A point scanning confocal adjustment system and method

Abstract

The present application relates to the field of microscopic optics, and especially to a point scanning confocal adjustment system and method, a light source emits light as a parallel light beam, the divergence angle is less than 1 mrad, the light beam reaches a galvanometer through a beam splitter, reaches a target object through a scanning lens group and an objective lens, returns after being reflected by the target object, reaches an adjustable mirror through a rotatable beam splitter, reaches a confocal aperture after being reflected and passing through a focusing lens; the confocal aperture is located at the back focal point of the lens, and confocal imaging is realized. Through a rotatable beam splitter, the present application realizes simultaneous observation of the imaging conditions of the target object and the confocal aperture position by using one camera. The present application solves the technical problem that, in the adjustment process, because the confocal aperture is small, in order to clearly see the confocal aperture, a camera with a high magnification is needed, the observable image surface range of the camera is very small, and if the initial position of the confocal aperture is not in the field of view of the camera, the confocal aperture position will be difficult to find.

Description

Technical Field

[0001] This invention relates to the field of microscopic optics, specifically to a point scanning confocal adjustment system and method. Background Technology

[0002] Reflective confocal microscopy systems are among the most advanced cell biomedical analytical instruments available today. They perform optical tomography on samples along their axis, obtaining in vivo images at the cellular level with a depth of 200–350 μm, and reconstructing a three-dimensional image of the sample. However, adjusting the axial position of the pinhole in a reflective confocal microscopy system is a common challenge.

[0003] In related technologies, the general debugging scheme of a confocal system requires observing the convergence of the light spot at the target object's position to determine if the target object is in the correct working plane. It also requires observing the position of the confocal aperture to adjust the position of the returning light spot, enabling the system to achieve a confocal state. A confocal system typically achieves an object-side resolution of around 1µm and an image-side resolution of around 20µm. If the system's image-side convergence is 20µm, a 20µm or 25µm confocal aperture is generally chosen to achieve confocal imaging. An excessively large confocal aperture will reduce resolution, while an excessively small aperture will reduce image brightness; therefore, adjusting the position of the confocal aperture is a crucial step in confocal system debugging. During confocal debugging, the confocal aperture involves alignment in three dimensions (up / down, left / right, and front / back). If the confocal aperture is not concentric with the beam reflected back from the observed object, it will result in low image brightness or imaging failure. If the front / back position of the confocal aperture is not located on the focal plane of the focusing lens, the resolution will not reach the system's design value. Summary of the Invention

[0004] The purpose of this invention is to provide a point scanning confocal adjustment system and method to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A point-scan confocal adjustment method, comprising:

[0007] The light source emits a parallel beam of light, which passes through a beam splitter to a galvanometer, then through a scanning lens group and an objective lens to the target object. After being reflected by the target object, the beam returns along the original path to the beam splitter, where it is reflected again. Finally, it passes through a rotatable beam splitter located in the first position to the camera as the object-side ray.

[0008] For imaging at infinity, the camera observes the convergence of light rays on the object side and adjusts the position of the target object. When the light rays converge to form an Airy disk, the target object is located on the confocal plane, thus fixing the position of the target object.

[0009] A mounting base is pre-set at the back focal point of the focusing lens. A calibration plate with coordinate markings is installed on the mounting base. The calibration plate is illuminated with an auxiliary light source. The rotatable beam splitter is rotated from the first position to the second position. The light beam passes through the rotatable beam splitter, the reflector, and the focusing lens to reach the calibration plate. At this time, the camera is still imaging at infinity. The camera object plane is exactly located at the back focal point of the focusing lens. When the camera can clearly image the coordinates on the calibration plate, the front and rear positions of the mounting base are fixed.

[0010] Turn off the auxiliary light source, turn on the light source, and adjust the rotatable beam splitter so that the beam returning from the target is centered in the camera's field of view. Turn on the auxiliary light source again, and adjust the adjustable mirror so that the center of the calibration plate is centered in the camera's field of view. At this point, the spot of the beam reflected from the target surface coincides with the center of the calibration plate. Replace the calibration plate with a confocal aperture plate, ensuring that the confocal aperture of the confocal aperture plate coincides with the returning beam. Fix the position of the adjustable mirror, remove the auxiliary light source and the rotatable beam splitter, and the confocal adjustment is complete.

