48, so that the second end of the adapter 48 forms a second clamp, and the second clamp is locked to the crossbar 45 through a fastener 43. For example, the fastener 43 can be a bolt, etc.

[0060] This embodiment can easily lock and unlock the uprights 44 and the crossbar 45. When locked, the uprights 44 and the crossbar 45 can be stably supported and fixed. When unlocked, the height of the crossbar 45 or the lateral position of the support 46 can be flexibly adjusted to match the position of the target imaging position 5, or the distance between the two uprights 44 can be adjusted to match different animal body shapes.

[0061] In some embodiments, when the first clamp and mounting base are unlocked, the adapter 48 is rotatably adjustable relative to the upright 44; when the second clamp is unlocked, the crossbar 45 is rotatably adjustable relative to the adapter 48.

[0062] Specifically, the upright 44 rotates within the first hole 481, and with the bottom end of the upright 44 fixed, the crossbar 45 and the support seat 46 can swing in the horizontal plane. The crossbar 45 swings within the second hole 482, which can adjust the tilt angle of the support seat 46 relative to the horizontal plane.

[0063] For example, if you want to image the head of an animal, which is curved, you can adjust the bearing 46 by rotating it so that it fits well with the target imaging position 5.

[0064] This embodiment allows the support 44 and the crossbar 45 to be rotated and adjusted relative to the adapter 48. This enables the support 46 to be initially positioned by adjusting its height and lateral position. Furthermore, it allows the support 46 to be swung in the horizontal plane and its tilt angle relative to the horizontal plane, thereby better fitting with the target imaging position 5 and obtaining a better imaging angle.

[0065] In some embodiments, such as Figure 5 and Figure 6 As shown, the mounting base includes a base plate 42 and a third clamp 41. The third clamp 41 includes a surrounding portion 411 and two engaging portions 412. The surrounding portion 411 surrounds the outer wall of the upright 44 and has an opening. The side of the surrounding portion 411 away from the opening is fixed to the base plate 42. The two engaging portions 412 are respectively connected to the two ends of the surrounding portion 411, and the two engaging portions 412 are connected by fasteners 43 to lock the upright 44.

[0066] The substrate 42 may be rectangular and has multiple fourth fixing holes 421 thereon to fix the substrate 42 in a preset position by fasteners, for example, when imaging primates, the mounting base is fixed to a monkey chair.

[0067] The mounting base of this embodiment can easily and quickly lock the bottom of the upright 44, making it easy to assemble and disassemble. It can also provide stable restraint, prevent the fixing frame from shaking, improve imaging stability, and can be adapted to uprights 44 with different outer diameters, making it more versatile.

[0068] In some embodiments, such as Figure 6 As shown, a cut is provided between the third clamp 41 and the substrate 42, and the third clamp 41 is fixed to the substrate 42 through the part other than the cut; the inner sidewall of the surrounding part 411 is provided with a first recess 413 at the inner end of the cut, and / or the outer sidewall of the surrounding part 411 is provided with a second recess 414 at the inner end of the cut.

[0069] like Figure 5 and Figure 6 As shown, in order to achieve the elastic expansion and contraction of the surrounding part 411, a cut is provided between the bottom of the third clamp 41 and the base plate 42, which can increase the elasticity of the surrounding part 411 at this position and can more reliably lock the upright 44. The outer diameter of the surrounding part 411 is D, the inner diameter is E, and the cut extends from the outer end of the engaging part 412 to position B of the surrounding part 411.

[0070] The first recess 413 includes arcuate segments A1, A2, and A3, wherein A1 and A3 are tangent to the inner wall of the surrounding portion 411, and the height of A2 is ≤ (E+D) / 2. The second recess 414 includes arcuate segments C1, C2, and C3, wherein C1 and C3 are tangent to the outer wall of the surrounding portion 411, and the height of C2 is ≤ (E+D) / 2.

