Rapid automatic focusing module and method suitable for fluorescence microscope

Abstract

The invention discloses a rapid automatic focusing module and method suitable for a fluorescence microscope. The automatic focusing module comprises a laser, a diaphragm, a collimating lens group, a reflector, a dichroic mirror, an objective lens, a sample to be measured, a telescope lens group, a CCD2 detector, a control system and a displacement platform which are sequentially arranged along a focusing light path, and the sample to be measured, the objective lens, the dichroic mirror, a tube lens and the CCD2 detector which are sequentially arranged along an imaging light path; the collimated laser beam is emitted to the plane where the sample to be detected is located through the objective lens, and then the laser beam is reflected back to the telephoto lens group and converged to the CCD2 detector. According to the offset of the center of the reflected light beam, the relative distance between the objective lens and the to-be-tested sample is moved through a control system, and automatic focusing of a single view field of the to-be-tested sample is achieved; the to-be-measured sample is moved to the focal planes of different object view fields in the X direction and the Y direction and is quickly focused in real time, so that the large-size to-be-measured sample is always kept at the focal plane position, and the image quality and the working efficiency are improved.

Description

Technical Field

[0001] The invention relates to a fast automatic focusing module and method suitable for a fluorescence microscope, belonging to the technical field of microscopic imaging. Background Art

[0002] At present, the sample to be tested can receive the laser signal of the illumination module and form a fluorescence signal under the excitation of the laser signal. The objective lens collects the fluorescence signal and transmits it to the imaging system for collection to obtain the corresponding image quality. In view of the high image magnification of the microscope, the depth of field is relatively small, which requires the user to spend a lot of time to adjust the relative distance between the objective lens and the sample to be tested so that the objective lens can converge the fluorescence signal to the imaging system to obtain clear image quality.

[0003] However, there are many limitations to the imaging method of the sample to be tested. For example, due to the large size of the sample to be tested, the focal planes of the various object fields of view are easily not coplanar, and the relative distance between the objective lens and the sample to be tested needs to be readjusted each time after the sample is replaced. Accordingly, it still takes time to continuously adjust the relative distance between the objective lens and the sample to be tested. In the related art, the imaging speed of the imaging system scanning the sample to be tested is slow.

[0004] In addition, given that the molecules to be tested have certain activity, in order to protect their activity, there is generally a glue layer on the substrate, which is covered with a cover glass. There are thickness deviations in different batches of samples to be tested, and the surface flatness of the samples is different, which has a great impact on the clarity of the image quality. If the autofocus system on the market is used, it is difficult to achieve the purpose of fast focusing, so it is necessary to develop a fast autofocus module suitable for fluorescence microscopy.

[0005] In response to the above problems, a fast autofocus module suitable for fluorescence microscope is provided, which aims to control the relative position of the objective lens and the sample to be tested according to the focal plane of each object field of view of the sample to be tested, and perform fast real-time focusing, thereby improving image quality and work efficiency, while reducing direct contact with the sample and avoiding other risks to a certain extent to ensure stable and reliable results. It has broad application prospects in life sciences and semiconductor fields. Summary of the invention

[0006] The invention provides a fast automatic focusing module and method suitable for a fluorescence microscope, which has the characteristics of fast sampling speed, high stability and real-time focusing.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] A fast autofocus module suitable for a fluorescence microscope, comprising a laser, an aperture, a collimating lens group, a reflector, a dichroic mirror, an objective lens, a displacement platform, a sample to be tested, a telephoto lens group, a CCD2 detector, a tube lens, a CCD1 detector and a control system;

[0009] The laser, the aperture, the collimating lens group, the reflector, the dichroic mirror, the objective lens, the sample to be tested and the displacement platform are arranged in sequence along the propagation direction of the laser to form a first optical path; the sample to be tested is located on the displacement platform;

[0010] The sample to be tested, the objective lens, the dichroic mirror, the reflector, the telescopic lens group and the CCD2 detector are arranged in sequence along the reflection direction of the laser and the reflection direction of the dichroic mirror to form a second optical path; the CCD2 detector, the control system and the displacement platform are connected in sequence;

[0011] The sample to be tested, the objective lens, the dichroic mirror, the tube lens and the CCD1 detector are arranged in sequence along the emission direction (the reflection direction of the laser) and the transmission direction of the dichroic mirror to form a third optical path;

[0012] The coating of the dichroic mirror needs to be selected according to the actual situation. The first optical path and the second optical path constitute the focusing optical path, and the third optical path is the imaging optical path. The laser wavelength of the focusing optical path, the excitation light wavelength of the imaging optical path, and the emission light wavelength of the imaging optical path do not overlap. The dichroic mirror reflects the laser in the focusing optical path (the laser emitted by the laser), and transmits the excitation light and emission light in the imaging optical path (the excitation light irradiates the sample to be tested, and the sample to be tested emits emission light after being excited by the laser).

[0013] The laser light emitted by the laser passes through the aperture and collimating lens group in sequence, and then the propagation direction of the light beam is changed by the reflector and the dichroic mirror, and the light beam is introduced into the objective lens, and is focused on the sample to be tested by the objective lens. After being reflected by the sample to be tested, the reflected light beam is collected by the objective lens again, and passes through the dichroic mirror, the reflector and the telephoto lens group in sequence, and then enters the CCD2 detector. The CCD2 detector receives the offset of the reflected light beam spot on the horizontal position generated in the telephoto lens group caused by the offset of the reflected light beam. The control system controls the relative distance between the objective lens and the sample to be tested according to the offset d, so as to realize the automatic focusing of a single field of view of the sample to be tested.

