CN116107074A - Overfocal scanning microscopic system based on acousto-optic modulator and measuring method - Google Patents

Abstract

The disclosure provides an over-focus scanning microscope system based on an acousto-optic modulator and a measuring method. The scanning microscope system includes: the device comprises a light source module, a light splitting module, a sample stage, an acquisition module and a measurement module. The light source module comprises an acousto-optic modulator for generating a series of monochromatic collimated illumination beams with adjustable wavelengths; the beam splitting module is used for splitting the monochromatic collimated illumination beam into a first illumination beam and a second illumination beam; the sample table is used for fixing the sample to be tested and realizing microscopic focusing of the sample to be tested; the acquisition module is used for acquiring microscopic images formed after light beams irradiate the sample to be detected from the front and the back; the measuring module is used for measuring the wavelength value of the first illumination beam, transmitting the wavelength value to the external upper computer, and enabling the external upper computer to feed back and regulate 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 with the set wavelength.

Description

Overfocal scanning microscopic system based on acousto-optic modulator and measuring method

Technical Field

The disclosure relates to the technical field of confocal scanning optical microscopic measurement, in particular to an acousto-optic modulator-based confocal scanning microscopic system and a measuring method.

Background

The traditional over-focus scanning microscopic image acquisition system uses piezoelectric ceramics to control the sample to generate z-axis displacement, so that the sample is positioned at different set optical axis positions, and further an over-focus scanning microscopic image sequence of the sample is acquired. But is limited by the mechanical property of the piezoelectric ceramic, and when the piezoelectric ceramic is continuously used for a period of time, the displacement control efficiency is obviously reduced, and the acquisition rate of the over-focus scanning image sequence is restricted. In the aspect of position regulation precision, the position precision of a mechanical regulation method is inversely related to a regulation range, and the increasingly-improved high-precision wide-range confocal microscopic image sequence acquisition requirement is difficult to meet. In addition, high-precision mechanical displacement control is complex, complex position calibration is needed, and debugging cost and hardware cost are high.

The method is characterized in that the spatial position of a sample is unchanged during measurement based on a wavelength-regulated scanning microscopic image sequence acquisition mode, monochromatic illumination light is generated by a monochromator, and microscopic images corresponding to different monochromatic lights are acquired to form a scanning microscopic image sequence. According to the chromatic aberration effect of the optical microscopy system, microscopic images with different wavelengths correspond to different states of focusing, negative overfocusing and positive overfocusing of the sample. The monochromator can be used for realizing higher wavelength regulation resolution and finer focus excess regulation, but the wavelength regulation speed of the monochromator is low, so that the acquisition efficiency of the optical microscopic image is limited.

Disclosure of Invention

Accordingly, it is a primary object of the present disclosure to provide an over-focus scanning microscope system and a measuring method based on an acousto-optic modulator, so as to at least partially solve at least one of the above-mentioned technical problems.

To achieve the above object, as an embodiment of one aspect of the present disclosure, there is provided an over-focus scanning microscopy system based on an acousto-optic modulator, including: the light source module comprises an acousto-optic modulator for generating a series of monochromatic collimated illumination beams with adjustable wavelengths; the beam splitting module is used for splitting the monochromatic collimated illumination beam into a first illumination beam and a second illumination beam; the sample table is used for fixing the sample to be measured and achieving microscopic focusing of the sample to be measured, the first illumination beam irradiates the sample to be measured from the front, and the second illumination beam irradiates the sample to be measured from the back; the acquisition module is used for acquiring microscopic images formed after light beams irradiate the sample to be detected from the front and the back; the measuring module is used for measuring the wavelength value of the first illumination beam, transmitting the wavelength value to the external upper computer, and enabling the external upper computer to feed back and regulate 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 with the set wavelength.

Embodiments according to the present disclosure, wherein the acousto-optic modulator employs high quality TeO ₂ The crystal has a clear aperture of 6-10 mm.

According to an embodiment of the present disclosure, the light source module further includes a broad spectrum highlight light source, a multimode optical fiber, and an optical fiber collimator sequentially disposed along the light path, and the broad spectrum highlight light source, the multimode optical fiber, and the optical fiber collimator are all located before the acousto-optic modulator, wherein: a broad spectrum high brightness light source for providing an illumination beam; the multimode optical fiber is used for transmitting the illumination light beam to the optical fiber collimator; the optical fiber collimator is used for collimating the illumination light beam and transmitting the illumination light beam to the acousto-optic modulator.

According to the embodiment of the disclosure, a xenon lamp light source, a laser pumping xenon lamp or an SLED combined light source is adopted as the wide-spectrum high-brightness light source; the multimode fiber adopts an armored high-quality multimode fiber with the wavelength application range of 350-800 nm and the fiber caliber of 600-2 mm and the interface of SMA or FC; the optical fiber collimator adopts an aspheric lens, a multimode optical fiber collimator, an apochromatic objective lens or an off-axis parabolic mirror.

According to an embodiment of the disclosure, the light splitting module employs an optical beam splitting prism.

According to an embodiment of the present disclosure, the system further comprises 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 illumination beam; the second beam splitter is used for dividing the first illumination beam converged by the first lens into a third illumination beam and a fourth illumination beam, wherein the third illumination beam enters the measurement module, the fourth illumination beam irradiates the sample to be measured from the front, a uniform illumination light field is formed on the surface of the sample to be measured, and the epi-illumination type kohler illumination is formed; the second lighting module includes: a first mirror for receiving the second illumination beam; the second reflector is positioned behind the first reflector and is used for adjusting the propagation direction of the second illumination light beam together with the first reflector so as to obtain a second illumination light beam with the propagation direction changed; a second lens for receiving and converging a second illumination beam having a changed propagation direction; and the third lens is used for receiving the second illumination light beams converged by the second lens so as to enable the converged second illumination light beams to irradiate the sample to be detected from the back surface to form transmission type Kohler illumination.

According to an embodiment of the present disclosure, the second illumination beam, which changes the propagation direction, is converged to the focal plane of the third lens through the second lens.

