Spot scanning confocal adjusting system and method

Abstract

The invention relates to the technical field of microscopy optics, in particular to a spot scanning confocal adjusting system and method, light emitted by a light source is parallel light beams, the divergence angle is smaller than 1mrad, the light beams reach a galvanometer through a spectroscope, reach a target object through a scanning mirror set and an objective lens, return after being reflected by the target object, pass through the spectroscope capable of rotating, and then pass through a scanning mirror set. After being reflected, the laser reaches the confocal hole through the focusing lens; the confocal hole is located at the rear focus of the lens, and confocal imaging is achieved. According to the invention, through the rotatable spectroscope, the imaging condition of the target object at the confocal hole position can be observed by one camera at the same time. The technical problem that the position of the confocal hole is difficult to find if the initial position of the confocal hole is not within the view field range of the camera due to the fact that the confocal hole is small, a camera with a high amplification factor needs to be selected for clearly seeing the confocal hole and the image plane range which can be observed by the camera is very small in the debugging process is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microscopic optics, in particular to a point scanning confocal adjustment system and method. BACKGROUND

[0002] The reflection confocal system is one of the advanced cell biology medical analysis instruments in recent times, which can perform optical tomography on the axial direction of a sample, thereby obtaining a living image of a cell level with a depth of 200-350 mu m, and reconstructing a three-dimensional image of the sample. Adjustment of the pinhole axial position of the reflection confocal system is a common difficulty.

[0003] In the related art, in a general debugging scheme of a confocal system, it is necessary to observe the converging condition of a target object position light spot to determine whether the target object is in a correct working plane, and it is also necessary to observe the confocal hole position to adjust the return light spot position, so that the system reaches a confocal state. The confocal system generally has a resolution of about 1 um in the object side and about 20 um in the image side. If the converging in the image side is 20 um, a confocal hole of 20 or 25 um is generally selected to realize confocal imaging. A confocal hole that is too large will result in a decrease in resolution, and a confocal hole that is too small will result in a decrease in imaging brightness. Therefore, adjustment of the position of the confocal hole is a key step of the confocal system. In confocal debugging, the confocal hole involves three-dimensional direction alignment (up and down, left and right, and front and back). If the confocal hole is not concentric with the light beam reflected back after reaching the observed object in the up-down and left-right directions, it will result in low imaging brightness or imaging failure. If the confocal hole is not located on the focal plane of the focusing lens in the front-back direction, the resolution will not reach the designed value of the system. SUMMARY

[0004] The present application aims to provide a point scanning confocal adjustment system and method to solve the problems in the background art.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0006] A point scanning confocal adjustment method, comprising:

[0007] A light source emits parallel light beams, the light beams pass through a beam splitter to reach a galvanometer mirror, then pass through a scanning mirror group and an objective lens to reach a target object, are reflected along the original path to the beam splitter after passing through the target object, are reflected again after passing through a rotatable beam splitter at a first position, and reach a camera as an object side light ray;

[0008] The camera images an object at infinity, the converging condition of the object side light ray is observed through the camera, the position of the target object is adjusted, the object side light ray converges into an Airy pattern, at this time the target object is located on a confocal plane, and the position of the target object is fixed;

[0009] A preset mounting seat is arranged at the back focal point of the focusing lens, a calibration board with coordinate marks is arranged on the mounting seat, an auxiliary light source is used to illuminate the calibration board, the rotatable beam splitter is turned from the first position to the second position, the light beam passes through the rotatable beam splitter, the mirror and the focusing lens to reach the calibration board, at this time, the camera still images the infinity, the camera object surface is just located at the back focal point of the focusing lens, when the camera can clearly image the coordinates on the calibration board, the mounting seat is fixed in position;

[0010] The auxiliary light source is turned off, the light source is turned on, the rotatable beam splitter is adjusted, the light beam returned from the target object is located at the center of the field of view of the camera, the auxiliary light source is turned on, the adjustable mirror is adjusted, the center of the calibration board is located at the center of the field of view of the camera, at this time, the light spot of the light beam reflected by the target object is coincided with the center of the calibration board, the calibration board is replaced by a confocal aperture plate, the confocal aperture of the confocal aperture plate is coincided with the returned light beam, the position of the adjustable mirror is fixed, the auxiliary light source and the rotatable beam splitter are removed, and the confocal adjustment is completed.

