Over-focus scanning microscopy system and measurement method based on acousto-optic modulator

Abstract

The present disclosure provides an over-focus scanning microscopy system and measurement method based on an acousto-optic modulator. The scanning microscopy system includes: a light source module, a spectroscopic module, a sample stage, a collection module and a measurement module. The light source module includes an acousto-optic modulator, which is used to generate a series of monochromatic collimated illumination beams with adjustable wavelengths; the spectroscopic module is used to divide the monochromatic collimated illumination beam into a first illumination beam and a second illumination beam; the sample stage is used to fix the sample to be tested and realize the microscopic focus of the sample to be tested; the collection module is used to collect the microscopic image formed after the light beam is irradiated from the front and back to the sample to be tested; the measurement module is used to measure the wavelength value of the first illumination beam and transmit it to an external host computer, and the external host computer feedbacks and adjusts the control parameters of the acousto-optic modulator in the light source module, so that the light source module outputs a monochromatic collimated illumination beam of a set wavelength.

Description

Technical Field

[0001] The present disclosure relates to the technical field of through-focus scanning optical microscopy measurement, and in particular to a through-focus scanning microscopy system and a measurement method based on an acousto-optic modulator. Background Art

[0002] The traditional through-focus scanning microscopic image acquisition system uses piezoelectric ceramics to control the z-axis displacement of the sample, so that the sample is located at different set optical axis positions, and then the through-focus scanning microscopic image sequence of the sample is acquired. However, due to the mechanical properties of the piezoelectric ceramics themselves, when the piezoelectric ceramics are used continuously for a period of time, their displacement control efficiency is significantly reduced, which restricts the acquisition rate of the through-focus scanning image sequence. In terms of position control accuracy, the position accuracy of the mechanical control method is negatively correlated with the control range, which makes it difficult to meet the increasing demand for high-precision and large-range through-focus microscopic image sequence acquisition. In addition, high-precision mechanical displacement control is complex and requires complex position calibration, and the debugging cost and hardware cost are high.

[0003] In the overfocus scanning microscopic image sequence acquisition method based on wavelength control, the spatial position of the sample remains unchanged during measurement. The monochromator generates monochromatic illumination light, and the microscopic images corresponding to different monochromatic lights are collected to form an overfocus scanning microscopic image sequence. According to the chromatic aberration effect of the optical microscope system, microscopic images of different wavelengths correspond to different states of the sample in focus, negative overfocus, and positive overfocus. The use of a monochromator can achieve higher wavelength control resolution and finer overfocus control, but the wavelength control speed of the monochromator is slow, which limits the efficiency of optical microscopic image acquisition. Summary of the invention

[0004] In view of this, the main purpose of the present disclosure is to provide an over-focus scanning microscopy system and a measurement method based on an acousto-optic modulator, in order to at least partially solve at least one of the above-mentioned technical problems.

[0005] In order to achieve the above-mentioned purpose, as an embodiment of one aspect of the present disclosure, a through-focus scanning microscopy system based on an acousto-optic modulator is provided, comprising: a light source module comprising an acousto-optic modulator, which is used to generate a series of monochromatic collimated illumination beams with adjustable wavelengths; a spectroscopic module is used to divide the monochromatic collimated illumination beam into a first illumination beam and a second illumination beam; a sample stage is used to fix the sample to be tested and realize the microscopic focus of the sample to be tested, the first illumination beam is irradiated to the sample to be tested from the front, and the second illumination beam is irradiated to the sample to be tested from the back; an acquisition module is used to collect microscopic images formed after the light beams are irradiated to the sample to be tested from the front and back; a measurement module is used to measure the wavelength value of the first illumination beam and transmit it to an external host computer, and the external host computer feeds back and adjusts the control parameters of the acousto-optic modulator in the light source module, so that the light source module outputs a monochromatic collimated illumination beam of a set wavelength.

[0006] According to an embodiment of the present disclosure, the acousto-optic modulator uses high-quality TeO2 crystal with a clear aperture of 6 to 10 mm.

[0007] According to an embodiment of the present disclosure, the light source module further includes a wide-spectrum high-brightness light source, a multimode optical fiber, and a fiber collimator which are sequentially arranged along the optical path, and the wide-spectrum high-brightness light source, the multimode optical fiber, and the fiber collimator are all located before the acousto-optic modulator, wherein: the wide-spectrum high-brightness light source is used to provide an illumination beam; the multimode optical fiber is used to transmit the illumination beam to the fiber collimator; the fiber collimator is used to collimate the illumination beam and transmit it to the acousto-optic modulator.

[0008] According to the embodiments of the present disclosure, the wide-spectrum high-brightness light source adopts a xenon lamp light source, a laser-pumped xenon lamp or an SLED combined light source; the multi-mode optical fiber adopts an armored high-quality multi-mode optical fiber with a wavelength application range of 350 to 800 nm, an optical fiber diameter of 600 μm to 2 mm, and an SMA or FC interface; the optical fiber collimator adopts an aspheric lens, a multi-mode optical fiber collimator, an achromatic objective lens or an off-axis parabolic mirror.

[0009] According to an embodiment of the present disclosure, the light splitting module adopts an optical beam splitting prism.

[0010] According to an embodiment of the present disclosure, the system further includes a first lighting module and a second lighting module after the light splitting module, wherein: the first lighting module includes: a first lens for receiving and converging a first lighting beam; a second beam splitter for dividing the first lighting beam converged by the first lens into a third lighting beam and a fourth lighting beam, wherein the third lighting beam enters the measurement module, and the fourth lighting beam irradiates the sample to be measured from the front, forming a uniform illumination light field on the surface of the sample to be measured, forming a falling Kohler illumination; the second lighting module includes: a first reflector for receiving a second lighting beam; a second reflector, located after the first reflector, and used together with the first reflector to adjust the propagation direction of the second lighting beam to obtain a second lighting beam with a changed propagation direction; a second lens for receiving and converging the second lighting beam with a changed propagation direction; and a third lens for receiving the second lighting beam after being converged by the second lens, so that the converged second lighting beam irradiates the sample to be measured from the back, forming a transmissive Kohler illumination.

[0011] According to an embodiment of the present disclosure, the second illumination light beam with a changed propagation direction is converged to a focal plane of the third lens through the second lens.

[0012] According to an embodiment of the present disclosure, the first lens adopts a cemented lens, an apochromatic objective lens or an aspherical lens; the second beam splitter adopts a flat beam splitter or a thin film beam splitter; the first reflector and the second reflector adopt UV-enhanced protected aluminum film plane reflectors; the second lens and the third lens adopt a cemented lens, an apochromatic objective lens and an aspherical lens.