According to an embodiment of the disclosure, wherein the first lens is 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 reflecting mirror and the second reflecting mirror adopt ultraviolet enhanced protection aluminum film plane reflecting mirrors; the second lens and the third lens adopt a cemented lens, an apochromatic objective lens and an aspheric lens.

According to the embodiment of the disclosure, the measuring module comprises an optical fiber and a spectrometer, the optical fiber is coupled to the spectrometer, the spectrometer is used for measuring the wavelength value of the third illumination beam and transmitting the wavelength value to the external upper computer, and the external upper computer feeds back and regulates control parameters of the acousto-optic modulator in the light source module, so that the light source module outputs the monochromatic collimated illumination beam with the set wavelength.

According to the embodiment of the disclosure, the optical fiber adopts an armored high-quality multimode optical fiber with the wavelength application range of 350-800 nm, the optical fiber caliber of 600-2 mm and an interface of SMA or FC; the spectrometer adopts an optical fiber spectrometer or a high-order grating high-resolution spectrometer, the measurement wavelength range is 380 nm-720 nm, the wavelength measurement precision is 0.5nm, and the wavelength resolution is 1nm.

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

According to the embodiment of the disclosure, the sample stage adopts a triaxial sample displacement stage, including a microscope triaxial displacement stage or a manual triaxial displacement stage, microscopic focusing of a sample to be measured is achieved through z-axis displacement, and searching and positioning of a measurement area of the sample to be measured are achieved through xy-axis displacement.

According to an embodiment of the present disclosure, wherein the acquisition module comprises a microscope objective, an imaging barrel, and an area camera, wherein: the microscope objective is used for receiving a microscopic image formed by the sample to be detected; the imaging lens cone is used for transmitting the microscopic image received by the microscopic objective lens to the area array camera; and the area array camera is used for recording microscopic images transmitted by the imaging lens barrel.

As an embodiment of another aspect of the present disclosure, there is also provided a measurement method applied to the above-mentioned confocal scanning microscopy system, including: the light source module generates monochromatic collimated illumination light beams with adjustable wavelengths through the acousto-optic modulator; the beam splitting module splits the monochromatic collimated illumination beam into a first illumination beam and a second illumination beam; the first illumination beam irradiates the sample to be measured from the front side, and the second illumination beam irradiates the sample to be measured from the back side, wherein the sample to be measured is fixed on the sample table and is subjected to microscopic focusing through movement of the sample table; the acquisition module acquires microscopic images formed after the first illumination light beam and the second illumination light beam are respectively irradiated to the sample to be detected from the front and the back; the measuring module measures the wavelength value of the first illumination beam and transmits the wavelength value to the external upper computer, and the external upper computer feeds back and regulates the control parameters of the acousto-optic modulator in the light source module to enable the light source module to output the monochromatic collimated illumination beam with the set wavelength; and

Repeatedly executing the steps of generating monochromatic collimated illumination beams with adjustable wavelengths by the light source module and collecting corresponding microscopic images by the collecting module, so that the collecting module collects microscopic image sequences of samples to be measured under the monochromatic collimated illumination beams with all the set monochromatic wavelengths.

According to an embodiment of the present disclosure, wherein the light source module includes a broad spectrum highlight light source, a multimode optical fiber, an optical fiber collimator, and an acousto-optic modulator sequentially disposed along an optical path, the light source module generates a wavelength-tunable monochromatic collimated illumination beam through the acousto-optic modulator, comprising: a broad spectrum high brightness light source outputs illumination light beams; the multimode optical fiber inputs the illumination beam into an optical fiber collimator; the optical fiber collimator outputs a collimated illumination beam and transmits the collimated illumination beam to the acousto-optic modulator; and modulating the collimated illumination beam by the acousto-optic modulator to produce a wavelength-tunable monochromatic collimated illumination beam.

According to an embodiment of the present disclosure, wherein the first illumination module includes a first lens and a second beam splitter, the second illumination module includes a first mirror, a second lens, and a third lens, the first illumination beam irradiates the sample to be measured from the front side, and the second illumination beam irradiates the sample to be measured from the back side, comprising: the first illumination beam irradiates the second beam splitter through the convergence of the first lens, and forms a third illumination beam and a fourth illumination beam through the beam splitting of the second beam splitter, wherein the third illumination beam enters the measuring module, and the fourth illumination beam irradiates the sample to be measured from the front; the second illumination beam is reflected by the first reflecting mirror and the second reflecting mirror in sequence, and irradiates the second lens after changing the propagation direction; the second illumination beam irradiates to the focal plane of the third lens through the convergence of the second lens, and irradiates to the sample to be detected from the back surface through the divergence of the third lens in parallel beams.

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

According to an embodiment of the present disclosure, wherein a sample to be measured is fixed to a sample stage and is brought into microscopic focusing by movement of the sample stage, comprising: the sample to be measured is fixed on the sample stage through the triaxial sample displacement stage; the triaxial sample displacement platform realizes the searching and positioning of the measuring area of the sample to be measured through xy axis displacement; the triaxial sample displacement table realizes micro focusing through z-axis displacement adjustment.

According to an embodiment of the present disclosure, wherein the collection module includes a microscope objective, an imaging lens barrel, and an area array camera, and the collection module collects microscopic images formed after the first illumination beam and the second illumination beam are respectively irradiated to the sample to be measured from the front and the back, and includes: the microscope objective receives microscopic images formed after the first illumination light beam and the second illumination light beam are respectively irradiated to the sample to be detected from the front and the back; the imaging lens cone transmits the microscopic image to an area array camera; and acquiring the microscopic image by an area array camera, wherein the area array camera acquires the microscopic image formed by the sample to be detected through exposure time adjustment.

According to an embodiment of the disclosure, a control parameter of an acousto-optic modulator in a light source module is feedback-controlled by an external host computer, so that the light source module outputs a monochromatic collimated illumination beam with a set wavelength, including: the external upper computer compares the wavelength value of the first illumination beam measured by the measuring module with the wavelength of the monochromatic collimated illumination beam output by the light source module; and the external upper computer feeds back and regulates control parameters of the acousto-optic modulator in the light source module according to the comparison result, so that the light source module outputs monochromatic collimated illumination beams with set wavelengths.