[0071] This embodiment, by providing a slit between the bottom of the third clamp 41 and the base plate 42, allows the two engaging portions 412 to be elastically extended and retracted, enabling the locking and unlocking of the upright rod 44 via the fastener 43. Furthermore, by providing a first recess 413 and / or a second recess 414 at the inner end of the slit, the elasticity of the third clamp 41 is increased, thereby increasing the clamping force on the upright rod 44 during locking and achieving stable and reliable fixation.

[0072] In some embodiments, such as Figure 5 As shown, the crossbar 45 has a flat surface 451 for mounting the support seat 46. The position where the support seat 46 connects to the crossbar 45 is provided with a set of mounting holes, which includes a positioning hole 463 and an arc-shaped adjustment hole 464. The support seat 46 is mounted on the crossbar 45 by fasteners 43 that pass through the positioning hole 463 and the adjustment hole 464 respectively.

[0073] For example, the adjustment hole 464 may be arc-shaped, with its center coinciding with the center of the positioning hole 463, and the adjustment hole 464 is located inside the positioning hole 463. A mounting hole group is provided on each of the two extensions 462 of the support 46, and the two mounting hole groups are symmetrically arranged with respect to the window assembly 1.

[0074] This embodiment allows the support base 46 to be stably fixed to the crossbar 45 by providing a plane 451 on the crossbar 45. Furthermore, by fixing the support base 46 to the crossbar 45 through positioning holes 463 and adjustment holes 464, reliable fixing is achieved, and the installation angle of the support base 46 relative to the crossbar 45 can be finely adjusted according to the target imaging position 5 to adapt to its shape. After the mounting hole groups on both sides are fixed, the support base 46 can be limited in position.

[0075] For example, when imaging the brain of a primate, since the imaging area is usually curved, the fixing method needs to be flexible and stable. Adjustment is made through the arc-shaped adjustment hole 464 and the position is adjusted by the adapter 48, so that universal adjustment can be achieved to adapt to the curved surface.

[0076] In some embodiments, such as Figure 5 As shown, the support base 46 is provided with multiple fixing hole groups, which are located on the outer periphery of the window assembly 1. The position adjustment assembly 2 can be selectively installed in one of the fixing hole groups.

[0077] For example, a group of fixing holes is located on the outer periphery of the opening 466. Two groups of fixing holes are provided between the opening 466 and the mounting hole groups on each side. These two groups are spaced apart longitudinally perpendicular to the transverse direction within the extending plane of the support 46. Each group of fixing holes may include two second fixing holes 465. Thus, the opening 466 has four second fixing holes 465 on each side of the transverse direction, spaced apart longitudinally. By providing fixing hole groups at the upper left, lower left, upper right, and lower right, the position adjustment component 2 is installed in one of the fixing hole groups according to actual imaging requirements. This minimizes the adjustment range of the position adjustment component 2, reducing shaking caused by position adjustment and improving imaging stability.

[0078] In this embodiment, multiple fixing hole groups are provided around the outer periphery of the window component 1 on the support 46. The position adjustment component 2 can be installed in one of the fixing hole groups according to the actual imaging requirements, so as to minimize the adjustment range of the position adjustment component 2, reduce the shaking caused by position adjustment, and improve imaging stability.

[0079] In some embodiments, such as Figure 2 and Figure 5 As shown, the window assembly 1 includes a transparent window sheet 11. An opening 466 is provided on the support 46, and multiple fasteners 43 are arranged around the opening 466 to fix the window assembly 1. The window sheet 11 is located within the opening 466. This structure fixes the window assembly 1 to the back of the support 46, and the transparent window sheet 11 can be exposed through the opening 466, allowing the two-photon imaging assembly 7 to perform imaging through the window sheet 11.

[0080] In some embodiments, the support 46 is provided with a positioning groove, and the window assembly 1 is embedded in the positioning groove. For example, the window assembly 1 is disc-shaped, the positioning groove has a circular cross-section, and its size matches the outer periphery of the window assembly 1.

[0081] For example, the support 46 has an annular step to form a positioning groove.

