Non-parallel optical axis coupled multi-site two-photon imaging stimulation device

Abstract

The application discloses a multi-site two-photon imaging stimulation device coupled by non-parallel light axes, which comprises a femtosecond laser, a laser scanning device and at least two optical branches; the optical branch comprises a first focusing optical element, a mirror and a second focusing optical element; the femtosecond laser emitted by the femtosecond laser enters the laser scanning device, the laser scanning device repeatedly scans the femtosecond laser between multiple scanning intervals, and the femtosecond laser of each scanning interval reaches a corresponding target site after passing through a corresponding optical branch; the femtosecond laser entering the optical branch is reflected to the second focusing optical element after being focused for the first time by the first focusing optical element, and reaches the corresponding target site visual field plane after being focused for the second time. The multi-site two-photon imaging stimulation device coupled by non-parallel light axes provided by the application is suitable for two-photon imaging or optical stimulation on multiple planar visual field regions with spatial angles located on a curved sample.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biophotonics, in particular to a multi-site two-photon imaging stimulation device with non-parallel optical axis coupling. BACKGROUND

[0002] Two-photon fluorescence imaging technology has the advantages of thick tissue optical penetration and good tomographic imaging capability, and has been well applied in the fields of neuroscience, embryonic development, tumor pathology and other biomedical fields. The imaging field of the traditional conventional two-photon microscope is limited to 1mm 2 range, which limits many experiments, such as simultaneous recording of the functional activity of neuron groups in multiple brain functional regions. With the advancement of technology, a variety of two-photon microscopic imaging and optical stimulation methods have been developed, which allow the imaging field to be not limited to a single region, greatly promoting the application potential of two-photon microscope in related biomedical fields.

[0003] One method of multi-site two-photon imaging is to develop a large-aperture large-field mesoscope. For example, in the journal article "A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging", N.J. Sofroniew, eLife, 2016, a mesoscope with an aperture of 25.6 mm, a numerical aperture of 0.6, and a field diameter of 5 mm was developed, and simultaneous imaging of 4 regional sites was achieved. The disadvantage of this continuous large-field two-photon microscope based on mesoscope is that the field is limited by the level of optical design and manufacturing, and it is difficult to realize a field diameter of more than 8 mm while maintaining cell resolution. The most commonly used method recently is a multi-site two-photon imaging stimulation method based on discrete regions, such as the journal article "MATRIEX imaging: multiarea two-photon real-time in vivo explorer", Yang M, Light: Science & Applications, 2019, or Chinese patent ZL201811306921.5, which proposes a multi-layer compound lens structure based on two-stage magnification and multi-optical axis coupling. The upper air objective provides a large imaging field, and the lower micro-lens (usually a self-focusing lens) is responsible for two-stage magnification to increase the numerical aperture to meet the requirements of cell imaging resolution. Usually, an array of multiple micro-lenses is combined to divide the imaging field into multiple discrete sub-fields, achieving multi-site imaging. The aperture of the micro-lens is small (such as 2 mm), which allows the spacing between multiple sites to be small, and the micro-lens can be easily moved or even implanted through a connecting rod, with strong regional selectivity. Moreover, this multi-layer optical structure based on two-stage magnification allows a larger selection range of multiple sites compared to a single mesoscope, and a 10 mm diameter selection range has been achieved in the literature, and allows for a certain drop in depth between regions, making it more widely applicable. However, the optical coupling between the upper air objective and the lower micro-lens in this method is based on parallel optical axis coupling. Specifically, although the incident angles of the incident beams corresponding to multiple site regions are different, the optical axes (central axes) of the focused beams after the first layer of objective lenses are parallel to each other, which is caused by the design principle of the flat-field scanning objective. The optical axes of each micro-lens in the micro-lens array are also parallel to each other, and the optical axis of each micro-lens coincides with the optical axis of the focused beam after the air objective, thereby achieving optical axis coupling.The problem caused by parallel optical axis coupling is that if the imaging or stimulation target is two (or multiple) sites on a surface with large curvature, the object field planes are not parallel, and the actual laser scanning into the object field plane can only be multiple parallel planes under the parallel optical axis coupling method, causing the actual object plane to have an angle with the target object plane, which will cause uneven imaging in the region and easily cause physical interference (collision) between the micro objective and the sample. How to improve the multi-layer objective compound optical structure based on the secondary amplification principle to adapt to the double-photon imaging or light stimulation of multiple non-parallel planes on the curved sample is a hot issue internationally, but there is currently a lack of reliable solutions. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a multi-site double-photon imaging and stimulation device with non-parallel optical axis coupling to solve the above problems in the prior art.

[0005] To solve the above technical problems, the technical scheme adopted by the present application is as follows: a multi-site double-photon imaging and stimulation device with non-parallel optical axis coupling, comprising: a femtosecond laser, a laser scanning device, and at least two optical branches.

