A fast autofocus module and method suitable for fluorescence microscope

Abstract

The present invention discloses a fast autofocus module and method suitable for a fluorescence microscope. The autofocus module includes a laser, an aperture, a collimating lens group, a reflector, a dichroic mirror, an objective lens, a sample to be tested, a telephoto lens group, a CCD2 detector, a control system, and a displacement platform, which are sequentially arranged along a focusing optical path; and a sample to be tested, an objective lens, a dichroic mirror, a tube lens, and a CCD2 detector, which are sequentially arranged along an imaging optical path; a collimated laser beam is emitted from the objective lens to the plane where the sample to be tested is located, and then reflected back to the telephoto lens group and converged onto the CCD2 detector. According to the offset position of the center of the reflected beam, the relative distance between the objective lens and the sample to be tested is moved by the control system to achieve automatic focusing of a single field of view of the sample to be tested; by moving the sample to be tested to the focal plane of different object fields of view in the XY direction and quickly focusing in real time, a large-sized sample to be tested is always kept in the focal plane position, thereby improving image quality and work efficiency.

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] Currently, the sample under test receives the laser signal from the illumination module and, under the stimulation of the laser signal, generates a fluorescent signal. The objective lens collects the fluorescent signal and transmits it to the imaging system for collection, thereby obtaining the corresponding image quality. Given the high image magnification characteristics of microscopes, the depth of field is relatively small, requiring the user to spend a considerable amount of time adjusting the relative distance between the objective lens and the sample under test so that the objective lens can focus the fluorescent signal to the imaging system, thereby obtaining a clear image quality.

[0003] However, this imaging method for the sample under test has numerous limitations. For example, due to the large size of the sample under test, the focal planes of the various object fields of view can easily become non-coplanar. Furthermore, the relative distance between the objective lens and the sample under test must be readjusted each time the sample is changed, which, in turn, requires time-consuming and continuous adjustment of the relative distance between the objective lens and the sample under test. Related art imaging systems typically have slow imaging speeds for scanning the sample under test.

[0004] Furthermore, given the active nature of the analyte molecules, a layer of glue, typically applied to the substrate and then covered with a coverslip, is required to protect this activity. Thickness variations and surface flatness can vary between batches of analyte samples, significantly impacting image clarity. Commercially available autofocus systems struggle to achieve fast focusing, necessitating the development of a fast autofocus module suitable for fluorescence microscopy.

[0005] To address the above issues, a fast autofocus module suitable for fluorescence microscopes is provided. It is designed 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. At the same time, it reduces direct contact with the sample and avoids 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 present 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 measured, 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 measured 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 measured 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 based on actual conditions. The first and second optical paths form 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 light in the focusing optical path (the laser light emitted by the laser) and transmits the excitation light and emission light in the imaging optical path (the excitation light illuminates 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 the direction of the beam propagation is changed by the reflector and dichroic mirror. The beam is then introduced into the objective lens and focused on the sample to be tested. After being reflected by the sample to be tested, the reflected beam is collected again by the objective lens and passes through the dichroic mirror, reflector and telephoto lens group in sequence before entering the CCD2 detector. The CCD2 detector receives the horizontal position offset of the reflected beam spot in the telephoto lens group caused by the offset of the reflected beam. The control system controls the relative distance between the objective lens and the sample to be tested according to the offset d, realizing 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. After being excited by the laser, the sample to be tested emits emission light. 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 laser emits a monochromatic beam in addition to the excitation and emission light. The aperture is used to adjust the size of the laser beam. A collimating lens assembly collimates the beam passing through the aperture. A reflector changes the direction of light propagation. The objective lens converges the focused light path onto the sample under test. The sample's plane reflects the light. The objective lens also collects the reflected beam and, through a dichroic mirror and the reflector, transmits it to the telescope lens assembly. The telescope lens assembly focuses and amplifies the beam, enhancing the visibility of sample details. The CCD2 detector receives the reflected beam signal and displays it as the horizontal offset of the reflected beam spot in the telescope lens assembly caused by the offset. The control system adjusts the relative distance between the objective lens and the sample under test based on the offset of the reflected beam spot in the telescope lens assembly to find the optimal focal plane for the sample within the object field of view. The objective lens's movement direction and position are precisely controlled by the Z-axis of the displacement stage.