[0082] This embodiment uses a positioning groove on the support 46 to position the window component 1 and the support 46, enabling quick installation. During assembly, alignment can be achieved without repeated adjustments. Furthermore, the sidewall of the positioning groove can constrain the window component 1, ensuring the installation position accuracy of the window component 1.

[0083] In some embodiments, such as Figure 2 As shown, the window assembly 1 includes: a base 13, a locking ring 12, and a transparent window piece 11. The window piece 11 is embedded in the locking ring 12. The base 13 is disc-shaped and has a mounting hole. The locking ring 12 is threaded into the mounting hole.

[0084] For example, both the locking ring 12 and the base 13 can be made of titanium alloy, and the diameter of the imaging area of ​​the window 11 can be up to 25 mm. The target imaging position 5 can fix the window assembly 1, and the base 13 has a protrusion on the side away from the support 46, which can be embedded into the hole of the target imaging position 5. The window 11 is embedded in the locking ring 12 and can be fixed together by a biocompatible curing adhesive. The locking ring 12 and the base 13 are connected by threads.

[0085] In this embodiment, the window piece 11 is installed in the base 13 by a locking ring 12, which makes disassembly and assembly convenient. The window piece 11 can be removed for cleaning and replacement by loosening the locking ring 12. Moreover, the window piece 11 can be reliably fixed by the threaded engagement, and it is not easy to damage the window piece 11.

[0086] In some embodiments, such as Figure 2 As shown, the base 13 is provided with a plurality of through holes 133 at intervals around the outer periphery of the mounting hole, and the through holes 133 are configured to be mounted at the target imaging position 5 by means of the fastener 14.

[0087] Among them, multiple threaded holes 131 are provided at intervals on the outer periphery of multiple through holes 133, such as Figure 5 As shown, multiple fasteners 43 arranged around the outer periphery of the opening 466 pass through multiple threaded holes 131 in a one-to-one correspondence to fix the base 13 to the carrier 46. For example, the fastener 14 can be a bone screw or the like. When the window assembly 1 is not installed at the target imaging position 5, the window piece 11 can be protected by covering the surface of the base 13 with a protective sheet.

[0088] This embodiment can reliably fix the base 13 to the target imaging position 5 through multiple fasteners 14, which is beneficial to achieving stable imaging.

[0089] In some embodiments, such as Figure 2 As shown, the end face of the base 13 facing the two-photon imaging assembly 7 is provided with an annular inner conical surface 132.

[0090] The two-photon imaging assembly 7 includes a main body 72 and a lens 71. The lens 71 is mounted on the main body 72 and is used for imaging through the window assembly 1. In this embodiment, when the lens 71 images through the window 11, especially when it moves to an area near the edge of the window 11, the inner conical surface 132 can avoid the main body 72, so that the lens 71 can reliably image over the entire area of ​​the window 11.

[0091] In some embodiments, such as Figure 2 As shown, the locking ring 12 has an operation part 121 on its end face facing the two-photon imaging assembly 7. The operation part 121 is configured to receive external operation to unscrew the locking ring 12.

[0092] For example, the operating part 121 can be a groove, a boss, or other structure that facilitates the application of force by external auxiliary tools.

[0093] In this embodiment, by providing an operating part 121 on the end face of the locking ring 12, the locking ring 12 can be easily unscrewed from the base 13 by using an auxiliary tool to operate on the operating part 121, so as to clean the window piece 11.

[0094] In some embodiments, such as Figure 3 As shown, the position adjustment component 2 includes: Base 21; The first connecting seat 22 is fixed on the base 21; The first slider 23 is engaged with the first connecting seat 22 and its position is adjustable along the first direction X. The second connecting seat 25 is connected to the first slider 23; The second slider 26 is engaged with the second connecting seat 25 and its position is adjustable along the second direction Y, which is perpendicular to the first direction X. The third slider 28 cooperates with the second slider 26 and is adjustable in position along the third direction Z, which is perpendicular to the first direction X and the second direction Y; the two-photon imaging component 7 is installed at the end of the third slider 28 near the base 21.