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

[0012] Furthermore, when the rotatable beam splitter is in the first position, the mirror surface of the rotatable beam splitter is parallel to the mirror surface of the galvanometer.

[0013] Furthermore, when the rotatable beam splitter is in the second position, the mirror surface of the rotatable beam splitter is perpendicular to the mirror surface of the galvanometer.

[0014] Furthermore, the angles of the first and second positions differ by 90°.

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

[0016] Furthermore, the rotatable beam splitter is based on a rotatable semi-transparent and semi-reflective mirror.

[0017] To achieve the above objectives, the present invention also provides the following technical solution:

[0018] A point-scan confocal adjustment system, comprising:

[0019] Beam splitter, objective lens, scanning lens group, rotatable beam splitter, adjustable mirror, focusing lens and light source;

[0020] The light source emits a parallel beam of light, which passes through a beam splitter to a galvanometer, then through a scanning lens group and an objective lens to the target object. After being reflected by the target object, the beam returns along the original path to the beam splitter, where it is reflected again. It then passes through a rotatable beam splitter to an adjustable mirror, and after reflection, it passes through a focusing lens to a confocal aperture. The confocal aperture is located at the rear focal point of the lens, thus achieving confocal imaging.

[0021] Furthermore, the target object, objective lens, and scanning lens assembly are located on the same axis.

[0022] Furthermore, the beam splitter is located on the same axis as the galvanometer and the adjustable mirror, respectively, while the galvanometer and the adjustable mirror are not located on the same axis.

[0023] Furthermore, the adjustable reflector and the lens are located on the same axis.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] This invention utilizes a rotatable beam splitter to enable simultaneous observation of the target object and the confocal aperture image using a single camera. A calibration plate is designed to be mounted at the confocal aperture location. This calibration plate has coordinates to confirm its position on the focal plane, and the adjustable reflector angle is approximately correct without significant deviation.

[0026] The calibration board is used to confirm that the initial position of the confocal aperture does not deviate from the camera's field of view, thus reducing the difficulty of setup and adjustment. This solves the technical problem during debugging: because the confocal aperture is small, a high-magnification camera is required to see it clearly. However, the small image plane that the camera can observe makes it difficult to locate the confocal aperture if its initial position is outside the camera's field of view. Furthermore, even if the confocal aperture is found, its small size makes it difficult to determine if it is on the accurate confocal plane, resulting in resolution not meeting the system design values.

[0027] This invention solves the problem in related technologies where common debugging schemes for confocal systems require fine-tuning the angle of the reflector to find the location of the confocal aperture. Since the confocal aperture is very small, and when the confocal aperture is not located on the back focal plane of the focusing lens, the confocal aperture will not produce a clear image. This makes it difficult to determine whether the inability to find the confocal aperture is due to it not being on the focal plane or due to a problem with the reflector angle.

[0028] This invention uses a beam splitter to allow the light beam emitted by the light source to be transmitted when it first passes through the light source, and then reflected back to the camera when it is reflected by the target object. By using a mirror group consisting of a shared objective lens and a scanning lens group, a portion of the optical path is overlapped, thereby reducing the system size. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the optical path of the system for determining the position of the calibration plate in this invention.

[0030] Figure 2 This is a schematic diagram of the optical path of the system for determining the position of the confocal aperture in this invention.

[0031] Figure 3 This is a schematic diagram of the rotatable beam splitter of the present invention mounted on the frame.

[0032] Figure 4 This is a schematic diagram of the calibration plate structure of the present invention.

[0033] Figure 5 This invention illustrates the light convergence observed by the camera when adjusting the target object's position. Figure 1 .

[0034] Figure 6 This invention illustrates the light convergence observed by the camera when adjusting the target object's position. Figure 2 .

[0035] Figure 7 This is a schematic diagram showing the convergence of light rays into an Airy disk shape as observed by the camera when the target object's position is adjusted according to the present invention.

[0036] Figure 8 This invention illustrates the fine-tuning of the adjustable mirror to bring the confocal aperture closer to the beam spot (returning beam) position. Figure 1 .

[0037] Figure 9 This invention illustrates the fine-tuning of the adjustable mirror to bring the confocal aperture closer to the light spot position. Figure 2 .