[0013] According to an embodiment of the present disclosure, the measurement module includes an optical fiber and a spectrometer. The optical fiber is coupled to the spectrometer. The spectrometer is used to measure the wavelength value of the third illumination light beam and transmit it to an external host computer. The external host computer feedbacks and adjusts the control parameters of the acousto-optic modulator in the light source module so that the light source module outputs a monochromatic collimated illumination light beam of a set wavelength.

[0014] According to the embodiments of the present disclosure, the optical fiber adopts armored high-quality multimode optical fiber with a wavelength range of 350 to 800 nm, an optical fiber diameter of 600 μm to 2 mm, and an SMA or FC interface; the spectrometer adopts a fiber optic spectrometer or a high-order grating high-resolution spectrometer with a measurement wavelength range of 380 nm to 720 nm, a wavelength measurement accuracy of 0.5 nm, and a wavelength resolution of 1 nm.

[0015] According to an embodiment of the present disclosure, the control parameters of the acousto-optic modulator include the output wavelength and the half-peak width of the output wavelength, the output wavelength ranges from 400 to 700 nm, and the half-peak width of the output wavelength is 2 nm.

[0016] According to an embodiment of the present disclosure, the sample stage adopts a three-axis sample displacement stage, including a microscope three-axis displacement stage or a manual three-axis displacement stage, and the microscopic focusing of the sample to be tested is achieved through z-axis displacement, and the searching and positioning of the measurement area of ​​the sample to be tested is achieved through xy-axis displacement.

[0017] According to an embodiment of the present disclosure, the acquisition module includes a microscope objective, an imaging lens barrel and an area array camera, wherein: the microscope objective is used to receive a microscopic image formed by a sample to be tested; the imaging lens barrel is used to transmit the microscopic image received by the microscope objective to the area array camera; and the area array camera is used to record the microscopic image transmitted by the imaging lens barrel.

[0018] As an embodiment of another aspect of the present disclosure, a measurement method applied to the above-mentioned over-focus scanning microscope system is also provided, comprising: a light source module generates a monochromatic collimated illumination beam with adjustable wavelength through an acousto-optic modulator; a light splitting module divides the monochromatic collimated illumination beam into a first illumination beam and a second illumination beam; the first illumination beam is irradiated to the sample to be tested from the front, and the second illumination beam is irradiated to the sample to be tested from the back, wherein the sample to be tested is fixed to a sample stage and microscopic focusing is achieved by moving the sample stage; a collection module collects a microscopic image formed after the first illumination beam and the second illumination beam are irradiated to the sample to be tested from the front and back, respectively; a measurement module measures the wavelength value of the first illumination beam and transmits it to an external host computer, and the external host computer feeds back and adjusts the control parameters of the acousto-optic modulator in the light source module, so that the light source module outputs a monochromatic collimated illumination beam of a set wavelength; and

[0019] Repeat the steps of generating a monochromatic collimated illumination beam with adjustable wavelength by the light source module and acquiring corresponding microscopic images by the acquisition module, so that the acquisition module acquires a microscopic image sequence of the sample to be tested under the monochromatic collimated illumination beams of all set monochromatic wavelengths.

[0020] According to an embodiment of the present disclosure, the light source module includes a wide-spectrum high-brightness light source, a multimode optical fiber, a fiber collimator and an acousto-optic modulator arranged in sequence along the optical path. The light source module generates a wavelength-adjustable monochromatic collimated illumination beam through the acousto-optic modulator, including: the wide-spectrum high-brightness light source outputs an illumination beam; the multimode optical fiber inputs the illumination beam into the fiber collimator; the fiber collimator outputs a collimated illumination beam and transmits it to the acousto-optic modulator; and the acousto-optic modulator modulates the collimated illumination beam to generate a wavelength-adjustable monochromatic collimated illumination beam.

[0021] According to an embodiment of the present disclosure, the first lighting module includes a first lens and a second beam splitter, the second lighting module includes a first reflector, a second reflector, a second lens and a third lens, the first lighting beam is irradiated to the sample to be tested from the front, and the second lighting beam is irradiated to the sample to be tested from the back, including: the first lighting beam is converged by the first lens and irradiated to the second beam splitter, and is split by the second beam splitter to form a third lighting beam and a fourth lighting beam, wherein the third lighting beam enters the measurement module, and the fourth lighting beam is irradiated to the sample to be tested from the front; the second lighting beam is reflected by the first reflector and the second reflector in sequence, and then irradiated to the second lens after the propagation direction is changed; the second lighting beam is converged by the second lens and irradiated to the focal plane of the third lens, and then diverges through the third lens to irradiate the sample to be tested from the back as a parallel beam.

[0022] According to an embodiment of the present disclosure, the measurement module includes an optical fiber and a spectrometer. The measurement module measures the wavelength value of the first illumination beam and transmits it to an external host computer, including: the first illumination beam passes through a second beam splitter to form a third illumination beam; the third illumination beam is transmitted to the spectrometer through the optical fiber; the spectrometer measures the wavelength value of the third illumination beam, and transmits the wavelength value of the third illumination beam to the external host computer.

[0023] According to an embodiment of the present disclosure, the sample to be tested is fixed on the sample stage and microscopic focusing is achieved through the movement of the sample stage, including: the sample to be tested is fixed on the sample stage through a three-axis sample displacement stage; the three-axis sample displacement stage achieves searching and positioning of the measurement area of ​​the sample to be tested through xy-axis displacement; the three-axis sample displacement stage achieves microscopic focusing through z-axis displacement adjustment.

[0024] According to an embodiment of the present disclosure, the acquisition module includes a microscope objective, an imaging lens barrel and an area array camera. The acquisition module acquires a microscopic image formed after the first illumination light beam and the second illumination light beam are irradiated from the front and back sides of the sample to be tested respectively, including: the microscope objective receives the microscopic image formed after the first illumination light beam and the second illumination light beam are irradiated from the front and back sides of the sample to be tested respectively; the imaging lens barrel transmits the microscopic image to the area array camera; the area array camera acquires the microscopic image, wherein the area array camera acquires the microscopic image formed by the sample to be tested by adjusting the exposure time.