According to an embodiment of the present disclosure, after collecting the microscopic image sequence of the sample to be measured, further comprising: and carrying out data processing on the microscopic image sequence of the sample to be detected by using a database matching method based on the sample model data.

According to the over-focus scanning microscopic system and the measuring method based on the acousto-optic modulator, through fixing the sample stage, microscopic images corresponding to different wavelengths are collected to form an over-focus scanning microscopic image sequence, and the position of the sample stage can be prevented from being mechanically regulated and controlled to obtain position uncertainty generated by scanning images of a sample to be measured at different focuses.

According to the over-focus scanning microscopic system and the measuring method based on the acousto-optic modulator, the wavelength is regulated and controlled by the acousto-optic modulator, monochromatic light with different wavelengths is generated, the wavelength regulation and control speed can be improved to the millisecond level, the optical wavelength regulation and control speed is improved, and the over-focus scanning microscopic image sequence acquisition efficiency is further optimized.

Therefore, the over-focus scanning microscopic system and the measuring method based on the acousto-optic modulator can simplify the optical and control structure of the over-focus scanning microscopic system, and reduce the volume of the over-focus scanning microscopic system under the condition of not reducing the control precision.

Drawings

FIG. 1 is a schematic diagram of an acousto-optic modulator based over-focus scanning microscopy system in accordance with an embodiment of the present disclosure;

FIG. 2 is a schematic diagram of the optical elements of the acousto-optic modulator-based over-focus scanning microscopy system shown in FIG. 1;

fig. 3 is a flow chart of a measurement method applied to the acousto-optic modulator-based over-focus scanning microscopy system shown in fig. 1.

Reference numerals:

100-light source module

1-broad spectrum highlight light source 2-multimode optical fiber 3-optical fiber collimator 4-acousto-optic modulator

200-spectroscopic module

5-first beam splitter

300-first lighting module

6-first lens 7-second beam splitter

400-second lighting module

12-first mirror 13-second mirror 14-second lens 15-third lens

500-measuring module

16-optical fiber 17-spectrometer

600-sample stage

9-triaxial sample displacement platform

700-acquisition module

8-microscope objective 10-imaging barrel 11-area array camera

Detailed Description

For the purposes of promoting an understanding of the principles and advantages of the disclosure, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same.

It should be understood that the description is only exemplary and is not intended to limit the scope of the present application. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present application. Various structural schematic diagrams according to embodiments of the present application are shown in the accompanying drawings. The figures are not drawn to scale, wherein certain details are exaggerated for clarity of presentation and may have been omitted.

According to one aspect of the present disclosure there is provided an acousto-optic modulator based over-focus scanning microscopy system comprising: the light source module comprises an acousto-optic modulator for generating a series of monochromatic collimated illumination beams with adjustable wavelengths; the beam splitting module is used for splitting the monochromatic collimated illumination beam into a first illumination beam and a second illumination beam; the sample table is used for fixing the sample to be measured and achieving microscopic focusing of the sample to be measured, the first illumination beam irradiates the sample to be measured from the front, and the second illumination beam irradiates the sample to be measured from the back; the acquisition module is used for acquiring microscopic images formed after light beams irradiate the sample to be detected from the front and the back; the measuring module is used for measuring the wavelength value of the first illumination beam, transmitting the wavelength value to the external upper computer, and enabling the external upper computer to feed back and regulate 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 with the set wavelength.

Fig. 1 is a schematic diagram of the architecture of an acousto-optic modulator based over-focus scanning microscopy system in accordance with an embodiment of the present disclosure.

As shown in fig. 1, the structure schematic diagram of the over-focus scanning system based on the acousto-optic modulator includes: the device comprises a light source module 100, a light splitting module 200, a first illumination module 300, a second illumination module 400, a measurement module 500, a sample stage 600 and a collection module 700.

According to an embodiment of the present disclosure, the light source module 100 generates a wavelength-tunable monochromatic collimated illumination beam, and the beam splitting module 200 is located behind the light source module 100, 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 divides the first illumination beam into a third illumination beam and a fourth illumination beam. The measurement module 500 receives the third illumination beam split from the first illumination beam, measures a wavelength value of the third illumination beam, and feeds back the wavelength measurement value of the third illumination beam to the external host computer. The external upper computer regulates and controls 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 measurement module 500, and controls the light source module 100 to output a monochromatic collimated illumination beam with a fixed wavelength. The first illumination beam is split by the first illumination module 300 and then outputs a fourth illumination beam, the fourth illumination beam irradiates the sample stage 600 from the front surface, a uniform illumination light field is formed on the surface of the sample to be measured, and the epi-kohler illumination is formed. The second illumination module 400 receives the second illumination beam, and the second illumination beam irradiates the sample to be measured from the back surface through multiple reflection and transmission of the second illumination module 400 to form a uniform illumination light field, so as to form transmission type kohler illumination. The sample stage 600 is used for placing a sample to be measured, and the measuring area of the sample to be measured can be searched and positioned by controlling the sample stage 600, and the position of the sample stage is fixed after the microscopic focusing of the sample to be measured is realized. The acquisition module 700 can acquire microscopic images formed by light beams irradiated to the sample to be detected from the front and the back, and transmit all acquired microscopic images of the sample to be detected to an external upper computer to form an over-focus scanning microscopic image sequence. The external upper computer can process the over-focus scanning microscopic image sequence to obtain parameter information such as the size, contrast and the like of the sample to be detected.

According to embodiments of the present disclosure, wherein the acousto-optic modulator 4 may employ a high quality TeO ₂ The crystal has a clear aperture of 6-10 mm.

According to the over-focus scanning system based on the acousto-optic modulator, through the fixed sample stage, microscopic images of samples to be detected corresponding to different wavelengths are collected to form an over-focus scanning microscopic image sequence, so that position errors and uncertainties caused by mechanical regulation can be avoided, and meanwhile unavoidable loss differences caused by frequent change of position regulation devices can be avoided. The wavelength is regulated and controlled through the acousto-optic modulator, the wavelength regulation and control speed can be improved to the millisecond level, and the acquisition efficiency of the over-focus scanning microscopic image sequence is optimized. Under the condition of improving the wavelength regulation speed, the volume of the over-focus scanning microscope system can be reduced.