[0038] Figure 10 This invention illustrates the fine-tuning of the adjustable mirror to bring the confocal aperture closer to the light spot position. Figure 3 .

[0039] Figure 11 This invention illustrates the fine-tuning of the adjustable mirror to bring the confocal aperture closer to the light spot position. Figure 3 .

[0040] Figure 12 This is a schematic diagram illustrating the fine-tuning of the adjustable reflector in this invention to make the returned beam coincide with the confocal aperture.

[0041] In the figure: 1-Target object, 2-Objective lens, 3-Scanning lens group, 4-Galvanometer, 5-Beam splitter, 6-Rotable beam splitter, 7-Adjustable mirror, 8-Focusing lens, 9-Light source, 10-Camera, 11-Adjustment auxiliary light source, 12-Confocal aperture, 13-Calibration plate. Detailed Implementation

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

[0043] In the description of this invention, it should be noted that the terms "upper end," "lower end," "inner," "outer," "front end," "rear end," "both ends," "one end," and "the other end," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention. In addition, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0044] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "sleeved with," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0045] Please see Figures 1 to 12 The present invention provides a technical solution:

[0046] A point-scanning confocal adjustment method is presented, which is an easy-to-operate and accurate method for debugging confocal systems. By using a rotatable beam splitter, it enables simultaneous observation of the target object and the confocal aperture position using a single camera.

[0047] In related technologies, during the confocal tuning process, because the confocal aperture is small, a camera with a high magnification is required to see it clearly. The area of ​​the image plane that the camera can observe is very small, which makes it difficult to find the location of the confocal aperture if its initial position is not within the camera's field of view.

[0048] To address the difficulty in locating the confocal aperture, this invention designs a calibration plate that can be installed at the confocal aperture location. The calibration plate confirms that the initial position of the confocal aperture will not deviate from the camera's field of view, thereby reducing the difficulty of setup and adjustment.

[0049] Since there is no calibration board in the confocal debugging process, the position of the confocal aperture needs to be found by finely adjusting the angle of the reflector. Because the size of the confocal aperture is very small, and when the confocal aperture 12 is not located on the back focal plane of the focusing lens 8, the confocal aperture will not form a clear image. This makes it difficult to determine in most cases whether the inability to find the position of the confocal aperture is due to it not being on the focal plane, or due to the angle of the adjustable reflector 7.

[0050] Since the calibration plate 13 of the present invention has coordinates that can be confirmed to be on the focal plane, and the angle of the adjustable reflector 7 is approximately correct, it will not deviate too much.

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

[0052] The light source 9 emits a parallel beam with a divergence angle of less than 1 mrad. The beam passes through the beam splitter 5 to the galvanometer 4, then through the scanning mirror group 3 and the objective lens 2 to the target object 1. After being reflected by the target object 1, the beam passes through the objective lens 2, the scanning mirror group 3, and the galvanometer 4 again to the beam splitter 5, where it is reflected again. It then passes through a rotatable beam splitter (i.e., the rotatable beam splitter 6, also known as a half-transparent half-reflective mirror or a 50 / 50 beam splitter) to the adjustable reflector 7. After reflection, it passes through the focusing lens 8 and reaches the confocal aperture 12. The confocal aperture 12 is located at the back focal point of the lens 8, achieving confocal imaging.

[0053] The functions of the beam splitter 5 are: to allow the light beam emitted by the light source 9 to be transmitted when it first passes through, and to be reflected back to the detector (i.e., camera 10) when it is reflected back by the target object 1; and to use the shared lens group (objective lens 2 and scanning lens group 3) to overlap part of the optical path, thereby reducing the system size.

[0054] In this embodiment, as Figure 3 As shown, the rotatable beam splitter 6 can be manually adjusted. The lens is installed at the square hole c of the frame a. Rotating the frame a causes the lens to rotate along the axis c of the frame a. Alternatively, the frame a can be driven by a stepper motor shaft, causing it to rotate along the axis c of the frame a, thus rotating the rotatable beam splitter 6 and ultimately adjusting it to a suitable angle.