[0025] According to an embodiment of the present disclosure, the control parameters of the acousto-optic modulator in the light source module are feedback-regulated by an external host computer so that the light source module outputs a monochromatic collimated illumination beam of a set wavelength, including: the external host computer compares the wavelength value of the first illumination beam measured by the measurement module with the wavelength of the monochromatic collimated illumination beam output by the light source module; the external host computer feedback-regulates the control parameters of the acousto-optic modulator in the light source module according to the comparison result so that the light source module outputs a monochromatic collimated illumination beam of a set wavelength.

[0026] According to an embodiment of the present disclosure, after acquiring the microscopic image sequence of the sample to be tested, the method further includes: performing data processing on the microscopic image sequence of the sample to be tested using a library matching method based on sample model data.

[0027] The present disclosure provides an acousto-optic modulator-based through-focus scanning microscopy system and measurement method, which, by fixing the sample stage, collects microscopic images corresponding to different wavelengths to form a through-focus scanning microscopic image sequence, thereby avoiding the position uncertainty caused by mechanically adjusting the position of the sample stage to obtain scanning images of the sample to be measured at different focal points.

[0028] The present invention provides an acousto-optic modulator-based through-focus scanning microscopy system and measurement method. By using an acousto-optic modulator to control the wavelength and generate monochromatic light of different wavelengths, the wavelength control speed can be increased to millisecond level, thereby improving the optical wavelength control speed and optimizing the through-focus scanning microscopy image sequence acquisition efficiency.

[0029] Therefore, the through-focus scanning microscope system and measurement method based on the acousto-optic modulator provided by the present invention can simplify the optical and control structures of the through-focus scanning microscope system and reduce the volume of the through-focus scanning microscope system without reducing the control accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a structural schematic diagram of an over-focus scanning microscopy system based on an acousto-optic modulator according to an embodiment of the present disclosure;

[0031] Figure 2 yes Figure 1 Schematic diagram of optical components of an AOM-based transfocus scanning microscopy system;

[0032] Figure 3 Is applied to Figure 1 The flowchart of the measurement method of the through-focus scanning microscope system based on the acoustic-optic modulator is shown.

[0033] Reference numerals:

[0034] 100-Light source module

[0035] 1-Broad spectrum high brightness light source 2-Multimode optical fiber 3-Fiber collimator 4-Acousto-optic modulator

[0036] 200-Spectral Module

[0037] 5-First beam splitter

[0038] 300-First lighting module

[0039] 6-First lens 7-Second beam splitter

[0040] 400-Second lighting module

[0041] 12-first reflector 13-second reflector 14-second lens 15-third lens

[0042] 500-Measurement Module

[0043] 16- Fiber Optic 17- Spectrometer

[0044] 600-Sample Stage

[0045] 9-Three-axis sample translation stage

[0046] 700-Acquisition Module

[0047] 8-microscope objective lens 10-imaging tube lens 11-area array camera DETAILED DESCRIPTION

[0048] In order to make the objectives, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0049] However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present application. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present application. Various structural schematic diagrams according to embodiments of the present application are shown in the accompanying drawings. These figures are not drawn to scale, and some details are magnified and some details may be omitted for the purpose of clear expression.

[0050] According to the overall concept of one aspect of the present disclosure, there is provided a through-focus scanning microscopy system based on an acousto-optic modulator, comprising: a light source module comprising an acousto-optic modulator, for generating a series of monochromatic collimated illumination beams with adjustable wavelengths; a light splitting module for splitting the monochromatic collimated illumination beam into a first illumination beam and a second illumination beam; a sample stage for fixing a sample to be tested and achieving microscopic focusing of the sample to be tested, the first illumination beam irradiating the sample to be tested from the front, and the second illumination beam irradiating the sample to be tested from the back; a collection module for collecting microscopic images formed after the light beams irradiate the sample to be tested from the front and back; a measurement module for measuring the wavelength value of the first illumination beam and transmitting it to an external host computer, and the external host computer feedback-regulates the control parameters of the acousto-optic modulator in the light source module, so that the light source module outputs a monochromatic collimated illumination beam of a set wavelength.

[0051] Figure 1 is a schematic structural diagram of an over-focus scanning microscope system based on an acousto-optic modulator according to an embodiment of the present disclosure.

[0052] like Figure 1 As shown, the structural schematic diagram of the through-focus scanning system based on the acousto-optic modulator includes: a light source module 100, a spectroscopic module 200, a first lighting module 300, a second lighting module 400, a measuring module 500, a sample stage 600, and a collection module 700.

[0053] According to an embodiment of the present disclosure, the light source module 100 generates a monochromatic collimated illumination beam with adjustable wavelength, and the spectroscopic module 200 is located behind the light source module 100, and splits the monochromatic collimated illumination beam output from the light source module 100 into a first illumination beam and a second illumination beam. The first illumination module 300 receives the first illumination beam emitted from the spectroscopic module 200, and splits the first illumination beam into a third illumination beam and a fourth illumination beam. The measuring module 500 receives the third illumination beam split from the first illumination beam, measures the wavelength value of the third illumination beam, and feeds back the wavelength measurement value of the third illumination beam to an external host computer. The external host computer adjusts the parameters of the acousto-optic modulator 4 in the light source module 100 in real time according to the wavelength value of the third illumination beam fed back by the measuring module 500, and controls the light source module 100 to output a monochromatic collimated illumination beam with a fixed wavelength. After the first illumination beam is split by the first illumination module 300, a fourth illumination beam is output, and the fourth illumination beam is irradiated onto the sample stage 600 from the front, forming a uniform illumination light field on the surface of the sample to be tested, forming a falling Kohler illumination. The second lighting module 400 receives the second lighting beam, which is reflected and transmitted by the second lighting module 400 multiple times, and illuminates the sample to be tested from the back to form a uniform illumination light field, forming a transmissive Kohler illumination. The sample stage 600 is used to place the sample to be tested. By controlling the sample stage 600, the measurement area of ​​the sample to be tested can be found and located, and the position of the sample stage can be fixed after the microscopic focus of the sample to be tested is achieved. The acquisition module 700 can acquire the microscopic image formed after the light beam is irradiated to the sample to be tested from the front and back, and transmit the acquired microscopic images of all the samples to be tested to an external host computer to form an over-focus scanning microscopic image sequence. The external host computer can process the over-focus scanning microscopic image sequence to obtain parameter information such as the size and contrast of the sample to be tested.

[0054] According to an embodiment of the present disclosure, the acousto-optic modulator 4 may be made of high-quality TeO2 crystal with a clear aperture of 6 to 10 mm.