[0017] The aperture is circular or rectangular, and sufficient laser light must pass through to improve focusing accuracy. The shape and size of the aperture can be determined by combining the laser's output shape 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 improve the excitation light, so it will not be elaborated here.

[0019] This application is suitable for test samples of any size, especially large ones. It allows for rapid focusing regardless of sample size. The focus range and accuracy are controllable and can be customized. Even if a layer of water is present above the sample, rapid focusing is possible.

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

[0021] The displacement platform includes both XY and Z axes. The control system controls the displacement platform's movement along the XY axes and the objective lens's movement along the Z axis. Movement along the Z axis adjusts the relative distance between the objective lens and the sample under test, enabling automatic focusing of a single field of view of the sample under test. Movement along the XY axes allows the sample under test to be moved to the focal plane of different object-space fields of view and rapidly focused in real time. Closed-loop travel control ensures that the sample under test always remains in focus, improving image quality and increasing 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 optimal focal plane. The laser emits a laser beam and adjusts the spot size of the laser beam through the aperture. It then passes through the collimating lens group, the reflector and the dichroic mirror in sequence, and then exits through the objective lens to the plane where the sample to be tested is located. It is then 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 onto the CCD2 detector. It is presented as the offset of the reflected beam spot on the horizontal position generated in the telephoto lens group caused by the offset of the reflected beam. According to the offset, the control system is used to control the movement direction and position of the objective lens to adjust the relative distance between the objective lens and the sample to find the optimal 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 this application can refer to the existing mature technology. This application has no special improvements on this and will not be repeated.

[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 horizontal position offset of the reflected beam spot in the telephoto lens group caused by the offset of the reflected beam center on the CCD2 detector through wired or wireless data, and calculates the vertical displacement of the focal plane based on this (the horizontal position offset of the reflected beam spot in the telephoto lens group caused by the offset of the reflected beam center). Then, the computer 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 measured to find the optimal focal plane (the sample to be measured is on the focal plane);

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

[0027] The above-mentioned CCD2 detector 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. The specific process includes: first, the displacement of the focal plane is converted into an angular offset: specifically, when the sample to be measured 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 measured is not at the focal plane and has a certain displacement in the vertical direction with respect to the focal plane, the incident laser is incident on the objective lens and converges on the plane where the sample to be measured is located, then reflects through the objective lens and emits at a certain angle to the vertical emission direction. The laser beam is emitted in the direction of the focal plane. At this time, the displacement of the focal plane is converted into the angular offset of the reflected laser beam. 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 beam into a horizontal position offset (compared to the offset of the center of the spot distribution of the laser beam reflected when it is in the focal plane). 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 captures 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 from the horizontal position offset displayed in the telescope system.

[0028] The CCD2 detector captures 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 horizontal position of the reflected beam spot 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 output shape of the above-mentioned laser 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 horizontal position of the reflected beam spot in the telescope lens group caused by the offset of the reflected beam.

[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 telescopic lens group is too long, the number of reflectors can be increased to reduce the structural 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 reflecting surface of the reflector and the reflecting 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 with the movement of the displacement platform.

[0035] A fast autofocus method for a fluorescence microscope is implemented using the fast autofocus module 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 base plate according to the reserved holes. Adjust the dichroic mirror so that the angle between its reflective surface and the axis direction is exactly 45 degrees. 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. Adjust the horizontal position of the collimating lens group so that the laser beam emitted from the aperture position can be distributed in the center of the lens of the collimating lens group.