[0014] The sample to be tested is illuminated by the excitation light. The sample to be tested emits emission light after being excited by the laser. The objective lens collects the emission light of the sample to be tested, and the dichroic mirror transmits the emission light. The tube lens converges the emission light on the CCD1 detector and presents it as a clear image on the CCD1 detector.

[0015] The emitted light in this application refers to the emitted light emitted by the sample to be tested after being excited by laser.

[0016] The above laser will emit a monochromatic beam in addition to the excitation light and the emission light; the aperture is used to adjust the size of the laser beam. The collimating lens group collimates the light beam passing through the aperture. The reflector will change the propagation direction of the light. The objective lens converges the focusing light path beam on the sample to be tested, wherein the plane where the sample to be tested is located will reflect the light, and the objective lens is also used to collect the reflected light beam, and sequentially pass through the dichroic mirror and the reflector to send the reflected light beam to the telescope lens group, the telescope lens group is used to focus and amplify the light beam, so that the detailed information of the sample to be tested is clearer and more visible, the CCD2 detector will receive the reflected light beam signal, and present it as the offset of the reflected light beam spot on the horizontal position generated in the telescope lens group caused by the offset of the reflected light beam. The control system adjusts the relative distance between the objective lens and the sample to be tested according to the offset of the reflected light beam spot on the horizontal position generated in the telescope lens group caused by the offset of the reflected light beam to find the optimal focal plane of the sample to be tested in the object field of view. The moving direction and moving position of the objective lens are precisely moved by the Z axis in the displacement platform.

[0017] The aperture is circular or rectangular, and sufficient laser light needs to be allowed to pass through to improve the accuracy of focusing. The shape and size of the aperture can be determined by combining the output shape of the laser and the focal length of the collimating lens group.

[0018] For the excitation light of the sample to be tested, a 45-degree dichroic mirror can be added between the color separation mirror and the tube lens, and an illumination module can be introduced to excite the excitation light. After the excitation light passes through the objective lens, it reaches the sample to be tested. After being excited by the laser, the sample to be tested emits emission light. This application does not make any improvements to the excitation light, and will not be elaborated on here.

[0019] This application is suitable for samples of any size, especially large samples. No matter how large the sample is, it can achieve fast focusing. The focusing range and focusing accuracy are controllable and can be selected by yourself. Even if there is a layer of water on the sample to be tested, fast focusing can be achieved.

[0020] The present application performs real-time detection of focal plane deviation based on optical principles and realizes single-field and multi-field focusing through closed-loop control, which is simple, reliable and low-cost.

[0021] The above displacement platform includes both XY axis and Z axis. The control system can control the displacement platform to move along the XY axis and the objective lens to move along the Z axis. By moving along the Z axis, the relative distance between the objective lens and the sample to be tested can be adjusted to achieve automatic focusing of a single field of view of the sample to be tested. By moving along the XY axis, the sample to be tested can be moved to the focal plane of different object fields of view and quickly focused in real time. The closed-loop control of the stroke keeps the sample to be tested in the focal plane position at all times, improving image quality and work efficiency.

[0022] In order to improve the focusing accuracy, the objective lens is connected to the Z axis to achieve precise movement and adjustment of the relative distance between the objective lens and the sample to be tested to find the best focal plane. The laser emits a laser beam and adjusts the spot size of the laser beam through the aperture, then passes through the collimating lens group, the reflector and the dichroic mirror in sequence, and then is emitted to the plane where the sample to be tested is located through the objective lens, and then is reflected back to the objective lens. The returned reflected beam passes through the dichroic mirror, the reflector and the telephoto lens group again, and converges on the CCD2 detector, and is presented as the offset of the reflected beam spot caused by the offset of the reflected beam in the horizontal position generated in the telephoto lens group. According to the offset, the control system is used to control the moving direction and position of the objective lens to adjust the relative distance between the objective lens and the sample to be tested, and find the best focal plane of the sample to be tested in the object field.

[0023] The adjustment structure and method along the XY axis and Z axis of the present application can refer to the existing mature technology. The present application has no special improvement on this and will not elaborate on it.

[0024] As one specific implementation scheme, the control system includes a motor controller, a computer and a motion controller, and the computer, the motion controller and the motor controller are connected in sequence;

[0025] The computer collects the offset of the reflected light beam spot on the CCD2 detector caused by the offset of the center of the reflected light beam in the telephoto lens group by wired or wireless communication, and calculates the displacement of the focal plane in the vertical direction based on the offset of the reflected light beam spot on the telephoto lens group caused by the offset of the center of the reflected light beam, and then sends a command to the motion controller, which controls the motor controller to drive the objective lens to move precisely along the Z axis, changing the relative distance between the objective lens and the sample to be tested, so as to find the best focal plane (the sample to be tested is on the focal plane);

[0026] The motion controller controls the motor controller to drive the displacement platform to move along the XY axis, so that the sample to be tested can be moved to the focal plane of different object fields of view and quickly focused in real time.

[0027] The CCD2 detector receives the horizontal position offset of the reflected light beam spot in the telephoto lens group caused by the offset of the reflected light beam. The specific process includes: first, the displacement of the focal plane is converted into an angular offset: specifically, when the sample to be tested is at the focal plane, the incident laser is incident on the objective lens and converges on the focal plane, then reflects through the objective lens and emits vertically; when the sample to be tested is not at the focal plane and has a certain displacement in the vertical direction with the focal plane, the incident laser is incident on the objective lens and converges on the plane where the sample to be tested is located, then reflects through the objective lens and emits at a certain angle to the vertical emission direction. In this case, the displacement of the focal plane is converted into the angular offset of the reflected laser, where the change in the angle of the emitted light is related to the displacement in the vertical direction of the focal plane, the focal length of the objective lens, and the angle of the objective lens; secondly, the telescope system converts the angular offset of the reflected laser into a horizontal position offset (compared to the offset of the center of the spot distribution of the laser beam reflected at the focal plane), and the horizontal position offset in the telescope system is related to the magnification of the telescope system, the change in the angle of the reflected light, and the defocus; finally, the CCD2 detector collects the horizontal position offset displayed in the telescope system. Based on the above relationship, the computer can calculate the vertical displacement of the focal plane through the horizontal position offset displayed in the telescope system.