[0055] The through-focus scanning system based on an acousto-optic modulator proposed in the present disclosure collects microscopic images of the sample to be tested corresponding to different wavelengths to form a through-focus scanning microscopic image sequence by fixing the sample stage, which can avoid the position error and uncertainty caused by mechanical control, and can also avoid the inevitable loss difference caused by frequent changes in the position control device. The present disclosure also controls the wavelength by an acousto-optic modulator, which can increase the wavelength control speed to the millisecond level and optimize the collection efficiency of the through-focus scanning microscopic image sequence. While improving the wavelength control speed, the volume of the through-focus scanning microscopic system can also be reduced.

[0056] Figure 2 yes Figure 1 Schematic diagram of the optical components of the AOM-based transfocus scanning microscopy system shown.

[0057] like Figure 2 As shown, the over-focus scanning system based on the acousto-optic modulator includes: a wide-spectrum high-brightness light source 1, a multimode optical fiber 2, a fiber collimator 3, an acousto-optic modulator 4, a first beam splitter 5, a first lens 6, a second beam splitter 7, a microscope objective 8, a three-axis sample displacement stage 9, an imaging tube lens 10, an area array camera 11, a first reflector 12, a second reflector 13, a second lens 14, a third lens 15, an optical fiber 16, and a spectrometer 17.

[0058] According to an embodiment of the present disclosure, the light source module 100 includes a wide-spectrum high-brightness light source 1, a multimode optical fiber 2, a fiber collimator 3 and an acousto-optic modulator 4 arranged in sequence along the optical path. The wide-spectrum high-brightness light source 1, the multimode optical fiber 2, and the fiber collimator 3 are all located before the acousto-optic modulator 4, and the multimode optical fiber 2 couples the wide-spectrum high-brightness light source 1 to the fiber collimator 3. The wide-spectrum high-brightness light source 1 outputs an illumination beam, which is transmitted to the fiber collimator 3 by the multimode optical fiber 2. After the fiber collimator 3 collimates the illumination beam, it outputs a parallel illumination beam. The acousto-optic modulator 4 receives the parallel illumination beam, and changes the output parameters under the control of the external host computer, and outputs a plurality of monochromatic collimated illumination beams of set wavelengths.

[0059] According to an embodiment of the present disclosure, the wide spectrum high brightness light source 1 may include a xenon lamp light source, a laser pumped xenon lamp or an SLED combined light source. The multimode optical fiber 2 may adopt an armored high-quality multimode optical fiber with a wavelength application range of 350 to 800 nm, an optical fiber aperture of 600 μm to 2 mm, and an SMA or FC interface. The optical fiber collimator 3 may include an aspheric lens, a multimode optical fiber collimator, an apochromatic objective lens, and an off-axis parabolic mirror. The acousto-optic modulator 4 may include a clear aperture of 6 to 10 mm and adopt a high-quality TeO2 crystal. According to an embodiment of the present disclosure, the acousto-optic modulator 4 may adopt a high-quality TeO2 crystal with a clear aperture of 6 to 10 mm.

[0060] like Figure 2 As shown, the light splitting module 200 uses a first beam splitter 5. The first beam splitter 5 can split the monochromatic collimated illumination beam generated by the light source module 100 into a first illumination beam and a second illumination beam.

[0061] According to an embodiment of the present disclosure, the first beam splitter 5 used in the light splitting module 200 may be an optical beam splitting prism, and specifically may be a cubic beam splitter with a clear aperture of 25 mm.

[0062] like Figure 2 As shown, the light splitting module 200 further includes a first lighting module 300 and a second lighting module 400. The first lighting module 300 includes: a first lens 6 and a second beam splitter 7. The second lighting module 400 includes: a first reflector 12, a second reflector 13, a second lens 14 and a third lens 15.

[0063] According to an embodiment of the present disclosure, after the first beam splitter 5 splits the monochromatic collimated illumination beam output by the light source module 100 into a first illumination beam and a second illumination beam, the first lens 6 can receive and converge the first illumination beam, and transmit the first illumination beam to the second beam splitter 7. The second beam splitter 7 divides the first illumination beam into a third illumination beam and a fourth illumination beam. The first lens 6 and the second beam splitter 7 are matched and installed, and the fourth illumination beam can be converged to the back focal plane of the microscope objective 8. After passing through the microscope objective 8, the fourth illumination beam outputs a parallel beam, which is irradiated to the surface of the sample to be tested from the front, forming a uniform illumination light field on the surface of the sample to be tested, and forming a falling Kohler illumination.

[0064] After the first beam splitter 5 splits the monochromatic collimated illumination beam output by the light source module 100 into a first illumination beam and a second illumination beam, the first reflector 12 can receive the second illumination beam and reflect the second illumination beam onto the second reflector 13. The second reflector 13 is located behind the first reflector 12, and reflects the second illumination beam after being reflected by the first reflector 12 again. The second reflector 13 and the first reflector 12 are used together to adjust the propagation direction of the second illumination beam, and transmit the adjusted second illumination beam to the second lens 14. The second lens 14 converges the parallel second illumination beam with the changed propagation direction to the focal plane of the third lens 15, and after being diverged by the third lens 15, it irradiates the sample to be tested from the back as a parallel beam, forming a transmissive Kohler illumination.

[0065] According to an embodiment of the present disclosure, the first lens 6 can be a cemented lens, an apochromatic objective lens or an aspherical lens, the second beam splitter 7 can be a flat beam splitter or a thin film beam splitter, the first reflector 12 and the second reflector 13 can be ultraviolet-enhanced protected aluminum film plane reflectors, and the second lens 14 and the third lens 15 can be a cemented lens, an apochromatic objective lens or an aspherical lens.

[0066] like Figure 2 As shown, the measurement module 500 includes an optical fiber 16 and a spectrometer 17. The optical fiber 16 is coupled to the spectrometer 17, and the optical fiber 16 can transmit the third illumination light beam split by the second beam splitter 7 to the spectrometer 17. The spectrometer 17 can measure the wavelength value of the current third illumination light beam, and transmit the wavelength measurement value of the current third illumination light beam to the external host computer in real time. The external host computer adjusts the control parameters of the acousto-optic modulator 4 in the light source module 100 according to the feedback of the wavelength measurement value of the third illumination light beam, so that the light source module 100 outputs the wavelength of the monochromatic collimated illumination light beam of the set wavelength.

[0067] According to the embodiment of the present disclosure, the optical fiber 16 adopts an armored high-quality multimode optical fiber with a wavelength application range of 350-800nm, an optical fiber diameter of 600μm-2mm, and an SMA or FC interface. The spectrometer 17 adopts an optical fiber spectrometer or a high-order grating high-resolution spectrometer, with a measurement wavelength range of 380nm-720nm, a wavelength measurement accuracy of 0.5nm, and a wavelength resolution of 1nm.