Fig. 2 is a schematic diagram of the optical elements of the acousto-optic modulator-based over-focus scanning microscopy system shown in fig. 1.

As shown in fig. 2, the over-focus scanning system based on an acousto-optic modulator includes: a broad spectrum high brightness light source 1, a multimode optical fiber 2, an optical 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 triaxial sample displacement table 9, an imaging barrel lens 10, an area array camera 11, a first reflecting mirror 12, a second reflecting mirror 13, a second lens 14, a third lens 15, an optical fiber 16 and a spectrometer 17.

According to an embodiment of the present disclosure, the light source module 100 includes a broad spectrum highlight light source 1, a multimode optical fiber 2, an optical fiber collimator 3 and an acousto-optic modulator 4 sequentially arranged along an optical path, the broad spectrum highlight light source 1, the multimode optical fiber 2, the optical fiber collimator 3 being positioned before the acousto-optic modulator 4, the multimode optical fiber 2 coupling the broad spectrum highlight light source 1 to the optical fiber collimator 3. The wide-spectrum high-brightness light source 1 outputs an illumination light beam, the illumination light beam is transmitted to the optical fiber collimator 3 by the multimode optical fiber 2, and the optical fiber collimator 3 outputs a parallel illumination light beam after collimating the illumination light beam. The acousto-optic modulator 4 receives the parallel illumination light beams, changes output parameters under the control of an external upper computer, and outputs monochromatic collimated illumination light beams with a plurality of set wavelengths.

According to embodiments of the present disclosure, the broad spectrum high brightness light source 1 may comprise a xenon light source, a laser pumped xenon lamp, or a SLED combination light source. The multimode fiber 2 can be an armored high-quality multimode fiber with the wavelength application range of 350-800 nm, the fiber caliber of 600 mu m-2 mm and the interface of SMA or FC. The optical fiber collimator 3 may comprise an aspherical lens, a multimode optical fiber collimator, an apochromatic objective lens, an off-axis parabolic mirror. The acousto-optic modulator 4 may be packaged The aperture of the light with the light is 6-10 mm, and high quality TeO is adopted ₂ And (5) a crystal. According to embodiments of the present disclosure, wherein the acousto-optic modulator 4 may employ a high quality TeO ₂ The crystal has a clear aperture of 6-10 mm.

As shown in fig. 2, the spectroscopic module 200 employs a first beam splitter 5. The first beam splitter 5 may split the monochromatic collimated illumination beam generated by the light source module 100 into a first illumination beam and a second illumination beam.

According to an embodiment of the present disclosure, the first beam splitter 5 used by the beam splitting module 200 may be an optical beam splitter prism, and specifically may be a cube beam splitter with a clear aperture of 25 mm.

As shown in fig. 2, the first illumination module 300 and the second illumination module 400 are further included after the light splitting module 200. The first lighting module 300 includes: a first lens 6 and a second beam splitter 7. The second lighting module 400 includes: a first mirror 12, a second mirror 13, a second lens 14 and a third lens 15.

According to an embodiment of the present disclosure, after the first beam splitter 5 splits the monochromatic collimated illumination beam output from the light source module 100 into the first illumination beam and the second illumination beam, the first lens 6 may receive and converge the first illumination beam, and transmit the first illumination beam to the second beam splitter 7. The second beam splitter 7 splits 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 installed in a matched mode, fourth illumination light beams can be converged to a back focal plane of the micro objective 8, parallel light beams are output after the fourth illumination light beams pass through the micro objective 8, the fourth illumination light beams irradiate the surface of a sample to be detected from the front, uniform illumination light fields are formed on the surface of the sample to be detected, and the incident type kohler illumination is formed.

After the first beam splitter 5 splits the monochromatic collimated illumination beam output from the light source module 100 into the first illumination beam and the second illumination beam, the first mirror 12 may receive the second illumination beam and reflect the second illumination beam onto the second mirror 13. The second mirror 13 is located behind the first mirror 12, and reflects the second illumination beam reflected by the first mirror 12 again. The second mirror 13 and the first mirror 12 are used together to adjust the propagation direction of the second illumination beam, and the second illumination beam after the adjustment direction is transmitted to the second lens 14. The second lens 14 converges the parallel second illumination beam with the propagation direction changed to the focal plane of the third lens 15, and irradiates the sample to be measured with the parallel beam from the back after being diverged by the third lens 15, thereby forming a transmissive kohler illumination.

According to an embodiment of the present disclosure, the first lens 6 may be a cemented lens, an apochromatic objective lens, or an aspheric lens, the second beam splitter 7 may be a flat beam splitter or a thin film beam splitter, the first mirror 12 and the second mirror 13 may be ultraviolet-reinforced protective aluminum film flat mirrors, and the second lens 14 and the third lens 15 may be cemented lenses, apochromatic objective lenses, or aspheric lenses.

As shown in fig. 2, 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 is capable of transmitting the third illumination 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 beam and transmit the wavelength measurement value of the current third illumination beam to an external host computer in real time. The external upper computer feeds back and regulates the control parameters of the acousto-optic modulator 4 in the light source module 100 according to 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 with the set wavelength.

According to the embodiment of the disclosure, the optical fiber 16 adopts an armored high-quality multimode optical fiber with the wavelength application range of 350-800 nm, the optical fiber caliber of 600 mu m-2 mm and an interface of SMA or FC. The spectrometer 17 adopts an optical fiber spectrometer or a high-order grating high-resolution spectrometer, the measuring wavelength range is 380 nm-720 nm, the wavelength measuring precision is 0.5nm, and the wavelength resolution is 1nm.

According to an embodiment of the present disclosure, 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 nm to 700nm, and the output wavelength half-peak width is 2nm.