[0028] The CCD2 detector collects the horizontal position offset in the telescope system (compared to the image offset at the focal plane), and then infers the change in the angle of the reflected light, and then infers the displacement of the focal plane.

[0029] In order to improve the focus detection accuracy, the CCD2 detector receives the offset d0 of the reflected beam spot in the horizontal position produced in the telephoto lens group caused by the offset of the reflected beam center. The control system controls the movement of the objective lens according to the offset d0 to find the optimal focal plane.

[0030] For ease of use, the position of the above-mentioned CCD2 detector can be moved up and down to balance the focus range and focus accuracy. The movement amount is determined based on the focus range and focus accuracy. When the position of the CCD2 detector moves downward and closer to the telephoto lens group, the focus range of the focus module will become larger and the focus accuracy will become weaker. Conversely, when it moves upward, the focus range of the focus module will become smaller, but the focus accuracy will be higher.

[0031] The above-mentioned laser emission shape can be point-shaped or line-shaped; the CCD1 detector is an area array industrial camera, which can clearly image the sample to be tested; the CCD2 detector is an area array industrial camera or a line array camera, which can calibrate the offset of the reflected light beam spot in the horizontal position caused by the offset of the reflected light beam in the telescope lens group.

[0032] The number of reflectors can be increased or decreased according to actual needs. For example, if the optical path of the collimating lens group or the telephoto lens group is too long, the number of reflectors can be increased to reduce the structure volume. If the optical path length is appropriate, the number of reflectors can be reduced to reduce costs.

[0033] In order to facilitate assembly and use, the reflector and the dichroic mirror are both at an angle of 45° to the vertical direction, and the reflection surface of the reflector and the reflection surface of the dichroic mirror are parallel and arranged facing each other.

[0034] The sample to be tested is located on the displacement platform and moves along with the movement of the displacement platform.

[0035] A fast autofocus method suitable for a fluorescence microscope is implemented using the fast autofocus module suitable for a fluorescence microscope, comprising the following steps:

[0036] 1) Install the laser, aperture, collimating lens group, reflector, telephoto lens group and CCD2 detector in the autofocus module on the bottom plate according to the reserved hole position, adjust the dichroic mirror so that the angle between its reflection surface and the axis direction is precisely 45 degrees, and adjust the objective lens so that its center line coincides with the optical axis;

[0037] 2) Turn on the laser light source and rotate the direction of the laser beam to align it with the center of the aperture, and adjust the horizontal position of the collimating lens group so that the laser beam emitted from the aperture position can be distributed in the lens center of the collimating lens group;

[0038] 3) Between the reflector and the dichroic mirror, use the detection tool frequency doubling plate to check the relative position of the incident and reflected laser beams. Specifically, use the frequency doubling plate to check the relative position of the laser beam at the mechanical hole when it is incident on the dichroic mirror, which should be slightly to the right of the center of the mechanical hole. Use the frequency doubling plate to check the relative position of the laser beam at the mechanical hole when it is reflected on the reflector, which should be slightly to the left of the center of the mechanical hole. Fine-tune the angle of the reflector so that the distances of the incident and reflected beams that deviate from the center of the mechanical hole remain consistent, and then fix the position of the reflector. The mechanical hole is located between the dichroic mirror and the reflector. The mechanical hole refers to a circular hole on the machined part (lens barrel) of the dichroic mirror that is 45 degrees to the plane where the center of the dichroic mirror is located and is perpendicular to the incident direction of the laser.

[0039] 4) Adjust the center of the telephoto lens group to ensure that the reflected laser beam can be distributed in the center of the lens of the telephoto lens group. The CCD2 detector can be moved up and down to balance the focus range and focus accuracy;

[0040] 5) When the sample to be measured is at the focal plane, the spot distribution center of the reflected laser beam coincides with the center of the CCD2 detector, and the offset of the reflected beam spot in the horizontal position in the telephoto lens group caused by the offset of the reflected beam is 0; when the sample to be measured is not at the focal plane, the spot distribution center of the reflected laser beam is offset from the center of the CCD2 detector, and the offset of the reflected beam spot in the horizontal position in the telephoto lens group caused by the offset of the reflected beam is not 0. The control system controls the relative distance between the objective lens and the sample to be measured according to the offset of the reflected beam spot in the horizontal position in the telephoto lens group caused by the offset of the reflected beam received by the CCD2 detector, so that the spot distribution center of the reflected laser beam coincides with the center of the CCD2 detector, thereby realizing automatic focusing of a single field of view of the sample to be measured;

[0041] 6) The sample to be tested is illuminated with the excitation light, and the sample to be tested emits emission light after being excited by the laser. The objective lens collects the emission light of the sample to be tested, and the dichroic mirror transmits the emission light; the tube lens converges the emission light on the CCD1 detector and presents it as a clear image on the CCD1 detector;

[0042] 7) Move the sample to be tested to different object fields of view by moving along the XY axis, and repeat steps 5)-6) to achieve focusing of different object fields of view.