[0011] Further, the divergence angle of the parallel light beam emitted by the light source is less than 1 mrad.

[0012] Further, the mirror surface of the rotatable beam splitter is parallel to the mirror surface of the galvanometer when the rotatable beam splitter is located at the first position.

[0013] Further, the mirror surface of the rotatable beam splitter is perpendicular to the mirror surface of the galvanometer when the rotatable beam splitter is located at the second position.

[0014] Further, the angle between the first position and the second position is 90°.

[0015] Further, when the confocal aperture is coincided with the light spot of the light beam, the camera cannot see the shape of the light spot, and there is a relatively uniform halo around the confocal aperture.

[0016] Further, the rotatable beam splitter is based on a rotatable half-transmissive half-reflective mirror piece.

[0017] To achieve the above object, the application further provides the following technical scheme:

[0018] A point scanning confocal adjustment system comprises:

[0019] A beam splitter, an objective lens, a scanning mirror group, a rotatable beam splitter, an adjustable mirror, a focusing lens and a light source.

[0020] The light source emits a parallel light beam, the light beam passes through the beam splitter to reach the galvanometer, then passes through the scanning mirror group and the objective lens to reach the target object, is reflected along the original path to reach the beam splitter after reflection, then passes through the rotatable beam splitter to reach the adjustable mirror, is reflected after passing through the focusing lens to reach the confocal aperture, and the confocal aperture is located at the back focal point of the lens to realize confocal imaging.

[0021] Further, the target object, the objective lens and the scanning mirror group are located on the same axis.

[0022] Further, the beamsplitter is located on the same axis with the galvanometer mirror and the adjustable mirror respectively, and the galvanometer mirror and the adjustable mirror are not located on the same axis.

[0023] Further, the adjustable mirror is located on the same axis with the lens.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] The present application realizes the imaging of the target object and the confocal hole position by using one camera through a rotatable beamsplitter. A calibration board which can be installed on the confocal hole position is designed. The calibration board has coordinates which can be confirmed on the focal plane, and the angle of the adjustable mirror is approximately correct and will not deviate too much.

[0026] The initial position of the confocal hole is confirmed by the calibration board, so that the confocal hole will not deviate from the field of view of the camera, thereby reducing the difficulty of assembly and adjustment. In the debugging process, in order to clearly see the confocal hole, a camera with a high magnification is needed due to the small size of the confocal hole. The camera can only observe a small image plane, which leads to the technical problem that it is difficult to find the confocal hole position if the initial position of the confocal hole is not in the field of view of the camera, or it is difficult to determine whether the confocal hole is in the accurate focal plane after the confocal hole is found, which leads to the resolution not reaching the design value of the system.

[0027] The present application solves the technical problem in the related art that the confocal system commonly used in the debugging scheme needs to adjust the angle of the mirror to find the position of the confocal hole. Due to the small size of the confocal hole, the confocal hole will not be clearly imaged when it is not located on the back focal plane of the focusing lens, which leads to the difficulty in determining whether the confocal hole cannot be found due to not being on the focal plane or due to the problem of the angle of the mirror.

[0028] The present application uses a beamsplitter to make the light beam emitted by the light source be transmitted for the first time, reflected by the target object and then reflected to the camera, and uses a mirror group composed of a common objective lens and a scanning mirror group to overlap part of the optical path, thereby reducing the size of the system. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The system optical path schematic diagram for determining the position of the calibration board of the present application.