[0095] The base 21 has a rectangular structure, on which a first connecting seat 22 is fixedly mounted. A first slider 23 is disposed on the top of the first connecting seat 22 and engages with the first connecting seat 22 via a dovetail groove. The first slider 23 is slidable relative to the first connecting seat 22 along a first direction X. Fasteners are provided on the side of the first connecting seat 22, allowing the position of the first slider 23 to be adjusted when loosened, and locking the first slider 23 in place using the fasteners. A second connecting seat 25 may be directly or indirectly disposed on the top of the first slider 23. A second slider 26 is located on one side of the second connecting seat 25 along the first direction X, and engages with the second connecting seat 25 via a dovetail groove. The position of the second slider 26 relative to the second connecting seat 25 along a second direction Y is adjustable. Fasteners are provided on the side of the second connecting seat 25, allowing the position of the second slider 26 to be adjusted when loosened, and locking the second slider 26 in place using the fasteners. The second slider 26 has a U-shaped locking member on the side away from the second connecting seat 25 along the first direction X. The third slider 28 has a long rod-shaped structure. The third slider 28 is embedded in the U-shaped locking member and is adjustable in position along the third direction Z. The side of the U-shaped locking member has a fastener, which can adjust the position of the third slider 28 when it is loosened. A limiting member 27 can be provided at the top of the third slider 28. The limiting member 27 can be a flat plate structure to limit the upward displacement of the second slider 26. The bottom of the third slider 28 is equipped with a two-photon imaging assembly 7, and the main body 72 of the two-photon imaging assembly 7 is connected to the bottom of the third slider 28.

[0096] The center of gravity of the position adjustment component 2 and the two-photon imaging component 7 can be located between the second slider 26 and the third slider 28, ensuring the stability of the entire component when worn on the head and improving imaging quality. Furthermore, the dimensions of the first connecting seat 22 and the first slider 23 along the first direction X can be reduced, making the device more compact.

[0097] In this embodiment, the position adjustment component 2 can flexibly adjust the position of the two-photon imaging component 7 in three directions to obtain imaging of the entire range of the window component 1. Moreover, the displacement adjustment in the three directions is independent of each other, and the displacement in a specific direction can be precisely adjusted as needed. Furthermore, the overall structure is compact and can reduce weight.

[0098] In some embodiments, such as Figure 3 As shown, the position adjustment component 2 also includes: The three-dimensional micro-manipulation component 24, connected between the first slider 23 and the second connecting seat 25, is configured to finely adjust the position of the two-photon imaging component 7 along the first direction X, the second direction Y, and the third direction Z.

[0099] For example, the diameter of the window piece 11 is 25mm, the adjustment amount through the dovetail groove in the first direction X and the second direction Y can be ±12.5mm, the adjustment amount through the dovetail groove in the third direction Z is 30mm, the clearance tolerance at the dovetail groove mating is 0.02mm, and the travel of the three-dimensional micro-manipulation component 24 in the three directions is ±2mm.

[0100] like Figure 7 As shown, after assembly, the position adjustment component 2 can be placed on the support base 8. The support base 8 includes a fixed base 81 and a support column 82 connected to the fixed base 81. The support base 8 can be magnetically fixed to the platform, and the base 21 of the position adjustment component 2 can be installed on the top of the support column 82 using fasteners 43. When installation is required, the position adjustment component 2 can be removed from the support base 8.

[0101] Based on the initial adjustment of the position of the two-photon imaging component 7 by the first slider 23, the second slider 26 and the third slider 28, this embodiment further adjusts the position of the two-photon imaging component 7 precisely by the three-dimensional micro-manipulation component 24. It can accurately position the local area of ​​the target imaging position 5 for imaging, and can also stitch the images of multiple local areas to form a whole image with better quality.

[0102] In some embodiments, such as Figure 1 As shown, the two-photon imaging device also includes a wire clamp assembly 3, which is mounted on a mounting frame and configured to limit the fiber optic cable 6 of the two-photon imaging assembly 7.