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

[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 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 generated in the telephoto lens group due to 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 generated in the telephoto lens group due to 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 generated in the telephoto lens group due to 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) Irradiating the sample to be tested with 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 onto 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. Both the incident laser and the reflected laser 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 this 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 fluorescence microscopes, 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 focus 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 the samples to be tested in different batches can be overcome to a large extent, and the stability is high. At the same time, 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 1Schematic diagram of the structure of a fast autofocus module suitable for a fluorescence microscope of the present invention;

[0048] Figure 2 : This 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 an embodiment of the present invention;

[0049] Figure 3 This 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 This is a fitting curve diagram of the reflected light beam data of the CCD2 detector of the present invention, where the sample to be tested is located 1 μm above the focal plane;

[0051] Figure 5 This is a fitting curve diagram of the reflected light beam data of the CCD2 detector of the present invention when the sample to be tested 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 measured; 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 illustrated below in conjunction with the examples, but the content of the present invention is not limited to the following examples.

[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 measured 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 to be tested 7;

[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 measured 7 and the displacement platform 12 are arranged in sequence along the propagation direction of the laser to form a first optical path; the sample to be measured 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 the focusing optical path, and the third optical path is the imaging optical path; the laser wavelength of the focusing optical path and the emission light 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 outside the excitation spectrum and the emission spectrum, it will reach and pass through the aperture 2 to adjust the size of the laser beam, and then pass through the collimating lens group 3 to collimate the laser beam with a certain divergence angle. After collimation, the laser beam passes through the reflector 4 and the dichroic mirror 5 in turn, changing the propagation direction of the beam and introducing the beam into the objective lens. The objective lens 6 collects the focused light path beam and converges it 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, not the center of the reflector, the reflected light beam 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 in the telephoto lens group caused by the offset of the reflected light beam 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 based on the focus range and focus accuracy. For example, when the CCD2 detector 9 is moved forward 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 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 of the displacement platform 12 based on the offset d of the reflected light beam spot in the telephoto lens group caused by the offset of the reflected light beam displayed by the CCD2 detector 9 to accurately move the relative distance between the objective lens 6 and the sample to be tested, so that the sample to be tested 7 has the best focal plane in the object field, wherein the objective lens 6 is connected to the Z axis of 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, further comprising: a sample to be tested 7, an objective lens 6, a dichroic mirror 5, a tube lens 10, and a CCD1 detector 11 are sequentially arranged along the imaging light path, wherein the sample to be tested 7 is irradiated with excitation light, and after being excited, the sample to be tested 7 emits emission light, which is collected by the objective lens 6. After passing through the dichroic mirror 5, the tube lens 10 converges the emission spectrum onto the CCD1 detector 11. 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 emission light; the CCD1 detector 11 will present a clear image quality, that is, detailed information of the sample to be tested 7.

[0064] Example 2

[0065] Based on 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 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 closed-loop control of the stroke ensures that the sample to be tested 7 always remains in the focal plane position, improving image quality and increasing 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 optimal focal plane. Laser 1 emits a laser beam and adjusts the spot size of the laser beam through aperture 2. The beam then passes through collimating lens group 3, reflector 4 and dichroic mirror 5 in sequence, and then exits through objective lens 6 to the plane where the sample to be tested 7 is located, and is reflected back to objective lens 6. The returned reflected beam passes through dichroic mirror 5, reflector 4 and telephoto lens group again, and converges onto CCD2 detector 9. It is presented as the offset of the reflected beam spot in the horizontal position in the telephoto lens group caused by the offset of the reflected 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 objective lens 6 to adjust the relative distance between objective lens 6 and sample to be tested 7, and find the optimal focal plane of sample to be tested 7 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 in the horizontal position in the telephoto lens group caused by the offset of the reflected light beam center through wired or wireless communication, and calculates the displacement of the focal plane in the vertical direction based on the offset (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 center), 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 to be measured 7, so as to find the optimal 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 the focal plane of different object fields and focus quickly in real time.