[0030] Figure 2 The system optical path schematic diagram for determining the position of the confocal hole of the present application.

[0031] Figure 3 The schematic diagram of the rotatable beamsplitter of the present application mounted on the lens holder.

[0032] Figure 4 The schematic diagram of the calibration plate structure of the present application.

[0033] Figure 5 The schematic diagram of the light convergence observed by the camera when the target position is adjusted in the present application. Figure 1 .

[0034] Figure 6 The schematic diagram of the light convergence observed by the camera when the target position is adjusted in the present application. Figure 2 .

[0035] Figure 7 The schematic diagram of the light convergence observed by the camera when the target position is adjusted in the present application.

[0036] Figure 8 The schematic diagram of the fine adjustment of the adjustable mirror to make the confocal hole position close to the spot (return light beam) position in the present application. Figure 1 .

[0037] Figure 9 The schematic diagram of the fine adjustment of the adjustable mirror to make the confocal hole position close to the spot position in the present application. Figure 2 .

[0038] Figure 10 The schematic diagram of the fine adjustment of the adjustable mirror to make the confocal hole position close to the spot position in the present application. Figure 3 .

[0039] Figure 11 The schematic diagram of the fine adjustment of the adjustable mirror to make the confocal hole position close to the spot position in the present application. Figure 3 .

[0040] Figure 12 The schematic diagram of the fine adjustment of the adjustable mirror to make the return light beam and the confocal hole coincide in the present application.

[0041] In the figure: 1-target, 2-objective lens, 3-scan mirror group, 4-vibrating mirror, 5-diffractive mirror, 6-rotatable diffractive mirror, 7-adjustable mirror, 8-focusing lens, 9-light source, 10-camera, 11-debugging auxiliary light source, 12-confocal hole, 13-calibration plate. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0043] In the description of the present application, it should be noted that the terms "upper end", "lower end", "inner", "outer", "front end", "rear end", "both ends", "one end", "the other end" 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 do not indicate or imply that the devices or elements 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. In addition, the terms "first", "second" are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0044] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "provided with", "provided with", "sleeved", "connected" and the like should be broadly understood, for example, "connected" can be fixedly connected, can also be detachably connected, or integrally connected; can be mechanically connected, can also be electrically connected; can be directly connected, can also be indirectly connected through an intermediate medium, can be internal communication of two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0045] Please refer to Figures 1 to 12 The present application provides a technical solution:

[0046] A point scanning confocal adjustment method is an easy-to-operate and accurate confocal system debugging method. By a rotatable beam splitter, the imaging condition of the target object and the confocal hole position is observed simultaneously by a camera.

[0047] In the related art, during the confocal debugging process, in order to clearly see the confocal hole, a camera with high magnification is needed due to the small size of the confocal hole. The camera can observe a very small image plane range, which makes it difficult to find the confocal hole position if the initial position of the confocal hole is not within the camera field of view.

[0048] In order to solve the problem of difficult to find the confocal hole position, the present application designs a calibration plate which can be installed at the confocal hole position. The initial position of the confocal hole is confirmed by the calibration plate to not deviate from the camera field of view, thereby reducing the difficulty of installation and adjustment.

[0049] Since there is no calibration plate in the related art during the confocal debugging process, the confocal hole position is found by adjusting the angle of the reflector. Since the confocal hole is very small, and when the confocal hole 12 is not located at the back focal plane of the focusing lens 8, the confocal hole will not be clearly imaged, which makes it difficult to determine whether the confocal hole is not in the focal plane or the angle of the adjustable reflector 7 is the problem.

[0050] Because the calibration plate 13 of the present application has coordinates that can be confirmed on the focal plane first, and the angle of the adjustable mirror 7 is roughly correct and will not deviate too much.

[0051] The confocal system mainly consists of: an objective lens 2, a scanning mirror group 3, a galvanometer 4, a beam splitter 5, a rotatable beam splitter 6, an adjustable mirror 7, a focusing lens 8, a light source 9, and a confocal aperture 12.