[0103] For example, the wire clamp assembly 3 is located at the top of the upright 44. Figure 4As shown, the wire clamp assembly 3 includes a first part 31 and a second part 32. One end of the first part 31 is provided with a groove 312, and one end of the second part 32 is provided with a protrusion 35 facing the first part 31. The protrusion 35 is embedded in the groove 312, and the protrusion 35 and the part of the first part 31 located on both sides of the groove 312 are connected by a hinge shaft 36.

[0104] The first part 31 has a cable tray 313 on the side facing the second part 32. The bottom of the cable tray 313 has a first fixing hole 311 for mounting to the top of the upright 44 with fasteners. The end of the second part 32 away from the hinge axis 36 has a pull rod 33. Magnets 34 are provided on the opposing surfaces of the first part 31 and the second part 32 away from the hinge axis 36. After the fiber optic cable 6 is inserted into the cable tray 313, the second part 32 is fastened to the first part 31 and attracted by the magnets 34. When it is necessary to remove the fiber optic cable 6, the second part 32 is opened relative to the first part 31.

[0105] Since the two-photon imaging component 7 has sub-micron resolution, even slight jitter has a significant impact on imaging quality. In this embodiment, by setting the wire clamp component 3 on the fixing frame, the fiber optic cable 6 of the two-photon imaging component 7 can be limited and fixed to prevent the fiber optic cable 6 from vibrating due to shaking during imaging, thereby improving imaging stability.

[0106] Secondly, this disclosure provides an imaging method based on the two-photon imaging device of the above embodiments, some embodiments of which include: Fix window component 1 at target imaging position 5; Fix the support 46 to the window component 1; Install the mounting bracket onto the bearing seat 46 and fix the mounting bracket in the preset position; The position adjustment component 2, which is equipped with the two-photon imaging component 7, is mounted on the support 46; Imaging is performed through window component 1 via two-photon imaging component 7.

[0107] The above steps are performed sequentially.

[0108] Specifically, such as Figure 2 As shown, the window component 1 is fixed to the target imaging position 5 by multiple fasteners 14; then, as... Figure 5 As shown, the support 46 is fixed to the base 13 of the window assembly 1 by a plurality of fasteners 43 and a plurality of threaded holes 131.

[0109] Next, the mounting bracket is installed on the support base 46. Specifically, two fasteners 43 are pre-fixed to the crossbar 45 by passing through the positioning hole 463 and the arc-shaped adjustment hole 464 on one side of the support base 46. Then, the adapter 48 and the upright 44 are installed and adjusted to the appropriate position. The bottom end of the upright 44 is then locked using the mounting bracket, which can be fixed in a preset position, such as on a monkey chair. Then, the crossbar 45, adapter 48, and upright 44 on the other side of the support base 46 are installed and fixed in the same way. After adjustment, the fasteners 43 at the positioning hole 463 and the arc-shaped adjustment hole 464 are tightened.

[0110] Then, the position adjustment component 2, on which the two-photon imaging component 7 is mounted, is installed on the appropriate fixing hole group on the carrier 46. Finally, imaging is performed through the window component 1 via the two-photon imaging component 7. During the imaging process, the position of the lens 71 can be adjusted by the position adjustment component 2.

[0111] This embodiment fixes the window assembly 1 at the target imaging position 5 and provides a support 46 on the mounting frame to install the two-photon imaging assembly 7. Because the support 46 is fixed to the window assembly 1, even if the animal moves at the target imaging position 5 while awake, the relative position between the two-photon imaging assembly 7 and the target imaging position 5 remains fixed, eliminating interference with image quality caused by body movement, thereby improving imaging stability and accuracy. In particular, when the mounting frame is fixed in a preset position, the movement of the target imaging position 5 is also restricted, resulting in more stable imaging.