[0070] The CCD2 detector 9 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, 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 on 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 respect to the focal plane, the incident laser is incident on 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. The laser beam is emitted in the direction of the focal plane. At this time, the displacement of the focal plane is converted into the angular offset of the reflected laser beam. 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 6, and the angle of the objective lens 6. Secondly, the telescope system converts the angular offset of the reflected laser beam into a horizontal position offset (compared to the offset of the center of the spot distribution of the laser beam reflected when it is in the focal plane). 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 9 captures 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 from the horizontal position offset displayed in the telescope system.

[0071] Example 4

[0072] Based on Example 3, the following improvements are further made: CCD2 detector receives the horizontal offset of the reflected beam spot in the telescopic lens group caused by the offset of the reflected beam center. Control system 13 controls the movement of objective lens 6 based on the horizontal offset of the reflected beam spot in the telescopic lens group caused by the offset of the reflected beam center received by CCD2 detector 9 to find the optimal focal plane. The laser 1 emits a point-like or line-like shape; CCD1 detector 11 is an area array industrial camera that clearly images the sample to be tested 7; CCD2 detector 9 is an area array industrial camera or a line array camera that calibrates the horizontal offset of the reflected beam spot in the telescopic lens group caused by the offset of the reflected 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 fluorescence microscopes described above, 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 base plate according to the reserved holes. Adjust the dichroic mirror 5 so that the angle between its reflective surface and the axis direction is precisely 45 degrees. Adjust the objective lens 6 so that its center line coincides with the optical axis.

[0076] 2) Turn on the laser light source 1 and rotate the direction of the laser beam to align it with the center of the aperture 2. Adjust the horizontal position of the collimating lens group 3 so that the laser beam emitted from the aperture 2 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 a frequency doubling plate as a detection tool to check the relative positions of the incident and reflected laser beams. Specifically, use the frequency doubling plate to check the relative position of the laser beam to the mechanical aperture when it is incident on the dichroic mirror 5. It should be slightly to the right of the center of the mechanical aperture. Use the frequency doubling plate to check the relative position of the laser beam to the mechanical aperture when it is reflected on the reflector 4. It should be slightly to the left of the center of the mechanical aperture. Fine-tune the angle of the reflector 4 so that the distances of the incident and reflected beams from the center of the mechanical aperture remain consistent, and then fix the position of the reflector 4. The mechanical aperture is located between the dichroic mirror 5 and the reflector 4. The mechanical aperture refers to a circular hole on the machined part (lens barrel) of the dichroic mirror 5, which is located at 45 degrees to the plane where the center of the dichroic mirror 5 is located and perpendicular to the direction of laser incidence.

[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 measured 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 generated in the telephoto lens group due to the offset of the reflected beam is 0; when the sample 7 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 9, and the offset of the reflected beam spot in the horizontal position generated in the telephoto lens group due to 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 measured according to the offset of the reflected beam spot in the horizontal position generated in the telephoto lens group due to 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 measured;

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

[0081] 7) The sample to be tested 7 is moved to different object fields of view by moving along the XY axis, and steps 5) to 6) are repeated 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, 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 measured is in the focal plane. If the edges of the rectangle are blurred and the size changes, it means that the sample to be measured is not in 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 edges of the rectangle become 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 edges of the rectangle become 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 blurrier the edges of the rectangle, the more obvious the size change, and the greater the center offset.

[0083] See also Figure 3, is the 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 at 1 μm above the focal plane of the sample 7 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 that of 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 measured is adjusted so that the center of the spot distribution of the reflected light beam coincides with the center of the CCD2 detector 9. The offset d0 is 0, and the sample to be measured 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 at a position 1 μm below the focal plane of the sample 7 in the embodiment. At this time, the center of the reflected light beam spot distribution 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 measured is adjusted so that the center of the spot distribution of the reflected light beam coincides with the center of the CCD2 detector 9. The offset d0 is 0, and the sample to be measured is located on the focal plane.

[0086] Each fast autofocus module suitable for fluorescence microscopy 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 at the level of hundreds of nanometers. Based on this, it is more important to quickly focus on different focal planes of each object field of view in the sample to be measured 7, especially in a large size range.