[0068] According to an embodiment of the present disclosure, the control parameters of the acousto-optic modulator 4 include the output wavelength and the half-peak width of the output wavelength. The output wavelength ranges from 400 to 700 nm, and the half-peak width of the output wavelength is 2 nm.

[0069] like Figure 2 As shown, the sample stage 600 adopts a three-axis sample displacement stage 9. The three-axis sample displacement stage 9 is used to place the sample to be measured, and can find the measurement area of ​​the sample to be measured through three-axis movement, and fix the sample to be measured at the microscope focus position.

[0070] According to the embodiment of the present disclosure, the three-axis sample displacement stage 9 may include a microscope three-axis displacement stage and a manual three-axis displacement stage. The three-axis sample displacement stage 9 realizes microscopic focusing of the sample to be measured through z-axis displacement and realizes searching and positioning of the measurement area of ​​the sample to be measured through xy-axis displacement.

[0071] like Figure 2 As shown, the acquisition module 700 includes a microscope objective lens 8 , an imaging tube lens 10 and an area array camera 11 .

[0072] According to the embodiment of the present disclosure, the microscope objective lens 8 can receive a microscopic image formed by the sample to be tested, and the area array camera 11 collects the microscopic image received by the microscope objective lens 8 through the imaging lens barrel 10. After the area array camera 11 collects the microscopic image of the sample to be tested, it transmits the microscopic image of the sample to be tested to an external host computer, so that the external host computer performs subsequent data processing on the microscopic pattern sequence of the sample to be tested.

[0073] According to the embodiment of the present disclosure, the microscope objective lens 8 can be a flat-field microscope objective lens, an aspheric lens, a cemented lens or a reflective converging lens, and the imaging tube lens 10 can be a tube lens with a focal length of 160 mm. The area array camera 11 can be a CCD camera or a CMOS camera.

[0074] based on Figure 1 and Figure 2 The schematic diagram of the structure of the over-focus scanning microscope system based on the acousto-optic modulator is shown in FIG. Figure 3 Shown is applied to Figure 1 The flow chart of the measurement method of the over-focus scanning microscope system based on the acousto-optic modulator shown in the figure comprises the following steps:

[0075] Step 301: The light source module generates a wavelength-adjustable monochromatic collimated illumination beam through an acousto-optic modulator;

[0076] Step 302: The light splitting module splits the monochromatic collimated illumination beam into a first illumination beam and a second illumination beam;

[0077] Step 303: a first illumination beam is irradiated to the sample to be tested from the front, and a second illumination beam is irradiated to the sample to be tested from the back, wherein the sample to be tested is fixed on a sample stage and microscopic focusing is achieved by moving the sample stage;

[0078] Step 304: The acquisition module acquires the microscopic image formed after the first illumination light beam and the second illumination light beam are irradiated to the sample to be tested from the front and back sides respectively;

[0079] Step 305: the measurement module measures the wavelength value of the first illumination light beam and transmits it to the external host computer, and the external host computer feeds back and adjusts the control parameters of the acousto-optic modulator in the light source module so that the light source module outputs a monochromatic collimated illumination light beam of a set wavelength; and

[0080] Step 306: Repeat the steps of generating a monochromatic collimated illumination beam with adjustable wavelength by the light source module and acquiring corresponding microscopic images by the acquisition module, so that the acquisition module acquires a microscopic image sequence of the sample to be tested under the monochromatic collimated illumination beams of all set monochromatic wavelengths.

[0081] According to an embodiment of the present disclosure, the sample to be tested is fixed on the sample stage 600 through a three-axis sample displacement stage 9. Before fixing the three-axis sample displacement stage, the parameters of the wide-spectrum high-brightness light source in the light source module are adjusted, and the light source module generates a monochromatic collimated illumination beam of a preset wavelength through an acousto-optic modulator, and the preset wavelength range is 500-600nm. Under the irradiation of the monochromatic collimated illumination beam of this preset wavelength, the three-axis sample displacement stage is fixed at a position where the sample is clearly focused. When the wavelength of the monochromatic collimated illumination beam meets the wavelength modulation range (400-700nm) of the acousto-optic modulator, the sample to be tested can present a microscopic image of different overfocus states at this fixed position. Specifically, when the wavelength of the monochromatic collimated illumination beam is less than the preset wavelength, the sample to be tested is in a negative overfocus state, and the overfocus amount generated in the z-axis is negative; when the wavelength of the monochromatic collimated illumination beam is greater than the preset wavelength, the sample to be tested is in a positive overfocus state, and the overfocus amount generated in the z-axis is positive.

[0082] The spectroscopic module splits the above-mentioned monochromatic collimated illumination beam into a first illumination beam and a second illumination beam. The first illumination beam irradiates the sample to be tested from the front, and the second illumination beam irradiates the sample to be tested from the back. By adjusting the three-axis sample displacement stage, a clear microscopic image of the sample to be tested appears on the area array camera. The clear image of the sample to be tested on the area array camera indicates that the sample to be tested is located at the microscopic focus position under the irradiation of the monochromatic collimated illumination beam. Lock the three-axis sample displacement stage, fix the sample to be tested at the microscopic focus position under the irradiation of the monochromatic collimated illumination beam, and do not move the three-axis sample displacement stage any more.

[0083] The first illumination beam and the second illumination beam are respectively irradiated to the sample to be tested from the front, and the integration time of the area array camera in the acquisition module is adjusted to match the monochromatic collimated illumination beam of the wavelength, so that the acquisition module collects the microscopic image of the sample to be tested under the irradiation of the monochromatic collimated illumination beam. The measurement module measures the wavelength value of the first illumination beam and transmits it to the external host computer, and the control parameters of the acousto-optic modulator in the light source module are fed back by the external host computer to adjust the control parameters of the acousto-optic modulator in the light source module, so that the light source module outputs the monochromatic collimated illumination beam of a stable wavelength. Repeat the steps for a predetermined number of times: after the parameters of the wide spectrum high brightness light source in the light source module are adjusted, the light source module generates a monochromatic collimated illumination beam at a set wavelength through the acousto-optic modulator and does not change the position of the three-axis sample displacement stage, and after the parameters of the area array camera in the acquisition module are changed, the acquisition module collects the microscopic image of the sample to be tested irradiated by the monochromatic collimated illumination beam of the set wavelength. After repeating the above steps, a microscopic image sequence of the sample to be tested under the irradiation of monochromatic collimated illumination beams of all monochromatic wavelengths is obtained, and the wavelengths of all the above monochromatic collimated illumination beams meet 400-700nm.