[0112] Furthermore, the two-photon imaging component 7 is mounted on the support 46 via the position adjustment component 2, allowing for adjustment of its position during imaging. This facilitates flexible adjustment to the target imaging position or the acquisition of imaging in different areas. The two-photon imaging component 7 and the position adjustment component 2 are mounted on the side of the support 46 away from the window component 1, making adjustment convenient and preventing interference with body parts during position adjustment. The support 46 also provides a transitional load, transferring most of the imaging device's weight to the mounting frame, thus distributing the load and preventing weight concentration on the window component 1. This reduces the risk of the window component 1 becoming loose, resulting in highly stable two-photon imaging images.

[0113] In some embodiments, the step of imaging through the window assembly 1 via the two-photon imaging assembly 7 includes: The position adjustment component 2 is used to position the lens 71 of the two-photon imaging component 7 at different positions of the window component 1 to obtain local images at different positions; The individual images are stitched together to form the overall image.

[0114] This embodiment can reduce the size of the lens 71, which is conducive to the lightweighting of the imaging device, thereby reducing the weight on the animal's head, reducing the stress response during the experiment, and obtaining more stable imaging quality. On the other hand, the small field of view lens has stronger fluorescence signal and higher imaging resolution in single-frame imaging. The position adjustment component 2 drives the lens 71 to scan and stitch images in sections, which can fully cover the entire observation area of ​​the window 11 while taking into account high-precision imaging.

[0115] In some embodiments, after the position adjustment component 2 on which the two-photon imaging component 7 is mounted is mounted on the support 46, the imaging method further includes: The fiber optic cable 6 of the two-photon imaging component 7 is fixed by the clamp assembly 3, which is mounted on the mounting bracket.

[0116] This embodiment uses a wire clamp assembly 3 on a mounting frame to limit and fix the fiber optic cable 6 of the two-photon imaging assembly 7, preventing vibration caused by shaking of the fiber optic cable 6 during imaging, thereby improving imaging stability.

[0117] In some embodiments, the imaging method of this disclosure further includes: After imaging is completed, the position adjustment component 2 is removed and placed on the support base 8; Remove the entire mounting bracket; Remove the support 46 from the window component 1.

[0118] The order in which the position adjustment component 2 is removed, followed by the removal of the support base 46 and the mounting bracket, is not restricted. Specifically, first, remove the fastener 43 between the position adjustment component 2 and the support base 46, and open the cable clamp assembly 3 to remove the fiber optic cable 6. Then, remove the position adjustment component 2 and place it on the support base 8. Next, remove the fastener between the support base 46 and the crossbar 45, and remove the fastener on the mounting base, then remove the mounting bracket. Next, remove the support base 46 from the window component 1. Finally, remove the window component 1 from the target imaging position 5, clean the window component 1, and protect the window panel 11 with a transparent protective sheet, allowing real-time monitoring of the window panel 11.

[0119] In some specific embodiments, such as imaging brain regions of primates, the device can be omnidirectionally fixed using the fixing component 4. This method is simple to install, highly stable, and reliable. The position adjustment component 2 can achieve full imaging of the entire target imaging position 5, and the device is lightweight, facilitating head-mounted installation and stable imaging. For example, it weighs only 32.9g.

[0120] For example, testing macaques using the head-mounted two-photon imaging device according to embodiments of this disclosure can achieve peak values ​​of: Δx = -1.5 μm, Δy = -1.4 μm, Δz = 0.1 μm, where Δx and Δy are the offsets of the observed target in the XY plane relative to the reference center of lens 71, used to characterize the degree of planar drift of the sample during long-term imaging, and Δz is a small offset in the third direction Z, such as... Figure 8 As shown. The imaging duration was 206 seconds, with a total of 2000 frames. The change of XY plane displacement over time is shown below. Figure 9 As shown.

[0121] The foregoing has provided a detailed description of a two-photon imaging device and imaging method provided in this disclosure. Specific embodiments have been used to illustrate the principles and implementation methods of this disclosure. The descriptions of these embodiments are merely for the purpose of helping to understand the method and its core ideas. It should be noted that those skilled in the art can make various improvements and modifications to this disclosure without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of this disclosure.