[0043] In step 1), precision machined parts are used for installation.

[0044] In the above step 4), the mechanical hole is a circular hole, and both the incident laser and the reflected laser will pass through this hole. The incident laser goes to the right of the center of the hole, and the reflected laser goes to the left of the center of the hole.

[0045] The technologies not mentioned in the present invention are all referred to the prior art.

[0046] The benefits of the implementation of the present invention are as follows: the present invention provides a fast autofocus module suitable for a fluorescence microscope, the module can realize real-time detection and focusing of the focal plane of the sample to be tested, and the stroke closed-loop control can ensure that the sample to be tested is always at the focal plane position. Especially for large-sized samples to be tested, the focusing of each object field can be achieved, and the advantage of its fast focusing speed is more obvious. At the same time, the autofocus accuracy of the present invention can be adjusted according to the design, and the focus detection and focusing accuracy can reach less than 10 nanometers. In addition, the influence of the deviation of the thickness of the water layer above different batches of samples to be tested can be overcome to a large extent, and the stability is high. At the same time, the direct contact with the sample is reduced, and other risks are avoided to a certain extent to ensure the stability and reliability of the results. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1It is a structural schematic diagram of a fast autofocus module suitable for a fluorescence microscope of the present invention;

[0048] Figure 2 It is a simulated distribution diagram of the offset position and offset direction of the reflected light beam corresponding to the CCD2 detector at different focal planes in the embodiment of the present invention;

[0049] Figure 3 It is a fitting curve diagram of the reflected light beam data of the CCD2 detector of the sample to be tested located at the focal plane of the present invention;

[0050] Figure 4 It is a fitting curve diagram of the reflected light beam data of the CCD2 detector of the sample to be tested of the present invention, which is located 1 μm above the focal plane;

[0051] Figure 5 It is a fitting curve diagram of the reflected light beam data of the CCD2 detector of the sample to be tested of the present invention, which is located 1 μm below the focal plane;

[0052] Reference numerals:

[0053] 1. Laser; 2. Aperture; 3. Collimating lens group; 4. Reflector; 5. Dichroic mirror; 6. Objective lens; 7. Sample to be tested; 8. Telescope system group; 9. CCD2 detector; 10. Tube lens; 11. CCD1 detector; 12. Displacement platform; 13. Control system. DETAILED DESCRIPTION

[0054] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the content of the present invention is not limited to the following embodiments.

[0055] The directional words such as up and down, left and right, horizontal, and vertical in this application are all based on the relative directions or positional relationships shown in the drawings or when used, and should not be understood as absolute limitations on this application.

[0056] Example 1

[0057] See also Figure 1 , is a schematic structural diagram of a fast autofocus module suitable for a fluorescence microscope in an embodiment, comprising a laser 1, an aperture 2, a collimating lens group 3, a reflector 4, a dichroic mirror 5, an objective lens 6, a displacement platform 12, a sample to be tested 7, a telephoto lens group, a CCD2 detector 9, a tube lens 10, a CCD1 detector 11 and a control system 13;

[0058] The dichroic mirror 5 reflects the laser light emitted by the laser 1 and transmits the emitted light of the sample 7 to be tested;

[0059] The laser 1, the aperture 2, the collimating lens group 3, the reflector 4, the dichroic mirror 5, the objective lens 6, the sample to be tested 7 and the displacement platform 12 are sequentially arranged along the propagation direction of the laser to form a first optical path; the sample to be tested 7 is located on the displacement platform 12;

[0060] The sample to be tested 7, the objective lens 6, the dichroic mirror 5, the reflector 4, the telescopic lens group and the CCD2 detector 9 are arranged in sequence along the reflection direction of the laser and the reflection direction of the dichroic mirror 5 to form a second optical path; the CCD2 detector 9, the control system 13 and the displacement platform 12 are connected in sequence;

[0061] The sample to be tested 7, the objective lens 6, the dichroic mirror 5, the tube lens 10 and the CCD1 detector 11 are arranged in sequence along the emission direction of the light (the reflection direction of the laser) and the transmission direction of the dichroic mirror 5 to form a third optical path; the first optical path and the second optical path constitute a focusing optical path, and the third optical path is an imaging optical path; the laser wavelength of the focusing optical path and the emission wavelength of the imaging optical path do not overlap; the dichroic mirror 5 reflects the laser in the focusing optical path and transmits the emission light in the imaging optical path.

[0062] Among them, after the laser 1 emits a monochromatic light beam between the excitation spectrum and the emission spectrum, it will reach and adjust the size of the laser light beam through the aperture 2, and then collimate the laser light beam with a certain divergence angle through the collimating lens group 3. After collimation, the laser light beam passes through the reflector 4 and the dichroic mirror 5 in turn, changing the propagation direction of the light beam and introducing the light beam into the objective lens. The objective lens 6 collects the focused light path light beam on the sample to be tested 7. The plane where the sample to be tested 7 is located reflects the laser light. After the reflected light beam is collected by the objective lens 6, it will pass through the dichroic mirror 5 and the reflector 4 in turn. Since the incident light beam is incident on the upper half of the reflector and not the center of the reflector, the reflected light beam reflected back will be reflected on the lower half of the reflector. The reflected light path will pass through the telephoto lens group 8 and display the offset d of the reflected light beam spot caused by the offset of the reflected light beam on the horizontal position generated in the telephoto lens group on the CCD2 detector 9, which is the specific information of the relative direction and relative distance of the sample to be tested 7 in the focal plane of the objective lens 6. The position of the CCD2 detector 9 can be moved up and down, and its position needs to be determined according to the focus range and focus accuracy. For example, when the position of the CCD2 detector 9 moves forward and is closer to the telephoto lens group, the focus range of the focus module will become larger and the focus accuracy will become weaker. On the contrary, when it moves backward, the focus range of the focus module will become smaller, but the focus accuracy will be higher. The control system 13 can control the Z axis in the displacement platform 12 to accurately move the relative distance between the objective lens 6 and the sample to be tested according to the offset d of the reflected light beam spot caused by the offset of the reflected light beam displayed by the CCD2 detector 9 in the horizontal position generated in the telephoto lens group, so as to make the sample to be tested 7 have the best focal plane under the object field of view, wherein the objective lens 6 is connected to the Z axis in the displacement platform 12.