As shown in fig. 2, the sample stage 600 employs a triaxial sample displacement stage 9. The triaxial sample displacement table 9 is used for placing a sample to be measured, and can search a measurement area of the sample to be measured through triaxial movement and fix the sample to be measured in a microscopic focusing position.

According to embodiments of the present disclosure, the three-axis sample displacement stage 9 may comprise a microscope three-axis displacement stage, a manual three-axis displacement stage. The triaxial sample displacement table 9 realizes microscopic focusing of the sample to be measured through z-axis displacement, and realizes searching and positioning of a measuring area of the sample to be measured through xy-axis displacement.

As shown in fig. 2, the acquisition module 700 includes a microscope objective 8, an imaging cylinder 10 and an area camera 11.

According to an embodiment of the present disclosure, the microscope objective 8 is capable of receiving a microscopic image formed by a sample to be measured, and the area camera 11 captures the microscopic image received by the microscope objective 8 through the imaging lens barrel 10. After the area array camera 11 collects the microscopic image of the sample to be measured, the microscopic image of the sample to be measured is transmitted to the external upper computer so that the external upper computer can perform subsequent data processing on the microscopic pattern sequence of the sample to be measured.

According to an embodiment of the present disclosure, the micro objective 8 may be a flat field micro objective, an aspherical lens, a cemented lens, or a reflective converging lens, and the imaging cylinder 10 may be a tube lens having a focal length of 160 mm. The area camera 11 may employ a CCD camera or a CMOS camera.

Based on the schematic structural diagrams of the over-focus scanning microscope system based on the acousto-optic modulator shown in fig. 1 and 2, fig. 3 shows a flow chart of a measurement method applied to the over-focus scanning microscope system based on the acousto-optic modulator shown in fig. 1, the method comprises the following steps:

Step 301: the light source module generates monochromatic collimated illumination light beams with adjustable wavelengths through the acousto-optic modulator;

step 302: the beam splitting module splits the monochromatic collimated illumination beam into a first illumination beam and a second illumination beam;

step 303: the first illumination beam irradiates the sample to be measured from the front side, and the second illumination beam irradiates the sample to be measured from the back side, wherein the sample to be measured is fixed on the sample table and is subjected to microscopic focusing through movement of the sample table;

step 304: the acquisition module acquires microscopic images formed after the first illumination light beam and the second illumination light beam are respectively irradiated to the sample to be detected from the front and the back;

step 305: the measuring module measures the wavelength value of the first illumination beam and transmits the wavelength value to the external upper computer, and the external upper computer feeds back and regulates the control parameters of the acousto-optic modulator in the light source module to enable the light source module to output the monochromatic collimated illumination beam with the set wavelength; and

step 306: repeatedly executing the steps of generating monochromatic collimated illumination beams with adjustable wavelengths by the light source module and collecting corresponding microscopic images by the collecting module, so that the collecting module collects microscopic image sequences of samples to be measured under the monochromatic collimated illumination beams with all the set monochromatic wavelengths.

According to an embodiment of the present disclosure, the sample to be measured is fixed to the sample stage 600 by the triaxial sample displacement stage 9. Before the triaxial sample displacement table is fixed, parameters of a wide-spectrum high-brightness light source in a light source module are adjusted, the light source module generates monochromatic collimated illumination light beams with preset wavelengths through an acousto-optic modulator, and the range of the preset wavelengths is 500-600 nm. Under the irradiation of the monochromatic collimation illumination beam with the preset wavelength, the triaxial sample displacement table is fixed at the position where the sample is clearly focused. When the wavelength of the monochromatic collimated illumination beam meets the wavelength modulation range (400-700 nm) of the acousto-optic modulator, the sample to be measured can present microscopic images in different over-focus states at the fixed position. Specifically, when the wavelength of the monochromatic collimated illumination beam is smaller than the preset wavelength, the sample to be measured is in a negative over-focus state, and the over-focus amount generated in the z axis is negative; when the wavelength of the monochromatic collimated illumination beam is larger than the preset wavelength, the sample to be measured is in a positive overfocal state, and the overfocal amount generated in the z axis is positive.

The beam splitting module splits the monochromatic collimated illumination beam into a first illumination beam and a second illumination beam. The first illumination beam irradiates the sample to be measured from the front side, and the second illumination beam irradiates the sample to be measured from the back side. And a clear microscopic image of the sample to be measured appears on the area array camera by adjusting the triaxial sample displacement table. The sample to be measured is clearly imaged on the area array camera, which indicates that the sample to be measured is positioned at a microscopic focusing position under the irradiation of the monochromatic collimated illumination beam. And locking the triaxial sample displacement platform, and fixing the sample to be detected at the microscopic focusing position under the irradiation of the monochromatic collimated illumination beam, so that the triaxial sample displacement platform is not moved any more.

The first illumination beam and the second illumination beam respectively irradiate the sample to be detected from the front side, and the integration time of the area array camera in the acquisition module is adjusted to match the monochromatic collimated illumination beam with the wavelength, so that the acquisition module acquires microscopic images of the sample to be detected under the irradiation of the monochromatic collimated illumination beam. The measuring module measures the wavelength value of the first illumination beam and transmits the value to the external upper computer, and the external upper computer feeds back and regulates the control parameters of the acousto-optic modulator in the light source module to enable the monochromatic collimated illumination beam with stable wavelength output by the light source module. Repeating the steps for a predetermined number of times: after the parameters of the broad spectrum high-brightness light source in the light source module are adjusted, the light source module generates monochromatic collimated illumination light beams with set wavelengths through the acousto-optic modulator, the position of the triaxial sample displacement table is not changed, and after the parameters of the area array camera in the acquisition module are changed, the acquisition module acquires microscopic images of the sample to be detected irradiated by the monochromatic collimated illumination light beams with the set wavelengths. And repeatedly executing the steps to obtain microscopic image sequences of the sample to be detected under the irradiation of all monochromatic collimated illumination beams with monochromatic wavelengths, wherein the wavelengths of all the monochromatic collimated illumination beams meet 400-700 nm.