Claims

1. A two-photon imaging device, characterized in that, include: The fixing component (4) includes a fixing frame and a support base (46), wherein the support base (46) is mounted on the fixing frame; A window assembly (1) is detachably mounted on the carrier (46), and the window assembly (1) is configured to be fixed at the target imaging position (5). A position adjustment component (2) is installed on the side of the support (46) away from the window component (1); and The two-photon imaging component (7), mounted on the position adjustment component (2), is configured to image through the window component (1) and the imaging position can be adjusted by the position adjustment component (2).

2. The two-photon imaging device according to claim 1, characterized in that, The fixing frame includes: Two uprights (44) are spaced apart along a predetermined direction in the horizontal plane; Two mounting bases, located in the bottom region of the upright (44), are configured to secure the upright (44); and Two horizontal bars (45) are respectively connected to the two vertical bars (44) near the top area; The two ends of the support base (46) are respectively installed on the two crossbars (45) and located between the two uprights (44), and the window assembly (1) is located between the two crossbars (45).

3. The two-photon imaging device according to claim 2, characterized in that, The mounting bracket also includes: Two adapters (48) are connected between one end of the crossbar (45) and the upright (44). The adapters (48) are provided with a first hole (481) and a second hole (482) that are perpendicular to each other. The upright (44) passes through the first hole (481) and the crossbar (45) passes through the second hole (482). The adapter (48) is adjustable in height along the upright (44) and / or the adapter (48) is adjustable in lateral position along the crossbar (45).

4. The two-photon imaging device according to claim 3, characterized in that, The first hole (481) has a first through groove (483) on the side wall near the first end of the adapter (48) so that the first end of the adapter (48) forms a first clamp, and the first clamp is connected and locked to the upright (44) by a fastener (43); the second hole (482) has a second through groove (484) on the side wall near the second end of the adapter (48) so that the second end of the adapter (48) forms a second clamp, and the second clamp is locked to the crossbar (45) by the fastener (43).

5. The two-photon imaging device according to claim 4, characterized in that, When the first clamp and the mounting base are released, the adapter (48) can be rotated relative to the upright (44); when the second clamp is released, the crossbar (45) can be rotated relative to the adapter (48).

6. The two-photon imaging device according to claim 2, characterized in that, The mounting base includes a base plate (42) and a third clamp (41). The third clamp (41) includes a surrounding portion (411) and two engaging portions (412). The surrounding portion (411) surrounds the outer wall of the upright (44) and has an opening. The side of the surrounding portion (411) away from the opening is fixed to the base plate (42). The two engaging portions (412) are respectively connected to the two ends of the surrounding portion (411) and the two engaging portions (412) are connected by fasteners (43) to lock the upright (44).

7. The two-photon imaging device according to claim 6, characterized in that, A cut is provided between the third clamp (41) and the substrate (42), and the third clamp (41) is fixed to the substrate (42) through the portion other than the cut; the inner sidewall of the surrounding part (411) is provided with a first recess (413) at the inner end of the cut, and / or the outer sidewall of the surrounding part (411) is provided with a second recess (414) at the inner end of the cut.

8. The two-photon imaging device according to claim 2, characterized in that, The crossbar (45) has a flat surface (451) for mounting the support seat (46). The support seat (46) is provided with a mounting hole group at the position where it connects to the crossbar (45). The mounting hole group includes a positioning hole (463) and an arc-shaped adjustment hole (464). The support seat (46) is mounted on the crossbar (45) by fasteners (43) that pass through the positioning hole (463) and the adjustment hole (464) respectively.

9. The two-photon imaging apparatus according to any one of claims 1 to 8, characterized in that, The support (46) is provided with a plurality of fixing hole groups, which are located on the outer periphery of the window assembly (1). The position adjustment assembly (2) can be selectively installed in one of the fixing hole groups; and / or The window assembly (1) includes a transparent window panel (11), the support (46) has an opening (466), and a plurality of fasteners (43) are provided on the support (46) around the opening (466) to fix the window assembly (1), the window panel (11) being located within the opening (466); and / or The support (46) is provided with a positioning groove, and the window component (1) is embedded in the positioning groove.