[0055] When adjusting the confocal aperture, place a target object 1 with a finely adjustable distance at the working surface of objective lens 2. When the rotatable beam splitter 6 is in the first position (i.e., Figure 1 , Figure 2 At the position indicated by the dotted line in the image, the parallel beam emitted by the light source 9 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 reach the target object 1. The reflected light then passes through the objective lens 2 and the scanning lens group 3 again, and the galvanometer 4, which is not activated and is in its initial position, is reflected by the beam splitter 5 to the rotatable beam splitter 6 located at the dotted line position, reaching the camera 10. At this time, the camera 10 can image at infinity. The camera 10 can observe the convergence of the object-side light rays through the optical system and adjust the position of the target object 1 (along the dotted line). Figure 1 , Figure 2 Move the horizontal arrow in the middle to converge the light. Figure 5 , Figure 6 and Figure 7 As shown, when converged into Figure 7 Airy spot morphology ( Figure 7 In the middle, the central bright spot is the Airy disk. At this time, 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 reflector.

[0057] like Figure 1 and Figure 4 The mounting base d is equipped with a calibration plate 13 bearing coordinate markings or a calibration pattern f. The calibration plate 13 is located at the position where the confocal aperture 12 is installed (at this time, the confocal aperture 12 is not installed). The rotatable beam splitter 6 is rotated 90° to the first position (i.e., Figure 1 , Figure 2 (At the solid line position in the image), due to the parallel beam emitted by the light source 9, it 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 reach the target object 1. The reflected light passes again through the objective lens 2 and the scanning lens group 3, and the galvanometer 4, which is not activated and is in its initial position, is reflected by the beam splitter 5, passes through the rotatable beam splitter 6 located at the solid line position, then passes through the adjustable mirror 7 and the focusing lens 8, and reaches the calibration plate 13, which is approximately located at the back focal point of the focusing lens 8. The calibration plate is illuminated by the auxiliary light source 11. At this time, the camera 10 is still imaging at infinity. After passing through the rotatable mirror 6, the adjustable mirror 7, and the focusing lens 8, the camera object plane is exactly located at the back focal point of the focusing lens 8, as shown in the image. Figure 1 As shown, the calibration plate 13 is moved by moving the mounting base d in the direction of the vertical arrow. Therefore, when the camera can clearly image the coordinates on the calibration plate 13, it proves that the calibration plate is exactly at the back focal point of the lens 8, and the calibration plate position adjustment is considered complete. Fix the position of the mounting base d, and then turn off the auxiliary light source 11.

[0058] Turn on the light source 9 and adjust the rotatable reflector 6 so that the beam of light returning from the target object 1 is at the center of the camera's field of view; turn on the auxiliary light source 11 and adjust the adjustable reflector 7 so that the center of the calibration plate is located at the center of the camera's field of view. At this time, the light spot reflected back from the surface of the target object 1 by the light source 9 should roughly coincide with the center e of the calibration plate 13.

[0059] Remove calibration plate 13 from mounting base d and replace it with a confocal aperture plate of the same shape and size. At this time, the confocal aperture 12 should be within the field of view of camera 10 and have a clear outline. Figures 8 to 12 The white circle marks the location of the confocal aperture 12. The returned beam basically coincides with the aperture, or as shown in the image. Figure 8 As shown, the light spot may be located to the right of the confocal aperture or in other positions. Fine-tune the adjustable mirror 7 to make the returned beam coincide with the confocal aperture. Figures 9 to 12 During adjustment, the confocal aperture position moves closer to the light spot position. When the confocal aperture coincides with the light spot, the camera 10 no longer sees the light spot shape, and a relatively uniform halo appears around the confocal aperture: such as Figure 12In the center, the brightest main spot is completely inside the confocal aperture 12, and the area around it that is illuminated is considered to be a halo.

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

[0061] In this embodiment, during the debugging process, galvanometer 4 does not need to be started, and the galvanometer target surface remains stationary at zero position. Figure 2 In the image, the four dashed lines on the galvanometer indicate that it can vibrate. During vibration, the angle between the solid line and the dashed line is about 5°.

[0062] In this embodiment, the light beam emitted by the light source 9 illuminates a relatively weak bright spot on the calibration plate 13, which is barely visible and difficult to observe. In contrast, the light emitted by the auxiliary light source 11 illuminates a brighter bright spot on the calibration plate 13, which is clearly visible and easy to observe. Therefore, the brightness of the light beam emitted by the light source 9 is less than the brightness of the light beam emitted by the auxiliary light source 11. Throughout the entire debugging process, the light source 9 is in the open state, and is finally turned off after the confocal aperture 12 is debugged.