[0084] According to an embodiment of the present disclosure, the light source module includes a wide-spectrum high-brightness light source, a multimode optical fiber, a fiber collimator, and an acousto-optic modulator arranged in sequence along the optical path. In step 301, the light source module generates a wavelength-adjustable monochromatic collimated illumination beam through the acousto-optic modulator, including: the wide-spectrum high-brightness light source outputs an illumination beam, the illumination beam passes through the multimode optical fiber and then enters the fiber collimator, and then enters the acousto-optic modulator after collimation by the fiber collimator; the acousto-optic modulator modulates the collimated illumination beam to generate a wavelength-adjustable monochromatic collimated illumination beam. Adjusting the wide-spectrum high-brightness light source so that the output brightness of the wide-spectrum high-brightness light source matches the acousto-optic modulator can optimize the image efficiency of the over-focus scanning microscope system.

[0085] According to an embodiment of the present disclosure, the light splitting module includes a first beam splitter, and in step 302, the light splitting module splits the monochromatic collimated illumination beam into a first illumination beam and a second illumination beam, including: the first beam splitter splits the monochromatic collimated illumination beam output by the acousto-optic modulator into the first illumination beam and the second illumination beam. According to an embodiment of the present disclosure, the first illumination module includes a first lens and a second beam splitter, and the second illumination module includes a first reflector, a second reflector, a second lens, and a third lens. In step 303, the first illumination beam is irradiated to the sample to be tested from the front, and the second illumination beam is irradiated to the sample to be tested from the back, wherein the sample to be tested is fixed to the sample stage and microscopic focusing is achieved by moving the sample stage, including:

[0086] The first illumination beam is converged by the first lens and irradiated to the second beam splitter, and is split by the second beam splitter to form a third illumination beam and a fourth illumination beam; by changing the positions of the first lens and the second beam splitter, the fourth illumination beam can be irradiated on the back focal plane of the microscope objective lens, and the fourth illumination beam is irradiated to the sample to be tested from the front as a parallel beam after passing through the microscope objective lens, forming a falling Kohler illumination; by changing the positions of the first reflector and the second reflector, the second illumination beam is reflected by the first reflector and the second reflector, and the propagation direction is changed and is incident on the second lens in parallel; by changing the positions of the second lens and the third lens, the second illumination beam is converged by the second lens and irradiated to the focal plane of the third lens, and then diverged by the third lens and irradiated to the sample to be tested from the back as a parallel beam; the sample to be tested is fixed to the sample stage through a three-axis sample displacement stage, and the measurement area of ​​the sample to be tested is found and the measurement area is located through the xy axis displacement of the three-axis sample displacement stage; the microscopic focusing of the sample to be tested is achieved through the z axis displacement of the three-axis sample displacement stage.

[0087] According to an embodiment of the present disclosure, the acquisition module includes a microscope objective lens, an imaging lens barrel, and an area array camera. In step 304, the acquisition module acquires a microscopic image formed after the first illumination beam and the second illumination beam are irradiated to the sample to be tested from the front and back, respectively, including: the first illumination beam and the second illumination beam are irradiated to the sample to be tested from the front and back, respectively, to form a microscopic image of the sample to be tested, and the microscope objective lens of the sample to be tested is transmitted to the area array camera by adjusting the position of the microscope objective lens and the imaging lens barrel; the microscopic image of the sample to be tested obtained by irradiating the current monochromatic collimated illumination beam is acquired by changing the exposure time of the area array camera

[0088] According to an embodiment of the present disclosure, a measurement module includes an optical fiber and a spectrometer. In step 305, the measurement module measures the wavelength value of the first illumination light beam and transmits it to an external host computer. The external host computer feeds back and adjusts the control parameters of the acousto-optic modulator in the light source module, so that the light source module outputs a monochromatic collimated illumination light beam of a set wavelength, including: the measurement module measures the wavelength value of the first illumination light beam and transmits it to the external host computer; the first illumination light beam passes through a second beam splitter to form a third illumination light beam, and the third illumination light beam is transmitted to the spectrometer through the optical fiber; the change in wavelength is measured by the spectrometer, so that the spectrometer can measure the wavelength value of the third illumination light beam; the spectrometer transmits the measured wavelength value of the third illumination light beam to the external host computer.

[0089] According to an embodiment of the present disclosure, the steps of repeatedly executing the light source module generating a monochromatic collimated illumination beam with adjustable wavelength and the acquisition module acquiring microscopic images in step 306, so that the acquisition module acquires a microscopic image sequence of the sample to be tested under the monochromatic collimated illumination beams of all set monochromatic wavelengths, include: repeatedly executing the light source module generating a monochromatic collimated illumination beam with adjustable wavelength for a predetermined number of times, and the predetermined number of times is related to the wavelength change amount. Within the wavelength control range of the acousto-optic modulator, after setting the wavelength change amount, the number of times the wavelength is generated within the wavelength control range, i.e., the predetermined number of times, can be determined. Specifically, for example, within the range of 400 to 700 nm, when the wavelength change amount is 20 nm, the light source module generates 16 monochromatic collimated illumination beams of different wavelengths, i.e., it is necessary to repeat the above steps 16 times, so that the acquisition module acquires microscopic images corresponding to all wavelengths within the range of 400 to 700 nm, and forms an over-focus microscopic image sequence. In practical applications, the wavelength change amount is changed as needed to ensure that the over-focus microscopic image corresponding to the desired sample over-focus state can be acquired, and the desired over-focus microscopic image sequence is obtained. When the light source module generates a monochromatic collimated illumination beam repeatedly for a predetermined number of times, the acquisition module also acquires microscopic images for a predetermined number of times to acquire microscopic images of the sample to be tested under the illumination of all monochromatic collimated illumination beams to form an overfocus microscopic image sequence. The overfocus microscopic image sequence includes overfocus microscopic images and focused microscopic images of the sample to be tested acquired by the acquisition module under the illumination of monochromatic collimated illumination beams of different wavelengths, and the overfocus microscopic images correspond to microscopic images in a negative overfocus state and microscopic images in a positive overfocus state.

[0090] According to an embodiment of the present disclosure, the measurement method of the through-focus scanning microscope system also includes using a library matching method based on sample model data to perform data processing on the microscopic image sequence of the sample to be tested, including: using a library matching method based on sample model data to perform data processing on the microscopic image sequence of the sample to be tested, matching the measured microscopic image sequence with a database, and searching and determining information such as the geometric size and refractive index of the sample to be tested.