[0063] See also Figure 1, is a structural schematic diagram of a fast autofocus module suitable for a fluorescence microscope in an embodiment, and also includes: a sample to be tested 7, an objective lens 6, a dichroic mirror 5, a tube lens 10, and a CCD1 detector 11 are arranged in sequence along the imaging light path, wherein the sample to be tested 7 is irradiated with excitation light, and the sample to be tested 7 will emit emission light after being excited, and the emission light is collected by the objective lens 6, and the emission spectrum is converged on the CCD1 detector 11 by the tube lens 10 after passing through the dichroic mirror 5, and the dichroic mirror 5 can be used not only to reflect the laser beam in the focusing light path, but also to transmit the excitation light and the emission light; the CCD1 detector 11 will present a clear image quality, that is, the detailed information of the sample to be tested 7.

[0064] Example 2

[0065] On the basis of Example 1, the following improvements are further made: the displacement platform 12 includes both an XY axis and a Z axis, and the control system 13 can control the displacement platform 12 to move along the XY axis and control the objective lens 6 to move along the Z axis; by moving along the Z axis, the relative distance between the objective lens 6 and the sample to be tested 7 is adjusted, and the automatic focusing of a single field of view of the sample to be tested 7 is achieved; by moving along the XY axis, the sample to be tested 7 is moved to the focal plane of different object fields of view and quickly focused in real time. The stroke closed-loop control allows the sample to be tested 7 to always remain in the focal plane position, improving image quality and work efficiency. The objective lens 6 is connected to the Z axis to achieve precise adjustment of the relative distance between the objective lens 6 and the sample to be tested 7 to find the best focal plane. The laser 1 emits a laser beam and adjusts the spot size of the laser beam through the aperture 2, and then passes through the collimating lens group 3, the reflector 4 and the dichroic mirror 5 in sequence, and then is emitted to the plane where the sample 7 to be tested is located through the objective lens 6, and is reflected back to the objective lens 6. The returned reflected light beam passes through the dichroic mirror 5, the reflector 4 and the telephoto lens group again, and converges on the CCD2 detector 9, and is presented as the offset of the reflected light beam spot on the horizontal position generated in the telephoto lens group caused by the offset of the reflected light beam. According to the offset direction and position offset of the reflected light beam, the control system 13 is used to control the moving direction and moving position of the objective lens 6 to adjust the relative distance between the objective lens 6 and the sample 7 to be tested, and find the best focal plane of the sample 7 to be tested in the object field.

[0066] Example 3

[0067] On the basis of Example 2, the following improvements are further made: the control system 13 includes a motor controller, a computer and a motion controller, and the computer, the motion controller and the motor controller are connected in sequence;

[0068] The computer collects the offset of the reflected light beam spot on the CCD2 detector 9 caused by the offset of the center of the reflected light beam in the telephoto lens group by wired or wireless communication, and calculates the displacement of the focal plane in the vertical direction based on the offset of the reflected light beam spot on the telephoto lens group caused by the offset of the center of the reflected light beam, and then sends an instruction to the motion controller, which controls the motor controller to drive the objective lens 6 to move precisely along the Z axis, changing the relative distance between the objective lens 6 and the sample 7 to be tested, so as to find the best focal plane (the displacement platform 12 is on the focal plane);

[0069] The motion controller controls the motor controller to drive the displacement platform 12 to move along the XY axis, so as to move the sample 7 to be tested to the focal plane of different object fields of view and quickly focus in real time.

[0070] The CCD2 detector 9 receives the horizontal position offset of the reflected light beam spot generated in the telephoto lens group caused by the offset of the reflected light beam, specifically: first, the displacement of the focal plane is converted into an angular offset: specifically, when the sample 7 to be tested is at the focal plane, the incident laser is incident through the objective lens 6 and converges on the focal plane, then reflects through the objective lens 6 and emits vertically; when the sample 7 to be tested is not at the focal plane and has a certain displacement in the vertical direction with the focal plane, the incident laser is incident through the objective lens 6 and converges on the plane where the sample 7 to be tested is located, then reflects through the objective lens 6 and emits vertically at a certain angle to the vertical emission direction. , at this time, the displacement of the focal plane is converted into the angular offset of the reflected laser, wherein the change of the angle of the emitted light is related to the displacement in the vertical direction of the focal plane, the focal length of the objective lens 6 and the angle of the objective lens 6; secondly, the telescope system converts the angular offset of the reflected laser into the horizontal position offset (compared to the offset of the center of the spot distribution of the laser beam reflected at the focal plane), and the horizontal position offset in the telescope system is related to the magnification of the telescope system, the change of the angle of the reflected light and the defocus; finally, the CCD2 detector 9 collects the horizontal position offset displayed in the telescope system. According to the above relationship, the computer can calculate the vertical displacement of the focal plane through the horizontal position offset displayed in the telescope system.