[0052] The light source 9 emits parallel light beams with a divergence angle less than 1 mrad. The light beams pass through the beam splitter 5 to the galvanometer 4, pass through the scanning mirror group 3 and the objective lens 2 to the target object 1, reflect after passing through the target object 1, pass through the objective lens 2 again, the scanning mirror group 3, and the galvanometer 4, reflect after passing through the beam splitter 5, pass through a rotatable beam splitter (i.e. the rotatable beam splitter 6, also known as a half-transmission half-reflection mirror or a 50-50 beam splitter), reach the adjustable mirror 7, reflect after passing through the focusing lens 8, and reach the confocal aperture 12; the confocal aperture 12 is located at the back focal point of the lens 8, achieving confocal imaging.

[0053] The beam splitter 5 has the following functions: when the light beams emitted by the light source 9 pass through for the first time, the beam splitter 5 transmits the light beams; when the light beams reflected by the target object 1 return, the beam splitter 5 reflects the light beams to the detector (i.e. the camera 10), and uses the common mirror group (the objective lens 2 and the scanning mirror group 3) to overlap part of the optical path, thereby reducing the size of the system.

[0054] In this embodiment, as shown in Figure 3 , the rotatable beam splitter 6 can be adjusted manually. The lens is installed in the square hole c of the lens holder a, and the lens is rotated along the axis c of the lens holder a by rotating the lens holder a. Of course, the lens holder a can also be driven by the rotating shaft of a stepping motor to rotate along the axis c of the lens holder a, so as to rotate the rotatable beam splitter 6 and finally adjust it to the appropriate angle.

[0055] When adjusting the confocal aperture, a target object 1 that can adjust the distance is placed at the working surface of the objective lens 2. When the rotatable beam splitter 6 is in the first position (i.e. the dotted line position in Figure 1 , Figure 2 , the parallel light beams emitted by the light source 9 pass through the beam splitter 5, reflect at the galvanometer 4, pass through the objective lens 2 and the scanning mirror group 3 to reach the target object 1, and the reflected light beams pass through the objective lens 2 and the scanning mirror group 3 again, do not start the galvanometer 4 in the initial position, reflect at the beam splitter 5 to the rotatable beam splitter 6 in the dotted line position, and reach the camera 10. At this time, the camera 10 is imaging at infinity, and the camera 10 can observe the convergence of the object-side light beams through the optical system. The position of the target object 1 is adjusted (moved along the horizontal arrow direction in Figure 1 , Figure 2 , so that the light beams converge as shown in Figure 5 , Figure 6 , and Figure 7 . When the convergence is Figure 7 Airy disk patternFigure 7 In this case, the center bright spot is an Airy disk, and the target object 1 is located on the confocal plane, and the position of the target object is fixed.

[0056] In this embodiment, the target object 1 can be a mirror.

[0057] As shown in Figure 1 and Figure 4 , a calibration plate 13 with coordinate marks or calibration patterns f is mounted on the mounting seat d, and the calibration plate 13 is located at the position where the confocal hole 12 is mounted (at this time, the confocal hole 12 is not mounted) : the rotatable beam splitter 6 is rotated by 90° to the first position (i.e. the solid line position in Figure 1 , Figure 2 , due to the parallel light beam emitted by the light source 9, the light beam passes through the beam splitter 5, is reflected by the galvanometer 4, passes through the objective lens 2 and the scanning lens group 3 to the target object 1, the reflected light beam passes through the objective lens 2, the scanning lens group 3, the galvanometer 4 which is not activated and is located at the initial position, is reflected by the beam splitter 5, passes through the rotatable beam splitter 6 which is located at the solid line position, and then passes through the adjustable mirror 7 and the focusing lens 8 to the calibration plate 13 which is located at the back focal point of the focusing lens 8 approximately. The calibration plate is illuminated by the auxiliary light source 11, and at this time, the camera 10 still images an infinite distance. The camera object plane is located at the back focal point of the focusing lens 8, as shown in Figure 1 , the calibration plate 13 is moved by moving the mounting seat d in the vertical arrow direction, so when the camera can clearly image the coordinates on the calibration plate 13, it is proved that the calibration plate is located at the back focal point of the lens 8, and at this time, it is considered that the calibration plate position adjustment is completed. The position of the mounting seat d is fixed, and then the auxiliary light source 11 is turned off.