According to an embodiment of the present disclosure, the light source module includes a broad spectrum high brightness light source, a multimode optical fiber, a fiber collimator, and an acousto-optic modulator sequentially disposed along an optical path, and the light source module generates a wavelength-tunable monochromatic collimated illumination beam through the acousto-optic modulator in step 301, including: the wide-spectrum high-brightness light source outputs an illumination light beam, and the illumination light beam is input into the optical fiber collimator after passing through the multimode optical fiber and then is input into the acousto-optic modulator after being collimated by the optical fiber collimator; the acousto-optic modulator modulates the collimated illumination beam to produce a monochromatic collimated illumination beam with an adjustable wavelength. The wide spectrum highlight light source is adjusted, so that the output brightness of the wide spectrum highlight light source is matched with that of the acousto-optic modulator, and the image efficiency of the over-focus scanning microscope system can be optimized.

According to an embodiment of the present disclosure, the beam splitting module includes a first beam splitter, the beam splitting module splitting a monochromatic collimated illumination beam into a first illumination beam and a second illumination beam in step 302, comprising: the first beam splitter splits the monochromatic collimated illumination beam output by the acousto-optic modulator into a first illumination beam and a second illumination beam. According to an embodiment of the present disclosure, the first illumination module includes a first lens and a second beam splitter, the second illumination module includes a first mirror, a second lens, and a third lens, the first illumination beam irradiates the sample to be measured from the front side in step 303, the second illumination beam irradiates the sample to be measured from the back side, wherein the sample to be measured is fixed to the sample stage and is brought into micro-focus by movement of the sample stage, and the method includes:

The first illumination beam is converged by the first lens and irradiates to the second beam splitter, and the third illumination beam and the fourth illumination beam are formed by beam splitting of the second beam splitter; the positions of the first lens and the second beam splitter are changed, so that a fourth illumination beam can irradiate on the back focal plane of the microscope objective, and the fourth illumination beam irradiates on the sample to be detected from the front side through the microscope objective by a parallel beam after passing through the microscope objective, so that an incident type kohler illumination is formed; the second illumination light beam is reflected by the first reflector and the second reflector to generate a change of the propagation direction and is parallel to the second lens after being reflected by the first reflector and the second reflector; the second illumination beam irradiates to the focal plane of the third lens through the convergence of the second lens and irradiates to the sample to be detected from the back through the divergence of the third lens by the parallel beam through the change of the positions of the second lens and the third lens; the sample to be measured is fixed on the sample table through a triaxial sample displacement table, and a measuring area of the sample to be measured is searched and positioned through xy axis displacement of the triaxial sample displacement table; and through the z-axis displacement of the triaxial sample displacement table, the microscopic focusing of the sample to be measured is realized.

According to an embodiment of the present disclosure, the acquisition module includes a microscope objective, an imaging barrel, and an area camera. In step 304, the collecting module collects microscopic images formed after the first illumination beam and the second illumination beam are respectively irradiated to the sample to be tested from the front and the back, including: the first illumination beam and the second illumination beam respectively irradiate the sample to be detected from the front side and the back side to form a microscopic image of the sample to be detected, and the microscopic objective of the sample to be detected is transmitted to the area array camera through position adjustment of the microscopic objective and the imaging lens cone; acquiring microscopic images of a sample to be detected, which are obtained by irradiating the current monochromatic collimated illumination beam, through changing the exposure time of an area-array camera

According to an embodiment of the disclosure, the measurement module includes an optical fiber and a spectrometer, in step 305, the measurement module measures a wavelength value of the first illumination beam and transmits the wavelength value to an external host computer, and the external host computer feeds back and controls a control parameter of an acousto-optic modulator in the light source module, so that the light source module outputs a monochromatic collimated illumination beam with a set wavelength, including: the measuring module measures the wavelength value of the first illumination beam and transmits the wavelength value to an external upper computer; the first illumination beam passes through the second beam splitter to form a third illumination beam, and the third illumination beam is transmitted to the spectrometer through the optical fiber; measuring the change in wavelength by the spectrometer such that the spectrometer is capable of measuring a wavelength value of the third illumination beam; the spectrometer transmits the measured wavelength value of the third illumination beam to an external host computer.

According to an embodiment of the disclosure, the step 306 of repeatedly performing the step of generating the monochromatic collimated illumination beam with the adjustable wavelength by the light source module and the step of collecting the microscopic image by the collecting module, so that the collecting module collects the microscopic image sequence of the sample to be measured under the set monochromatic collimated illumination beam with all monochromatic wavelengths, includes: the light source module repeatedly performs a predetermined number of times to generate a wavelength-tunable monochromatic collimated illumination beam, the predetermined number of times being related to the amount of wavelength change. After setting the wavelength change amount within the wavelength adjustment range of the acousto-optic modulator, the number of times of generating the wavelength within the wavelength adjustment range, that is, the predetermined number of times can be determined. Specifically, for example, in the range of 400-700 nm, when the wavelength change amount is 20nm, the light source module generates 16 monochromatic collimated illumination beams with different wavelengths, that is, the steps are required to be repeatedly executed 16 times, so that the acquisition module acquires microscopic images corresponding to all wavelengths in the range of 400-700 nm to form an over-focus microscopic image sequence. In practical application, the wavelength change amount is changed according to the requirement, so that the required excessive-focus microscopic image corresponding to the excessive-focus state of the sample can be acquired, and the required excessive-focus microscopic image sequence is acquired. When the light source module repeatedly executes the preset times to generate the monochromatic collimation illumination light beams, the acquisition module also executes the acquisition of microscopic images of the preset times so as to acquire microscopic images of the sample to be detected under the irradiation of all the monochromatic collimation illumination light beams, and an over-focus microscopic image sequence is formed. The confocal microscopic image sequence comprises a confocal microscopic image and a focusing microscopic image of the sample to be detected, which are acquired by the acquisition module under the irradiation of monochromatic collimated illumination beams with different wavelengths, wherein the confocal microscopic image corresponds to a microscopic image in a negative confocal state and a microscopic image in a positive confocal state.