[0006] The optical branch comprises a first focusing optical element, a mirror, and a second focusing optical element.

[0007] The femtosecond laser emitted by the femtosecond laser enters the laser scanning device, and the laser scanning device repeatedly scans the femtosecond laser between multiple scanning intervals. The femtosecond laser in each scanning interval reaches a corresponding target site after passing through a corresponding optical branch. The femtosecond laser entering the optical branch is first focused by the first focusing optical element and then reflected to the second focusing optical element, and after the second focusing, it reaches the corresponding target site field plane.

[0008] Preferably, the optical axis direction of the second focusing optical element in the optical branch is parallel to the normal of the corresponding target site field plane.

[0009] Preferably, the second focusing optical elements in different optical branches are in a non-parallel relationship with each other.

[0010] Preferably, the optical axis of the first focusing optical element in the optical branch intersects with the optical axis of the second focusing optical element at the mirror in the optical branch.

[0011] Preferably, the multi-site double-photon imaging and stimulation device with non-parallel optical axis coupling further comprises a first 5-axis displacement stage for adjusting the three-dimensional position and two-dimensional swing angle of the mirror.

[0012] Preferably, the laser scanning device is a two-axis scanner, realizing two-dimensional deflection and scanning of the femtosecond laser direction.

[0013] Preferably, the non-parallel optical axis coupled multi-site two-photon imaging stimulation device further comprises a second 5-axis displacement table for adjusting the three-dimensional position and two-dimensional swing angle of the second focusing optical element.

[0014] Preferably, the number of optical branches is two.

[0015] Preferably, the first focusing optical element is a low-power objective or a long-focus lens.

[0016] Preferably, the second focusing optical element is a micro objective or a self-focusing lens.

[0017] The beneficial effects of the present application are: the non-parallel optical axis coupled multi-site two-photon imaging stimulation device provided by the present application adopts a double-layer objective array composite structure for two-stage focusing magnification and multi-site imaging, the optical axis direction of each micro objective is parallel to the normal line of the respective target field plane, but is in a non-parallel relationship with each other; each branch realizes the direction conversion of the low-power objective optical axis to the micro objective optical axis through a mirror, and the device is suitable for two-photon imaging or optical stimulation on multiple planar field regions with spatial angles on a curved sample. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The figure is a structural schematic diagram of the non-parallel optical axis coupled multi-site two-photon imaging stimulation device of the present application.

[0019] Explanation of reference signs:

[0020] 1 - femtosecond laser; 2 - femtosecond laser; 3 - laser scanning device; 21, 22 - femtosecond laser; 41, 42 - low-power objective; 51, 52 - mirror; 61, 62 - micro objective; 7 - sample; 71, 72 - target site field plane. DETAILED DESCRIPTION

[0021] The present application will be further described in detail below with reference to examples, so that those skilled in the art can implement the present application according to the description.

[0022] It should be understood that the terms such as "have", "contain" and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0023] Example 1

[0024] As Figure 1As shown, the non-parallel optical axis coupling multi-site two-photon imaging and stimulation device of the embodiment comprises a femtosecond laser 1, a laser scanning device 3 and two optical branches;

[0025] The optical branch comprises a first focusing optical element, a mirror and a second focusing optical element; in the embodiment, the first focusing optical element is a low-power objective lens (or a long focal length lens), and the second focusing optical element is a micro objective lens (or a self-focusing lens). For example, in an embodiment, the low-power objective lens can adopt products of the following model: MPlan Apo 2x (Mitutoyo), the long focal length lens can adopt products of the following model: AC254-100-A (thorlabs), and the micro objective lens can adopt products of the following model: D0224-7X / 0.7 (Nanjing Dongli Le Optical).

[0026] The femtosecond laser emitted by the femtosecond laser 1 enters the laser scanning device 3, and the laser scanning device 3 repeatedly scans the femtosecond laser between multiple scanning intervals. The femtosecond laser of each scanning interval reaches a corresponding target site for imaging or stimulation after passing through a corresponding optical branch. In the embodiment, the two femtosecond lasers 21 and 22 are the central optical axis directions of the two selected scanning intervals in the embodiment.

[0027] The femtosecond laser entering the optical branch is first focused by the first focusing optical element and then reflected to the second focusing optical element, and then reaches the corresponding target site view plane after the second focusing. For example, the femtosecond laser 21 is first focused by the low-power objective lens 41, then reflected by the mirror 51 to adjust the central optical axis direction, and then secondly focused by the micro objective lens 61, so as to convert the low numerical aperture of the low-power objective lens into the high numerical aperture of the micro objective lens, thereby improving the resolution of imaging or light stimulation; the femtosecond laser 22 is first focused by the low-power objective lens 42, then reflected by the mirror 52 to adjust the central optical axis direction, and then secondly focused by the micro objective lens 62.