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

[0064] The parts of this invention not described herein are prior art, or may be the same as prior art, or may be known art, or may be implemented using prior art, and will not be described in detail here.

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

Claims

1. A point scanning confocal adjustment method, characterized by, The light source (9) emits parallel light beams, the light beams pass through the beam splitter (5) to reach the galvanometer mirror (4), then pass through the scanning mirror group (3) and the objective lens (2) to reach the target object (1), are reflected by the target object (1) to return to the beam splitter (5), are reflected by the beam splitter (5) again, and then pass through the rotatable beam splitter (6) at the first position to reach the camera (10) as the object-side light rays. The camera (10) is used for imaging at infinity, the convergence of the object-side light rays is observed through the camera (10), the position of the target object (1) is adjusted, the object-side light rays converge into an Airy pattern, and at this time, 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, the light beams reflected by the target object (1) to return to the beam splitter (5) are reflected by the rotatable beam splitter (6), pass through the reflector (7) and the focusing lens (8), reach the calibration plate (13) installed on the mounting seat at the approximate back focal point of the focusing lens (8), the calibration plate (13) has coordinates, the calibration plate (13) is illuminated by the auxiliary light source (11), at this time, the camera (10) is still used for imaging at infinity, the object plane of the camera (10) is located on the back focal point of the focusing lens (8), and when the camera (10) can clearly image the coordinates on the calibration plate (13), the positions of the mounting seat are fixed. The auxiliary light source (11) is turned off, the light source (9) is turned on, the rotatable beam splitter (6) is adjusted, the light beams returned by the light source (9) to the target object (1) are located at the center of the field of view of the camera (10), the auxiliary light source (11) is turned on, the adjustable reflector (7) is adjusted, 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 light spot of the light beams returned by the light source (9) through the surface of the target object (1) coincides with the center of the calibration plate (13), the calibration plate (13) is replaced by a confocal aperture plate, when the confocal aperture (12) of the confocal aperture plate coincides with the returned light beams, the position of the adjustable reflector (7) is fixed, the auxiliary light source (11) and the rotatable beam splitter (6) are removed, and the confocal adjustment is completed. The divergence angle of the parallel light beams emitted by the light source (9) is less than 1 mrad.

2. A method of point scanning confocal regulation as claimed in 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).

3. A method of point scanning confocal regulation as claimed in 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).

4. A method of point scanning confocal regulation as claimed in claim 1, wherein, When the confocal aperture (12) coincides with the light spot of the light beams, the camera (10) cannot see the shape of the light spot, and there is a relatively uniform halo around the confocal aperture (12).

5. A method of point scanning confocal regulation as claimed in claim 1, wherein, The rotatable beam splitter (6) is based on a rotatable half-transmissive half-reflective lens.

6. A method of point scanning confocal regulation as claimed in claim 1, wherein, The beam splitter (5), the objective lens (2), the scanning mirror group (3), the rotatable beam splitter (6), the adjustable reflector (7), the focusing lens (8) and the light source (9) are included.

7. A point scanning confocal adjustment system, the system implements confocal adjustment by using the method of any one of claims 1 to 6, characterized in that the system ​ ​ The light source (9) emits parallel light beams, the light beams pass through the beam splitter (5) to the galvanometer (4), then pass through the scanning lens group (3) and the objective lens (2) to the target object (1), reflect along the original path to the beam splitter (5) after passing through the target object (1), reflect again after passing through the rotatable beam splitter (6), reach the adjustable mirror (7), reflect after passing through the focusing lens (8), reach the confocal aperture (12), and the confocal aperture (12) is located at the back focal point of the lens (8), so that confocal imaging is realized.

8. A point scanning confocal regulation system as claimed in 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. A point scanning confocal regulation system as claimed in claim 7, characterized in that, The beam splitter (5) is located on the same axis with the galvanometer (4) and the adjustable mirror (7), and the galvanometer (4) and the adjustable mirror (7) are not located on the same axis.

10. A point scanning confocal regulation system as claimed in claim 7, characterized in that, The adjustable mirror (7) is located on the same axis with the lens (8).

Images