[0071] Example 4

[0072] On the basis of Example 3, the following improvements are further made: the CCD2 detector receives the offset of the reflected light beam spot in the horizontal position in the telescope lens group caused by the offset of the reflected light beam center, and the control system 13 controls the movement of the objective lens 6 according to the offset of the reflected light beam spot in the horizontal position in the telescope lens group caused by the offset of the reflected light beam center received by the CCD2 detector 9 to find the best focal plane. The laser 1 emits in a point or line shape; the CCD1 detector 11 is a planar array industrial camera, which clearly images the sample 7 to be tested; the CCD2 detector 9 is a planar array industrial camera or a line array camera, which calibrates the offset of the reflected light beam spot in the horizontal position in the telescope lens group caused by the offset of the reflected light beam.

[0073] The reflector 4 and the dichroic mirror 5 both form an angle of 45° with the vertical direction, and the reflective surface of the reflector 4 and the reflective surface of the dichroic mirror 5 are parallel and arranged facing each other.

[0074] A fast autofocus method for a fluorescence microscope is implemented using the fast autofocus modules for a fluorescence microscope in the above examples, comprising the following steps:

[0075] 1) Install the laser 1, aperture 2, collimating lens group 3, reflector 4, telephoto lens group and CCD2 detector 9 in the autofocus module on the bottom plate according to the reserved hole position, adjust the dichroic mirror 5 so that the angle between its reflection surface and the axis direction is precisely 45 degrees, and adjust the objective lens 6 so that its center line coincides with the optical axis;

[0076] 2) Turn on the laser light source 1, rotate the direction of the laser beam to align it with the center of the aperture 2, and adjust the horizontal position of the collimating lens group 3 so that the laser beam emitted from the aperture 2 position can be distributed in the lens center of the collimating lens group 3;

[0077] 3) Between the reflector 4 and the dichroic mirror 5, use the detection tool frequency doubling plate to check the relative position of the incident and reflected laser beams. Specifically, use the frequency doubling plate to check the relative position of the laser beam at the mechanical hole when it is incident on the dichroic mirror 5, which should be slightly to the right of the center of the mechanical hole. Use the frequency doubling plate to check the relative position of the laser beam at the mechanical hole when it is reflected on the reflector 4, which should be slightly to the left of the center of the mechanical hole. Fine-tune the angle of the reflector 4 so that the distances of the incident and reflected deviations from the center of the mechanical hole can be kept consistent, and then fix the position of the reflector 4. The mechanical hole is located between the dichroic mirror 5 and the reflector 4. The mechanical hole refers to a circular hole on the machined part (lens barrel) of the dichroic mirror 5 that is located at 45 degrees to the plane where the center of the dichroic mirror 5 is located and is perpendicular to the incident direction of the laser.

[0078] 4) Adjust the center of the telephoto lens group to ensure that the reflected laser beam can be distributed in the center of the lens of the telephoto lens group. The CCD2 detector 9 can be moved up and down to balance the focusing range and focusing accuracy;

[0079] 5) When the sample 7 to be tested is at the focal plane, the spot distribution center of the reflected laser beam coincides with the center of the CCD2 detector 9, and the offset of the reflected beam spot in the horizontal position in the telephoto lens group caused by the offset of the reflected beam is 0; when the sample 7 to be tested is not at the focal plane, the spot distribution center of the reflected laser beam is offset from the center of the CCD2 detector 9, and the offset of the reflected beam spot in the horizontal position in the telephoto lens group caused by the offset of the reflected beam is not 0. The control system 13 controls the relative distance between the objective lens 6 and the sample 7 to be tested according to the offset of the reflected beam spot in the horizontal position in the telephoto lens group caused by the offset of the reflected beam received by the CCD2 detector 9, so that the spot distribution center of the reflected laser beam coincides with the center of the CCD2 detector 9, thereby realizing automatic focusing of a single field of view of the sample 7 to be tested;

[0080] 6) The sample 7 to be tested is irradiated with the excitation light, and the sample 7 to be tested emits emission light after being excited by the laser. The objective lens 6 collects the emission light of the sample 7 to be tested, and the dichroic mirror 5 transmits the emission light; the tube lens 10 converges the emission light on the CCD1 detector 11, and presents it as a clear image on the CCD1 detector 11;

[0081] 7) Move the sample 7 to different object fields of view by moving along the XY axis, and repeat steps 5)-6) to achieve focusing of different object fields of view.

[0082] join Figure 2 , are the offset directions and offset positions of the reflected light beams at different focal planes in the embodiment, specifically: the aperture 2 is selected to be rectangular in shape, which is equivalent to the object side in the object image being a rectangle. According to the conjugate imaging principle, the projection corresponding to the aperture 2 should be a rectangle. If the edges of the rectangle are clear and the center of the rectangle coincides with the center of the CCD2 detector 9, it means that the sample to be tested is already on the focal plane. If the edges of the rectangle are blurred and the size changes, it means that the sample to be tested is not on the focal plane. Figure 2 Attached is a simulation diagram of the offset direction and offset of the reflected light beam at different focal planes. When the sample 7 to be tested is above the focal plane, the edge of the rectangle becomes blurred, the size changes, and the center of the rectangle moves upward. At this time, the objective lens 6 needs to be moved upward. When the sample 7 to be tested is below the focal plane, the edge of the rectangle becomes blurred, the size changes, and the center of the rectangle moves downward. At this time, the objective lens 6 needs to be moved downward. In addition, the farther away from the focal plane, the blurred the edge of the rectangle, the more obvious the size change, and the greater the center offset.

[0083] See also Figure 3, is a fitting curve of the reflected light beam data on the CCD2 detector 9 at the focal plane of the sample 7 in the embodiment. At this time, the spot distribution center of the reflected light beam coincides with the center of the CCD2 detector 9.