[0028] In the embodiment, the laser scanning device 33 is a two-axis scanner, which realizes two-dimensional deflection and scanning of the femtosecond laser. The scanner types include galvanometer scanners, resonant scanners, rotating polygon mirrors and acousto-optic deflectors, etc. Each scanning interval is a small range of two-dimensional rectangular scanning in the two-dimensional space angle domain, and the central optical axis of each branch is the optical axis direction of the center of the scanning rectangle.

[0029] Among them, the optical axis direction of the second focusing optical element in the optical branch is basically parallel to the normal line of the corresponding target site view plane. For example, the optical axis direction of the micro objective lens 61 is perpendicular to the target view plane 71, and the optical axis direction of the micro objective lens 62 is perpendicular to the target view plane 72. The second focusing optical elements in different optical branches are in a non-parallel relationship.Figure 1 In this embodiment, the outer contour of sample 7 is a curve of a partial ellipsoid, and the target view planes 71 and 72 are two target sites with a certain distance located near the surface thereof. The normal directions of the two planes are non-parallel, so the optical axis directions of the micro-objective 61 and 62 are also non-parallel. Therefore, this embodiment provides a multi-site two-photon imaging stimulation device based on non-parallel optical axis coupling.

[0030] The optical axis of the first focusing optical element in the optical branch intersects the optical axis of the second focusing optical element at the mirror in the optical branch.

[0031] In a preferred embodiment, the adjustment of the three-dimensional position and two-dimensional swing angle of the mirror is realized by a first 5-axis displacement stage, so that the low-power objective optical axis direction and the micro-objective optical axis direction satisfy the reflection law. According to the position and normal direction of the target site, the three-dimensional position and two-dimensional swing angle of the second focusing optical element are adjusted by a second 5-axis displacement stage. In this embodiment, the first 5-axis displacement and the second 5-axis displacement stages are both conventional products (not shown in the figure), and will not be described in detail.

[0032] In this embodiment, for the convenience of description, a multi-site two-photon imaging stimulation mode of non-parallel optical axis coupling of two target sites is shown, and the number of corresponding optical branches is two. It can be understood that, according to the principle, it can be extended to three or more target sites. In summary, the embodiments of the present application can also have various transformations and modifications, and are not limited to the specific structures of the above embodiments.

[0033] In the description of the present application, it should be understood that the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application.

[0034] Although the embodiments of the present application have been disclosed as above, they are not limited to the application and implementation listed in the specification and embodiments, and can be fully applied to various fields suitable for the present application, and additional modifications can be easily realized by those skilled in the art, and therefore the present application is not limited to specific details, without departing from the general concept defined by the claims and equivalent scope.

Claims

1. A non-collinear optical axis coupled multi-site two-photon imaging stimulation device, characterized in that, Comprise: a femtosecond laser, a laser scanning device and at least two optical branches; the optical branches comprise a first focusing optical element, a mirror and a second focusing optical element; the femtosecond laser emits femtosecond laser into the laser scanning device, the laser scanning device repeatedly scans the femtosecond laser between multiple scanning intervals, the femtosecond laser of each scanning interval reaches a corresponding target site after passing through a corresponding optical branch; the femtosecond laser entering the optical branch is reflected to the second focusing optical element after the first focusing by the first focusing optical element, and reaches the corresponding target site view plane after the second focusing; the optical axis direction of the second focusing optical element in the optical branch is parallel to the normal of the corresponding target site view plane; the second focusing optical elements in different optical branches are in non-parallel relationship with each other.

2. The non-collinear optical axis coupled multi-site two-photon imaging stimulation device according to claim 1, wherein, the optical axis of the first focusing optical element in the optical branch intersects with the optical axis of the second focusing optical element at the mirror in the optical branch.

3. The non-collinear optical axis coupled multi-site two-photon imaging stimulation device of claim 1, wherein, It also comprises a first 5-axis displacement table for adjusting the three-dimensional position and two-dimensional swing angle of the mirror.

4. The non-collinear optical axis coupled multi-site two-photon imaging stimulation device of claim 1, wherein, It also comprises a second 5-axis displacement table for adjusting the three-dimensional position and two-dimensional swing angle of the second focusing optical element.

5. The non-collinear optical axis coupled multi-site two-photon imaging stimulation device of claim 1, wherein, The laser scanning device is a two-axis scanner, which realizes two-dimensional deflection and scanning of the direction of femtosecond laser.

6. The non-collinear optical axis coupled multi-site two-photon imaging stimulation device of claim 1, wherein, The number of optical branches is two.

7. The non-collinear optical axis coupled multi-site two-photon imaging stimulation device according to any one of claims 1-6, wherein, The first focusing optical element is a low-power objective or a long-focus lens.

8. The non-collinear optical axis coupled multi-site two-photon imaging stimulation device according to any one of claims 1-6, wherein, The second focusing optical element is a micro objective or a self-focusing lens.

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