[0091] The over-focus scanning microscopy system and measurement method based on an acousto-optic modulator proposed in the present disclosure measures the microscopic images of the sample to be tested under different wavelength conditions to form an over-focus scanning microscopic image sequence under the condition of a fixed sample stage, without moving the sample to be tested to the focusing position of the monochromatic collimated illumination light beam of different wavelengths, thereby avoiding the position uncertainty caused by adjusting the sample position and the low displacement control rate caused by frequent adjustment of the high-precision position measurement device. The present disclosure also adopts an acousto-optic modulator to change the wavelength of the monochromatic collimated illumination light beam, thereby increasing the wavelength control rate to the millisecond level, significantly improving the optical microscopic image acquisition efficiency. In addition, since the volume of the acousto-optic modulator is very small, the over-focus scanning microscopy system proposed in the present disclosure can not only simplify the displacement control device, but also simplify the optical structure of the high-precision control monochromator to change the wavelength, significantly reducing the volume of the over-focus scanning microscopy system.

[0092] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects of the present disclosure. It should be understood that the above description is only a specific embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure should be included in the protection scope of the present disclosure.

Claims

1. An over-focus scanning microscopy system based on an acousto-optic modulator, comprising: A light source module (100) comprises an acousto-optic modulator (4) for generating a series of monochromatic collimated illumination light beams with adjustable wavelengths; A light splitting module (200), used for splitting the monochromatic collimated illumination light beam into a first illumination light beam and a second illumination light beam; A sample stage (600) is used to fix the sample to be tested and realize microscopic focusing of the sample to be tested, wherein the first illumination light beam irradiates the sample to be tested from the front side, and the second illumination light beam irradiates the sample to be tested from the back side; A collection module (700) is used to collect microscopic images formed after the light beam is irradiated from the front and back sides of the sample to be tested; The measuring module (500) is used to measure the wavelength value of the first illumination light beam and transmit it to an external host computer, and the external host computer feedback-adjusts the control parameters of the acousto-optic modulator (4) in the light source module (100) so that the light source module (100) outputs a monochromatic collimated illumination light beam of a set wavelength.

2. The over-focus scanning microscopy system based on an acousto-optic modulator according to claim 1, wherein: The acousto-optic modulator (4) adopts high-quality TeO2 crystal, and the aperture of the light is 6-10 mm.

3. The over-focus scanning microscopy system based on an acousto-optic modulator according to claim 1, wherein: The light source module (100) further comprises a wide spectrum high brightness light source (1), a multimode optical fiber (2) and an optical fiber collimator (3) which are sequentially arranged along the optical path, and the wide spectrum high brightness light source (1), the multimode optical fiber (2) and the optical fiber collimator (3) are all located before the acousto-optic modulator (4), wherein: The wide spectrum high brightness light source (1) is used to provide an illumination beam; The multimode optical fiber (2) is used to transmit the illumination light beam to the optical fiber collimator (3); The optical fiber collimator (3) is used to collimate the illumination light beam and transmit it to the acousto-optic modulator (4).

4. The over-focus scanning microscopy system based on an acousto-optic modulator according to claim 3, wherein: The wide spectrum high brightness light source (1) adopts a xenon lamp light source, a laser pumped xenon lamp or a SLED combined light source; The multimode optical fiber (2) is an armored high-quality multimode optical fiber with a wavelength application range of 350 to 800 nm, an optical fiber diameter of 600 μm to 2 mm, and an SMA or FC interface; The optical fiber collimator (3) adopts an aspherical lens, a multimode optical fiber collimator, an apochromatic objective lens or an off-axis parabolic mirror.

5. The AOM-based over-focus scanning microscopy system according to claim 1, wherein: The light splitting module (200) adopts an optical beam splitting prism.

6. The over-focus scanning microscopy system based on an acousto-optic modulator according to claim 1, further comprising a first illumination module (300) and a second illumination module (400) after the light splitting module, wherein: The first lighting module (300) comprises: A first lens (6), used for receiving and focusing the first illumination light beam; a second beam splitter (7) for splitting the first illumination light beam converged by the first lens (6) into a third illumination light beam and a fourth illumination light beam, wherein the third illumination light beam enters the measurement module (500), and the fourth illumination light beam irradiates the sample to be measured from the front, forming a uniform illumination light field on the surface of the sample to be measured, thereby forming an epi-Köhler illumination; The second lighting module (400) comprises: A first reflector (12), configured to receive the second illumination light beam; a second reflector (13), located behind the first reflector (12), and used together with the first reflector (12) to adjust the propagation direction of the second illumination light beam to obtain a second illumination light beam with a changed propagation direction; A second lens (14) is used to receive and converge the second illumination light beam with a changed propagation direction; The third lens (15) is used to receive the second illumination light beam converged by the second lens (14), so that the converged second illumination light beam irradiates the sample to be tested from the back side to form transmissive Kohler illumination.

7. The AOM-based over-focus scanning microscopy system according to claim 6, wherein: The second illumination light beam with a changed propagation direction is converged to the focal plane of the third lens (15) through the second lens (14).

8. The AOM-based over-focus scanning microscopy system according to claim 6, wherein: The first lens (6) is a cemented lens, an apochromatic objective lens or an aspherical lens; The second beam splitter (7) adopts a flat beam splitter or a thin film beam splitter; The first reflector (12) and the second reflector (13) are ultraviolet enhanced protective aluminum film plane reflectors; The second lens (14) and the third lens (15) are cemented lenses, apochromatic objective lenses or aspherical lenses.

9. The AOM-based over-focus scanning microscopy system according to claim 6, wherein: The measurement module (500) comprises an optical fiber (16) and a spectrometer (17), wherein the optical fiber (16) is coupled to the spectrometer (17), and the spectrometer (17) is used to measure the wavelength value of the third illumination light beam and transmit the value to an external host computer, and the external host computer feedback-controls the control parameters of the acousto-optic modulator (4) in the light source module (100), so that the light source module (100) outputs a monochromatic collimated illumination light beam of a set wavelength.

10. The AOM-based over-focus scanning microscopy system according to claim 9, wherein: The optical fiber (16) is an armored high-quality multimode optical fiber with a wavelength application range of 350 to 800 nm, an optical fiber diameter of 600 μm to 2 mm, and an SMA or FC interface; The spectrometer (17) adopts a fiber spectrometer or a high-order grating high-resolution spectrometer, with a measurement wavelength range of 380nm to 720nm, a wavelength measurement accuracy of 0.5nm, and a wavelength resolution of 1nm.