According to an embodiment of the present disclosure, the measuring method of the confocal scanning microscope system further includes performing data processing on the microscopic image sequence of the sample to be measured by using a library matching method based on sample model data, including: and carrying out data processing on the microscopic image sequence of the sample to be detected by using a database matching method based on sample model data, matching the measured microscopic image sequence with a database, and searching and determining the information such as the geometric dimension, the refractive index and the like of the sample to be detected.

According to the over-focus scanning microscopic system and the measuring method based on the acousto-optic modulator, microscopic images of samples to be measured under different wavelength conditions are measured under the condition of fixing a sample table to form an over-focus scanning microscopic image sequence, the samples to be measured do not need to be moved to focusing positions of monochromatic collimation illumination light beams with different wavelengths, and position uncertainty caused by adjusting and controlling the positions of the samples and low displacement control rate caused by frequently adjusting and controlling a high-precision position measuring device can be avoided. The method also adopts the acousto-optic modulator to change the wavelength of the monochromatic collimated illumination beam, improves the wavelength regulation and control rate to the millisecond level, and remarkably improves the acquisition efficiency of the optical microscopic image. In addition, because the volume of the acousto-optic modulator is very small, the over-focus scanning microscopic system provided by the disclosure not only can simplify a displacement control device, but also can simplify an optical structure for controlling the wavelength change of the monochromator with high precision, and the volume of the over-focus scanning microscopic system is remarkably reduced.

While the foregoing is directed to embodiments of the present disclosure, other and further details of the invention may be had by the present application, it is to be understood that the foregoing description is merely exemplary of the present disclosure and that no limitations are intended to the scope of the disclosure, except insofar as modifications, equivalents, improvements or modifications may be made without departing from the spirit and principles of the present disclosure.

Claims (21)

1. An acousto-optic modulator based over-focus scanning microscopy system comprising:

a light source module (100) comprising an acousto-optic modulator (4) for generating a series of wavelength-tunable monochromatic collimated illumination beams;

a beam splitting module (200) for splitting the monochromatic collimated illumination beam into a first illumination beam and a second illumination beam;

a sample stage (600) for fixing a sample to be measured and achieving microscopic focusing of the sample to be measured, wherein the first illumination beam irradiates the sample to be measured from the front side, and the second illumination beam irradiates the sample to be measured from the back side;

the acquisition module (700) is used for acquiring microscopic images formed after light beams irradiate the sample to be detected from the front and the back;

and the measuring module (500) is used for measuring the wavelength value of the first illumination light beam, transmitting the wavelength value to an external upper computer, and enabling the external upper computer to feed back and regulate the control parameters of the acousto-optic modulator (4) in the light source module (100) so that the light source module (100) outputs the monochromatic collimated illumination light beam with the set wavelength.

2. The acousto-optic modulator based confocal scanning microscopy system according to claim 1, wherein said acousto-optic modulator (4) employs high quality TeO ₂ The crystal has a clear aperture of 6-10 mm.

3. The acousto-optic modulator-based over-focus scanning microscopy system according to claim 1, wherein the light source module (100) further comprises a broad spectrum highlight light source (1), a multimode optical fiber (2) and a fiber collimator (3) arranged in sequence along an optical path, and the broad spectrum highlight light source (1), the multimode optical fiber (2) and the fiber collimator (3) are all located before the acousto-optic modulator (4), wherein:

the broad spectrum highlighting light source (1) is used for providing an illumination light beam;

-said multimode optical fiber (2) for transmitting said illumination beam to said fiber collimator (3);

the optical fiber collimator (3) is used for collimating the illumination light beam and transmitting the illumination light beam to the acousto-optic modulator (4).

4. The acousto-optic modulator based confocal scanning microscopy system of claim 3, wherein,

the wide spectrum high brightness light source (1) adopts a xenon lamp light source, a laser pumping xenon lamp or an SLED combined light source;

the multimode optical fiber (2) adopts an armored high-quality multimode optical fiber with the wavelength application range of 350-800 nm, the optical fiber caliber of 600 mu m-2 mm and an interface of SMA or FC;

The optical fiber collimator (3) adopts an aspheric lens, a multimode optical fiber collimator, an apochromatic objective lens or an off-axis parabolic mirror.

5. The acousto-optic modulator based confocal scanning microscopy system of claim 1, wherein the beam splitting module (200) employs an optical beam splitting prism.

6. The acousto-optic modulator based over-focus scanning microscopy system of claim 1, further comprising a first illumination module (300) and a second illumination module (400) after the spectroscopy module, wherein:

the first lighting module (300) comprises:

a first lens (6) for receiving and converging the first illumination beam;

a second beam splitter (7) for splitting the first illumination beam converged by the first lens (6) into a third illumination beam and a fourth illumination beam, wherein the third illumination beam enters the measurement module (500), the fourth illumination beam irradiates the sample to be measured from the front side, a uniform illumination light field is formed on the surface of the sample to be measured, and an incident kohler illumination is formed;

the second lighting module (400) comprises:

-a first mirror (12) for receiving said second illumination beam;

a second mirror (13) located behind the first mirror (12) for adjusting the propagation direction of the second illumination beam together with the first mirror (12) to obtain a second illumination beam with a changed propagation direction;

A second lens (14) for receiving and converging the second illumination beam of varying propagation direction;

and the third lens (15) is used for receiving the second illumination light beam converged by the second lens (14) so as to enable the converged second illumination light beam to irradiate the sample to be detected from the back surface to form transmission type Kohler illumination.

7. The acousto-optic modulator based over-focus scanning microscopy system according to claim 6, wherein the second illumination beam changing the propagation direction is converged by the second lens (14) to the focal plane of the third lens (15).

8. The acousto-optic modulator based confocal scanning microscopy system of claim 6, wherein,

the first lens (6) adopts a cemented lens, an apochromatic objective lens or an aspheric lens;

the second beam splitter (7) adopts a flat beam splitter or a thin film beam splitter;

the first reflecting mirror (12) and the second reflecting mirror (13) adopt ultraviolet enhanced protection aluminum film plane reflecting mirrors;

the second lens (14) and the third lens (15) are cemented lenses, apochromatic objective lenses or aspheric lenses.