[0084] See also Figure 4 , is the fitting curve of the reflected light beam data on the CCD2 detector 9 where the sample 7 to be tested is located 1 μm above the focal plane in the embodiment. At this time, the spot distribution center of the reflected light beam is to the left of the center of the CCD2 detector 9, which is smaller than Figure 3 The center position of the CCD2 detector 9 is offset by 45 pixels, about 0.2475 mm, that is, the offset d0 is about -0.2475 mm. The relative distance between the objective lens and the sample to be tested is adjusted so that the spot distribution center of the reflected light beam coincides with the center of the CCD2 detector 9, the offset d0 is 0, and the sample to be tested is located on the focal plane.

[0085] See also Figure 5 , is the fitting curve of the reflected light beam data on the CCD2 detector 9 where the sample 7 to be tested is located 1 μm below the focal plane in the embodiment. At this time, the spot distribution center of the reflected light beam is to the right of the center of the CCD2 detector 9, which is higher than Figure 3 The center position of the CCD2 detector 9 is offset by 48 pixels, about 0.264 mm, that is, the offset d0 is about +0.264 mm. The relative distance between the objective lens and the sample to be tested is adjusted so that the spot distribution center of the reflected light beam coincides with the center of the CCD2 detector 9, the offset d0 is 0, and the sample to be tested is located on the focal plane.

[0086] The fast autofocus modules for fluorescence microscopes can achieve a focus detection and focusing accuracy of less than 10 nanometers.

[0087] In some specific embodiments, the depth of field of the microscope is relatively small, that is, the up and down movement range of the Z-axis in the displacement platform is very small, generally in the order of hundreds of nanometers. Based on this, it becomes more important to quickly focus on different focal planes of each object field of view in the sample 7 to be tested, especially in a large size range.

Claims

1. A fast autofocus module suitable for a fluorescence microscope, characterized in that: It includes laser, aperture, collimating lens group, reflector, dichroic mirror, objective lens, displacement platform, sample to be tested, telescope lens group, CCD2 detector, tube lens, CCD1 detector and control system; The laser, the aperture, the collimating lens group, the reflector, the dichroic mirror, the objective lens, the sample to be tested and the displacement platform are arranged in sequence along the propagation direction of the laser to form a first optical path; the sample to be tested is located on the displacement platform; The sample to be tested, the objective lens, the dichroic mirror, the reflector, the telescopic lens group and the CCD2 detector are arranged in sequence along the reflection direction of the laser and the reflection direction of the dichroic mirror to form a second optical path; the CCD2 detector, the control system and the displacement platform are connected in sequence; The sample to be tested, the objective lens, the dichroic mirror, the tube lens and the CCD1 detector are arranged in sequence along the emission direction of the light and the transmission direction of the dichroic mirror to form a third optical path; The first optical path and the second optical path form a focusing optical path, and the third optical path is an imaging optical path; the laser wavelength of the focusing optical path, the excitation light wavelength of the imaging optical path, and the emission light wavelength of the imaging optical path do not overlap; the dichroic mirror reflects the laser in the focusing optical path and transmits the excitation light and emission light in the imaging optical path; The laser light emitted by the laser passes through the aperture and collimating lens group in sequence, and then the propagation direction of the light beam is changed by the reflector and the dichroic mirror, and the light beam is introduced into the objective lens, and is focused on the sample to be tested by the objective lens. After being reflected by the sample to be tested, the reflected light beam is collected by the objective lens again, and passes through the dichroic mirror, the reflector and the telephoto lens group in sequence, and then enters the CCD2 detector. The CCD2 detector receives the offset d of the reflected light beam spot on the horizontal position generated in the telephoto lens group caused by the offset of the reflected light beam. The control system controls the relative distance between the objective lens and the sample to be tested according to the offset d, so as to realize the automatic focusing of a single field of view of the sample to be tested. The sample to be tested is irradiated with the excitation light, and the sample to be tested emits emission light after being excited by the laser. The objective lens collects the emission light of the sample to be tested, and the dichroic mirror transmits the emission light; The tube lens focuses the emitted light onto the CCD1 detector and presents it as a clear image on the CCD1 detector.

2. The fast autofocus module suitable for fluorescence microscopy according to claim 1, characterized in that: The displacement platform includes both an XY axis and a Z axis. The control system can control the displacement platform to move along the XY axis and control the objective lens to move along the Z axis. By moving along the Z axis, the relative distance between the objective lens and the sample to be tested can be adjusted to achieve automatic focusing of a single field of view of the sample to be tested. By moving along the XY axis, the sample to be tested can be moved to the focal plane of different object fields of view and quickly focused in real time.

3. The fast autofocus module suitable for fluorescence microscopy according to claim 2, characterized in that: The objective lens is connected on the Z-axis to achieve precise movement and adjustment of the relative distance between the objective lens and the sample to be measured in order to find the best focal plane.

4. The fast autofocus module for a fluorescence microscope according to any one of claims 1 to 3, characterized in that: The control system includes a motor controller, a computer and a motion controller, and the computer, the motion controller and the motor controller are connected in sequence; The computer collects the offset of the reflected light beam spot on the CCD2 detector caused by the offset of the center of the reflected light beam in the telescope lens group through wired or wireless communication, and calculates the displacement of the focal plane in the vertical direction based on the offset, and then sends instructions to the motion controller, which controls the motor controller to drive the objective lens to move precisely along the Z axis, changing the relative distance between the objective lens and the sample to be tested, so as to find the best focal plane; The motion controller controls the motor controller to drive the displacement platform to move along the XY axis, so that the sample to be tested can be moved to the focal plane of different object fields of view and quickly focused in real time.