11. The AOM-based over-focus scanning microscopy system according to claim 9, wherein: The control parameters of the acousto-optic modulator (4) include an output wavelength and an output wavelength half-peak width, the output wavelength ranges from 400 to 700 nm, and the output wavelength half-peak width is 2 nm.

12. The AOM-based over-focus scanning microscopy system according to claim 1, wherein: The sample stage (600) adopts a three-axis sample displacement stage (9), including a microscope three-axis displacement stage or a manual three-axis displacement stage, which realizes microscopic focusing of the sample to be measured through z-axis displacement and realizes searching and positioning of the measurement area of ​​the sample to be measured through xy-axis displacement.

13. The AOM-based over-focus scanning microscopy system according to claim 1, wherein: The acquisition module (700) comprises a microscope objective lens (8), an imaging lens barrel (10) and an area array camera (11), wherein: The microscope objective lens (8) is used to receive a microscopic image formed by the sample to be tested; The imaging lens barrel (10) is used to transmit the microscopic image received by the microscope objective lens (8) to the area array camera (11); The area array camera (11) is used to record the microscopic image transmitted by the imaging lens barrel (10).

14. A measurement method applied to the over-focus scanning microscope system according to any one of claims 1 to 13, comprising: The light source module generates a wavelength-adjustable monochromatic collimated illumination beam through an acousto-optic modulator; The light splitting module splits the monochromatic collimated illumination beam into a first illumination beam and a second illumination beam; The first illumination beam is irradiated to the sample to be tested from the front, and the second illumination beam is irradiated to the sample to be tested from the back, wherein the sample to be tested is fixed on the sample stage and microscopic focusing is achieved by moving the sample stage; The acquisition module acquires a microscopic image formed after the first illumination light beam and the second illumination light beam are irradiated to the sample to be tested from the front and back sides respectively; The measuring module measures the wavelength value of the first illumination light beam and transmits it to the external host computer, and the external host computer feedbacks and adjusts the control parameters of the acousto-optic modulator in the light source module, so that the light source module outputs a monochromatic collimated illumination light beam of a set wavelength; as well as The steps of generating a monochromatic collimated illumination beam with adjustable wavelength by the light source module and acquiring corresponding microscopic images by the acquisition module are repeated, so that the acquisition module acquires a microscopic image sequence of the sample to be tested under the monochromatic collimated illumination beams of all set monochromatic wavelengths.

15. The measuring method of the over-focus scanning microscope system according to claim 14, wherein: The light source module comprises a wide-spectrum high-brightness light source, a multimode optical fiber, an optical fiber collimator and an acousto-optic modulator arranged in sequence along the optical path. The light source module generates a wavelength-adjustable monochromatic collimated illumination beam through the acousto-optic modulator, comprising: The wide spectrum high brightness light source outputs an illumination beam; The multimode optical fiber inputs the illumination light beam into the optical fiber collimator; The optical fiber collimator outputs a collimated illumination beam and transmits it to the AOM; and The acousto-optic modulator modulates the collimated illumination light beam to generate a monochromatic collimated illumination light beam with adjustable wavelength.

16. The measuring method of the over-focus scanning microscope system according to claim 14, wherein: The first lighting module includes a first lens and a second beam splitter, the second lighting module includes a first reflector, a second reflector, a second lens and a third lens, the first lighting beam irradiates the sample to be tested from the front, and the second lighting beam irradiates the sample to be tested from the back, including: The first illumination beam is converged by the first lens and irradiated to the second beam splitter, and is split by the second beam splitter to form a third illumination beam and a fourth illumination beam, wherein the third illumination beam enters the measurement module, and the fourth illumination beam is irradiated to the sample to be measured from the front; The second illumination light beam is reflected by the first reflector and the second reflector in sequence, and then irradiates the second lens after changing the propagation direction; the second illumination light beam is converged by the second lens and irradiates the focal plane of the third lens, and then diverges through the third lens to irradiate the sample to be tested from the back as a parallel light beam.

17. The measuring method of the over-focus scanning microscope system according to claim 16, wherein: The measuring module includes an optical fiber and a spectrometer. The measuring module measures the wavelength value of the first illumination light beam and transmits it to an external host computer, including: The first illumination light beam passes through the second beam splitter to form a third illumination light beam; The third illumination light beam is transmitted to the spectrometer through the optical fiber; The spectrometer measures the wavelength value of the third illumination light beam and transmits the wavelength value of the third illumination light beam to an external host computer.

18. The measuring method of the over-focus scanning microscope system according to claim 14, wherein: The sample to be tested is fixed on a sample stage and microscopic focusing is achieved by moving the sample stage, including: The sample to be tested is fixed on the sample stage through a three-axis sample displacement stage; The three-axis sample displacement stage realizes the search and positioning of the measurement area of ​​the sample to be measured by xy axis displacement; The three-axis sample displacement stage realizes microscopic focusing through z-axis displacement adjustment.

19. The measuring method of the over-focus scanning microscope system according to claim 14, wherein: The acquisition module includes a microscope objective lens, an imaging lens barrel and an area array camera. The acquisition module acquires a microscopic image formed after the first illumination beam and the second illumination beam are irradiated from the front and back of the sample to be tested respectively, including: The microscope objective lens receives the first illumination light beam and the second illumination light beam and irradiates the sample to be tested from the front and back sides respectively to form a microscope image; The imaging lens barrel transmits the microscopic image to the area array camera; The area array camera collects the microscopic image, wherein the area array camera collects the microscopic image formed by the sample to be tested by adjusting the exposure time.

20. The method for measuring an over-focus scanning microscope system according to claim 14, wherein: The control parameters of the acousto-optic modulator in the light source module are adjusted by feedback from an external host computer so that the light source module outputs a monochromatic collimated illumination beam of a set wavelength, including: The external host computer compares the wavelength value of the first illumination light beam measured by the measurement module with the wavelength of the monochromatic collimated illumination light beam output by the light source module; The external host computer adjusts the control parameters of the acousto-optic modulator in the light source module according to the comparison result feedback, so that the light source module outputs a monochromatic collimated illumination beam with a set wavelength.

21. The measuring method according to claim 14, after collecting the microscopic image sequence of the sample to be measured, further comprising: The microscopic image sequence of the sample to be tested is processed using a library matching method based on sample model data.

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