[0058] The light source 9 is turned on, and the rotatable mirror 6 is adjusted so that the light beam returned from the target object 1 is located at the center of the field of view of the camera; the auxiliary light source 11 is turned on, and the adjustable mirror 7 is adjusted so that the center of the calibration plate is located at the center of the field of view of the camera, and at this time, the light spot reflected by the light source 9 from the surface of the target object 1 should be approximately coincident with the center e of the calibration plate 13.

[0059] The calibration plate 13 is removed from the mounting seat d, and the calibration plate 13 is replaced with a confocal hole plate which has the same shape and size as the calibration plate 13, and at this time, the confocal hole 12 should be in the field of view of the camera 10 and the outline should be clear Figures 8 to 12 , the white circle in the figure indicates the position of the confocal hole 12, and the returned light beam is basically coincident with the pinhole, or as shown in Figure 8 , the light spot can be located to the right of the confocal hole or in other directions, and the adjustable mirror 7 is finely adjusted so that the returned light beam is coincident with the confocal hole as shown in Figures 9 to 12 . During the adjustment process, the position of the confocal hole approaches the position of the light spot, and when the confocal hole is coincident with the light spot, the camera 10 no longer sees the light spot shape, and there is a relatively uniform halo around the confocal hole: as shown in Figure 12In the middle, the center of the brightest main spot is fully into the confocal hole 12, and the surrounding illuminated is considered to be halo.

[0060] Finally, the position of the adjustable mirror 7 is fixed, the auxiliary light source 11 and the rotatable mirror 6 are removed, and the system confocal adjustment is completed.

[0061] In this embodiment, the galvanometer 4 does not need to start during the debugging process, and the galvanometer target surface is stationary at zero position. Figure 2 In the middle, the center of the brightest main spot is fully into the confocal hole 12, and the surrounding illuminated is considered to be halo.

[0062] In this embodiment, the light beam brightness of the light source 9 irradiated on the calibration plate 13 is relatively weak, and it is basically not displayed and not easy to observe. In comparison, the light spot irradiated on the calibration plate 13 by the auxiliary light source 11 is relatively bright, clear and easy to observe. Therefore, the light beam brightness of the light source 9 is smaller than the light beam brightness of the auxiliary light source 11. During the entire debugging process, the light source 9 is in an open state, and after the confocal hole 12 is debugged, it is finally closed.

[0063] Of course, during the calibration plate position debugging process, the light source 9 can also be closed first to reduce or minimize the light beam image of the light source 9 on the light beam of the auxiliary light source 11, and the calibration plate is illuminated by the auxiliary light source 11. After the final position of the calibration plate is determined, the light source 9 is turned on.

[0064] The parts of the present application not described are prior art, or can be the same as prior art, or are known technology, or can be implemented using prior art, and will not be described in detail here.