10. The two-photon imaging apparatus according to any one of claims 1 to 8, characterized in that, The window assembly (1) includes: a base (13), a locking ring (12) and a transparent window piece (11), the window piece (11) being embedded in the locking ring (12), the base (13) being disc-shaped and having a mounting hole, and the locking ring (12) being threaded into the mounting hole.

11. The two-photon imaging device according to claim 10, characterized in that, The base (13) has a plurality of through holes (133) spaced apart around the outer periphery of the mounting hole, the through holes (133) being configured to be mounted at the target imaging position (5) by means of a fastener (14); and / or The base (13) has an annular inner conical surface (132) on its end face facing the two-photon imaging assembly (7); and / or The locking ring (12) has an operating part (121) on its end face facing the two-photon imaging assembly (7). The operating part (121) is configured to receive an external operation to unscrew the locking ring (12).

12. The two-photon imaging apparatus according to any one of claims 1 to 8, characterized in that, The window assembly (1) includes a transparent window (11), and the two-photon imaging assembly (7) includes a lens (71). The lens (71) images through the window (11), and the imaging area of ​​the lens (71) is smaller than the area of ​​the window (11). The two-photon imaging assembly (7) is configured to obtain local images of different positions of the window (11) by moving, so as to stitch together an overall image.

13. The two-photon imaging apparatus according to any one of claims 1 to 8, characterized in that, The position adjustment component (2) includes: Base (21); The first connecting seat (22) is fixed to the base (21); The first slider (23) engages with the first connecting seat (22) and its position is adjustable along the first direction (X); The second connecting seat (25) is connected to the first slider (23); The second slider (26) engages with the second connecting seat (25) and is position-adjustable along the second direction (Y), which is perpendicular to the first direction (X). The third slider (28) cooperates with the second slider (26) and is adjustable along a third direction (Z), which is perpendicular to the first direction (X) and the second direction (Y); the two-photon imaging component (7) is mounted on one end of the third slider (28) near the base (21).

14. The two-photon imaging device according to claim 13, characterized in that, The position adjustment component (2) further includes: A three-dimensional micro-manipulation component (24), connected between the first slider (23) and the second connector (25), is configured to fine-tune the position of the two-photon imaging component (7) along the first direction (X), the second direction (Y) and the third direction (Z).

15. The two-photon imaging device according to claim 1, characterized in that, It also includes a wire clamp assembly (3), which is mounted on the mounting frame and configured to limit the fiber optic cable (6) of the two-photon imaging assembly (7).

16. An imaging method based on the two-photon imaging device according to any one of claims 1 to 15, characterized in that, include: The window component (1) is fixed at the target imaging position (5); Fix the support (46) to the window assembly (1); Install the fixing frame onto the bearing seat (46) and fix the fixing frame in a preset position; The position adjustment component (2) on which the two-photon imaging component (7) is installed is mounted on the support (46); Imaging is performed through the window assembly (1) via the two-photon imaging component (7).

17. The imaging method according to claim 16, characterized in that, The steps of imaging through the window assembly (1) using the two-photon imaging assembly (7) include: The position adjustment component (2) is used to position the lens (71) of the two-photon imaging component (7) at different positions of the window component (1) to obtain local images at different positions; The individual images are stitched together to form the overall image.

18. The imaging method according to claim 16, characterized in that, After the position adjustment assembly (2) on which the two-photon imaging component (7) is mounted is placed on the carrier (46), the imaging method further includes: The fiber optic cable (6) of the two-photon imaging component (7) is fixed by a clamp assembly (3), which is mounted on the mounting frame.

19. The imaging method according to claim 16, characterized in that, Also includes: After imaging is completed, the position adjustment component (2) is removed and placed on the support base (8); Remove the support (46) from the fixture; Remove the entire mounting bracket.

Images