Claims

1. A fast autofocus module suitable for a fluorescence microscope, characterized by: 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 measured 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 measured 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 constitute 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 the direction of the beam propagation is changed by the reflector and dichroic mirror, and the 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 beam is collected again by the objective lens, and passes through the dichroic mirror, reflector and telephoto lens group in sequence, and enters the CCD2 detector. The CCD2 detector receives the horizontal position offset of the reflected beam spot caused by the offset of the reflected beam in the telephoto lens group. d , the control system is based on the offset d Control the relative distance between the objective lens and the sample to be tested to achieve automatic focusing of a single field of view of the sample to be tested; The sample to be tested is illuminated by the excitation light. After being excited by the laser, the sample to be tested emits emission light. 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, where it is presented as a clear image; 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, specifically including: first, converting the displacement of the focal plane into an angular offset: specifically, when the sample to be measured 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 measured is not at the focal plane and has a certain displacement in the vertical direction with respect to the focal plane, the incident laser is incident on the objective lens and converges on the plane where the sample to be measured is located, then reflects through the objective lens and emits 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 telephoto 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 telephoto system; The position of the CCD2 detector can be moved up and down, and the amount of movement is determined according to 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.

2. The fast autofocus module for fluorescence microscopy according to claim 1, characterized in that: The displacement platform includes both XY and Z axes. The control system can control the displacement platform to move along the XY axes 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, and automatic focusing of a single field of view of the sample to be tested can be achieved. By moving along the XY axes, 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 for fluorescence microscopy according to claim 2, characterized in that: 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 measured to find the optimal focal plane.

4. The fast autofocus module for fluorescence microscopy 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, motion controller and motor controller are connected in sequence; The computer collects the horizontal offset of the reflected beam spot in the telescope lens group caused by the offset of the reflected beam center on the CCD2 detector through wired or wireless communication, and calculates the vertical displacement of the focal plane based on this. Then, the computer 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 measured to find the optimal focal plane. The motion controller controls the motor controller to drive the displacement platform to move along the XY axis, moving the sample to be tested to the focal plane of different object fields of view and focusing quickly 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 horizontal position of the reflected beam spot caused by the offset of the reflected beam center in the telescope lens group. d 0 , the control system is based on the offset d 0 Control the movement of the objective lens to find the best focal plane.

6. 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 horizontal position of the reflected beam spot in the telescope lens group caused by the offset of the reflected beam.

7. 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 reflecting surface of the reflector and the reflecting surface of the dichroic mirror are parallel and arranged facing each other.

8. A fast autofocus method for a fluorescence microscope, implemented using the fast autofocus module for a fluorescence microscope according to any one of claims 1 to 7, 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 base plate according to the reserved holes. Adjust the dichroic mirror so that the angle between its reflective surface and the axis direction is 45 degrees. 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. Adjust the horizontal position of the collimating lens group so that the laser beam emitted from the aperture position can be distributed in the center of the collimating lens group. 3) Use a frequency doubling plate, a testing tool, to check the relative positions of the incident and reflected laser beams between the reflector and the dichroic mirror. Specifically, use the frequency doubling plate to check the relative position of the laser beam to the mechanical aperture when it is incident on the dichroic mirror. It should be slightly to the right of the center of the mechanical aperture. Use the frequency doubling plate to check the relative position of the laser beam to the mechanical aperture when it is reflected on the reflector. It should be slightly to the left of the center of the mechanical aperture. Fine-tune the reflector angle so that the distances of the incident and reflected beams from the center of the mechanical aperture remain consistent, and then fix the reflector position. The mechanical aperture is located between the dichroic mirror and the reflector. The mechanical aperture 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 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 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 generated in the telephoto lens group due to 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 generated in the telephoto lens group due to 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 generated in the telephoto lens group due to 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 excitation light. After being excited by the laser, the sample to be tested emits emission light. 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 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