5. The fast autofocus module for fluorescence microscopy according to any one of claims 1 to 3, characterized in that: The CCD2 detector receives the offset of the reflected light beam spot on the horizontal position in the telephoto lens group caused by the offset of the reflected light beam, specifically including: first, the displacement of the focal plane is converted into an angular offset: specifically, when the sample to be tested is at the focal plane, the incident laser is incident on the objective lens and converges on the focal plane, then reflects through the objective lens and emits vertically; when the sample to be tested is not at the focal plane and has a certain displacement in the vertical direction with the focal plane, the incident laser is incident on the objective lens and converges on the plane where the sample to be tested is located, then reflects through the objective lens, and will emit in a direction with a certain angle to the vertical emission direction. At this time, the displacement of the focal plane is converted into the angular offset of the reflected laser; secondly, the telescope system converts the angular offset of the reflected laser into a horizontal position offset; finally, the CCD2 detector collects the horizontal position offset displayed in the telescope system.

6. The fast autofocus module for fluorescence microscopy according to any one of claims 1 to 3, characterized in that: The CCD2 detector receives the offset d0 of the reflected light beam spot on the horizontal position generated in the telephoto lens group caused by the offset of the reflected light beam center. The control system controls the movement of the objective lens according to the offset d0 to find the best focal plane.

7. The fast autofocus module for fluorescence microscopy according to any one of claims 1 to 3, characterized in that: The position of the CCD2 detector can be moved up and down, and the movement amount is determined according to the focus range and focus accuracy. When the position of the CCD2 detector moves downward and is closer to the telephoto lens group, the focus range of the focus module will become larger and the focus accuracy will become weaker. Conversely, when it moves upward, the focus range of the focus module will become smaller, but the focus accuracy will be higher.

8. The fast autofocus module for fluorescence microscopy according to any one of claims 1 to 3, characterized in that: The laser emission shape can be point-shaped or line-shaped; the CCD1 detector is an area array industrial camera, which can clearly image the sample to be tested; the CCD2 detector is an area array industrial camera or a line array camera, which can calibrate the offset of the reflected light beam spot in the horizontal position in the telescope lens group caused by the offset of the reflected light beam.

9. The fast autofocus module for fluorescence microscopy according to any one of claims 1 to 3, characterized in that: The reflector and the dichroic mirror both form an angle of 45° with the vertical direction, and the reflection surface of the reflector and the reflection surface of the dichroic mirror are parallel and arranged facing each other.

10. A fast autofocus method for a fluorescence microscope, implemented by using the fast autofocus module for a fluorescence microscope according to any one of claims 1 to 9, characterized in that: The following steps are involved: 1) Install the laser, aperture, collimating lens group, reflector, telephoto lens group and CCD2 detector in the autofocus module on the bottom plate according to the reserved hole position, adjust the dichroic mirror so that the angle between its reflection surface and the axis direction is 45 degrees, and adjust the objective lens so that its center line coincides with the optical axis; 2) Turn on the laser light source and rotate the direction of the laser beam to align it with the center of the aperture, and adjust the horizontal position of the collimating lens group so that the laser beam emitted from the aperture position can be distributed in the lens center of the collimating lens group; 3) Between the reflector and the dichroic mirror, use the detection tool frequency doubling plate to check the relative position of the incident and reflected laser beams. Specifically, use the frequency doubling plate to check the relative position of the laser beam at the mechanical hole when it is incident on the dichroic mirror, which should be slightly to the right of the center of the mechanical hole. Use the frequency doubling plate to check the relative position of the laser beam at the mechanical hole when it is reflected on the reflector, which should be slightly to the left of the center of the mechanical hole. Fine-tune the angle of the reflector so that the distances of the incident and reflected beams that deviate from the center of the mechanical hole are consistent, and then fix the position of the reflector. The mechanical hole is located between the dichroic mirror and the reflector. The mechanical hole refers to a circular hole on the machined part of the dichroic mirror that is located at 45 degrees to the plane where the center of the dichroic mirror is located and is perpendicular to the direction of laser incidence. 4) Adjust the center of the telephoto lens group to ensure that the reflected laser beam can be distributed in the center of the lens of the telephoto lens group. The CCD2 detector can be moved up and down to balance the focus range and focus accuracy; 5) When the sample to be measured is at the focal plane, the spot distribution center of the reflected laser beam coincides with the center of the CCD2 detector, and the offset of the reflected beam spot in the horizontal position in the telephoto lens group caused by the offset of the reflected beam is 0; when the sample to be measured is not at the focal plane, the spot distribution center of the reflected laser beam is offset from the center of the CCD2 detector, and the offset of the reflected beam spot in the horizontal position in the telephoto lens group caused by the offset of the reflected beam is not 0. The control system controls the relative distance between the objective lens and the sample to be measured according to the offset of the reflected beam spot in the horizontal position in the telephoto lens group caused by the offset of the reflected beam received by the CCD2 detector, so that the spot distribution center of the reflected laser beam coincides with the center of the CCD2 detector, thereby realizing automatic focusing of a single field of view of the sample to be measured; 6) The sample to be tested is illuminated with the excitation light, and the sample to be tested emits emission light after being excited by the laser. The objective lens collects the emission light of the sample to be tested, and the dichroic mirror transmits the emission light; the tube lens converges the emission light on the CCD1 detector and presents it as a clear image on the CCD1 detector; 7) Move the sample to be tested to different object fields of view by moving along the XY axis, and repeat steps 5)-6) to achieve focusing of different object fields of view.

Images