[0065] Although embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and alterations can be made without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A point scanning confocal adjustment method, characterized in that: include: A light source (9) emits a parallel light beam, which passes through a beam splitter (5) to reach a galvanometer (4), then passes through a scanning lens group (3) and an objective lens (2) to reach a target object (1), is reflected by the target object (1), returns along the original path to the beam splitter (5), is reflected again, and then passes through a rotatable beam splitter (6) located at a first position to reach a camera (10) as an object side light beam; The camera (10) is used to image at infinity. The camera (10) observes the convergence of light on the object side and adjusts the position of the target object (1). When the light on the object side converges into an Airy disk shape, the target object (1) is located on the confocal plane, and the position of the target object (1) is fixed. The rotatable beam splitter (6) is rotated from the first position to the second position, and the light beam passes through the rotatable beam splitter (6) and the reflector (7) and the focusing lens (8) to reach the calibration plate (13) installed on the mounting seat approximately at the back focus of the focusing lens (8). The calibration plate (13) has coordinates, and the calibration plate (13) is illuminated by the auxiliary light source (11). At this time, the camera (10) still forms an image at infinity, and the object plane of the camera is exactly located at the back focus of the focusing lens (8). When the camera (10) can clearly form an image of the coordinates on the calibration plate (13), the front and rear positions of the mounting seat are fixed; Turn off the auxiliary light source (11), turn on the light source (9), adjust the rotatable spectroscope (6) so that the light beam returned from the light source (9) to the target object (1) is at the center of the field of view of the camera (10), turn on the auxiliary light source (11), adjust the adjustable reflector (7), so that the center of the calibration plate (13) is located at the center of the field of view of the camera (10), at this time, the spot of the light beam reflected back from the surface of the target object (1) by the light source (9) coincides with the center of the calibration plate (13), replace the calibration plate (13) with a confocal hole plate, when the confocal hole (12) of the confocal hole plate coincides with the returned light beam, fix the position of the adjustable reflector (7), remove the auxiliary light source (11) and the rotatable spectroscope (6), and the confocal adjustment is completed.

2. A point scanning confocal adjustment method according to claim 1, characterized in that: The light source (9) emits a parallel light beam with a divergence angle less than 1 mrad.

3. The point scanning confocal adjustment method according to claim 1, wherein: When the rotatable beam splitter (6) is located at the first position, the mirror surface of the rotatable beam splitter (6) is parallel to the mirror surface of the galvanometer mirror (4).

4. The point scanning confocal adjustment method according to claim 1, wherein: When the rotatable beam splitter (6) is located at the second position, the mirror surface of the rotatable beam splitter (6) is perpendicular to the mirror surface of the galvanometer mirror (4).

5. The point scanning confocal adjustment method according to claim 1, wherein: When the confocal hole (12) and the spot of the light beam coincide, the camera (10) cannot see the spot shape, and there is a relatively uniform halo around the confocal hole (12).

6. The point scanning confocal adjustment method according to claim 1, wherein: The rotatable beam splitter (6) is based on a rotatable semi-transparent and semi-reflective mirror.

7. A point scanning confocal adjustment system, characterized in that: include: A beam splitter (5), an objective lens (2), a scanning lens group (3), a rotatable beam splitter (6), an adjustable reflector (7), a focusing lens (8) and a light source (9); The light source (9) emits a parallel light beam, which passes through the beam splitter (5) to reach the galvanometer (4), then passes through the scanning lens group (3) and the objective lens (2) to reach the target object (1), is reflected by the target object (1), returns along the original path to reach the beam splitter (5), and then reflects again, passes through the rotatable beam splitter (6), reaches the adjustable reflector (7), passes through the focusing lens (8), and reaches the confocal hole (12). The confocal hole (12) is located at the back focus of the lens (8), thereby realizing confocal imaging.

8. The point scanning confocal adjustment system according to claim 7, characterized in that: The target object (1), the objective lens (2) and the scanning lens group (3) are located on the same axis.

9. The point scanning confocal adjustment system according to claim 7, characterized in that: The beam splitter (5) is located on the same axis as the galvanometer (4) and the adjustable reflector (7), respectively; the galvanometer (4) and the adjustable reflector (7) are not located on the same axis.

10. The point scanning confocal adjustment system according to claim 7, characterized in that: The adjustable reflector (7) and the lens (8) are located on the same axis.

Images