9. The acousto-optic modulator-based confocal scanning microscopy system according to claim 6, wherein the measurement module (500) comprises an optical fiber (16) and a spectrometer (17), the optical fiber (16) is coupled to the spectrometer (17), the spectrometer (17) is used for measuring the wavelength value of the third illumination beam and transmitting the wavelength value to an external upper computer, and the external upper computer feedback regulates and 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 beam with a set wavelength.

10. The acousto-optic modulator-based over-focus scanning microscopy system according to claim 9, wherein the optical fiber (16) adopts an armored high-quality multimode optical fiber with a wavelength application range of 350-800 nm, an optical fiber caliber of 600 μm-2 mm and an interface of SMA or FC;

the spectrometer (17) adopts an optical fiber spectrometer or a high-order grating high-resolution spectrometer, the measurement wavelength range is 380 nm-720 nm, the wavelength measurement precision is 0.5nm, and the wavelength resolution is 1nm.

11. The acousto-optic modulator based confocal scanning microscopy system according to claim 9, wherein the control parameters of the acousto-optic modulator (4) comprise an output wavelength in the range of 400-700 nm and an output wavelength half-peak width of 2nm.

12. The acousto-optic modulator-based confocal 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, microscopic focusing of the sample to be measured is achieved through z-axis displacement, and searching and positioning of the measurement area of the sample to be measured are achieved through xy-axis displacement.

13. The acousto-optic modulator based over-focus scanning microscopy system of claim 1, wherein the acquisition module (700) comprises a microscope objective (8), an imaging cylinder (10) and an area array camera (11), wherein:

The microscope objective (8) is used for receiving a microscopic image formed by the sample to be tested;

the imaging lens barrel (10) is used for transmitting the microscopic image received by the microscopic objective lens (8) to the area array camera (11);

the area-array camera (11) is used for recording the microscopic images transmitted by the imaging lens barrel (10).

14. A measurement method applied to the over-focus scanning microscopy system of any one of claims 1 to 13, comprising:

the light source module generates monochromatic collimated illumination light beams with adjustable wavelengths through the acousto-optic modulator;

the beam splitting module splits the monochromatic collimated illumination beam into a first illumination beam and a second illumination beam;

the first illumination beam irradiates the sample to be measured from the front side, and the second illumination beam irradiates the sample to be measured from the back side, wherein the sample to be measured is fixed on the sample table and is subjected to microscopic focusing through movement of the sample table;

the acquisition module acquires microscopic images formed after the first illumination light beam and the second illumination light beam are respectively irradiated to the sample to be detected from the front and the back;

the measuring module measures the wavelength value of the first illumination beam and transmits the wavelength value to the external upper computer, and the external upper computer feeds back and regulates the control parameters of the acousto-optic modulator in the light source module to enable the light source module to output the monochromatic collimated illumination beam with the set wavelength; and

Repeatedly executing the steps of generating monochromatic collimated illumination beams with adjustable wavelengths by the light source module and collecting corresponding microscopic images by the collecting module, so that the collecting module collects microscopic image sequences of samples to be measured under the monochromatic collimated illumination beams with all the set monochromatic wavelengths.

15. The method of claim 14, wherein the light source module comprises a broad spectrum high brightness light source, a multimode fiber, a fiber collimator, and an acousto-optic modulator sequentially arranged along the light path, the light source module generating a wavelength-tunable monochromatic collimated illumination beam by the acousto-optic modulator, comprising:

the broad spectrum high brightness light source outputs illumination light beams;

the multimode optical fiber inputs the illumination beam into the fiber collimator;

the optical fiber collimator outputs a collimated illumination beam and transmits the collimated illumination beam to the acousto-optic modulator; and

the acousto-optic modulator modulates the collimated illumination beam to produce a wavelength-tunable monochromatic collimated illumination beam.

16. The method of measuring an over-focus scanning microscope system according to claim 14, wherein the first illumination module includes a first lens and a second beam splitter, the second illumination module includes a first mirror, a second lens, and a third lens, the first illumination beam irradiates the sample to be measured from a front side, the second illumination beam irradiates the sample to be measured from a back side, comprising:

The first illumination beam irradiates the second beam splitter through the convergence of the first lens, and forms a third illumination beam and a fourth illumination beam through the beam splitting of the second beam splitter, wherein the third illumination beam enters the measurement module, and the fourth illumination beam irradiates the sample to be measured from the front;

the second illumination beam is reflected by the first reflecting mirror and the second reflecting mirror in sequence, changes the propagation direction and irradiates the second lens; the second illumination beam irradiates to the focal plane of the third lens through the convergence of the second lens, and irradiates to the sample to be detected from the back surface through the divergence of the third lens in parallel beams.

17. The method of claim 16, wherein the measurement module comprises an optical fiber and a spectrometer, and wherein the measurement module measures the wavelength value of the first illumination beam and transmits the same to an external host computer, comprising:

the first illumination beam passes through the second beam splitter to form a third illumination beam;

the third illumination beam is transmitted to the spectrometer via 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 upper computer.

18. The method for measuring an over-focus scanning microscope system according to claim 14, wherein the sample to be measured is fixed to a sample stage and is brought into microscopic focus by movement of the sample stage, comprising:

the sample to be measured is fixed on the sample stage through the triaxial sample displacement stage;

the triaxial sample displacement platform realizes the searching and positioning of a sample measurement area to be measured through xy axis displacement;

and the triaxial sample displacement table realizes microscopic focusing through z-axis displacement adjustment.

19. The method for measuring an over-focus scanning microscope system according to claim 14, wherein the acquisition module includes a microscope objective, an imaging lens barrel, and an area array camera, and the acquisition module acquires microscopic images formed after the first illumination beam and the second illumination beam are respectively irradiated to the sample to be measured from the front and the back, and includes:

the microscope objective receives microscopic images formed after the first illumination light beam and the second illumination light beam are respectively irradiated to the sample to be detected from the front and the back;

the imaging lens barrel transmits the microscopic image to an 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 through exposure time adjustment.

20. The method for measuring an over-focus scanning microscope system according to claim 14, wherein the step of controlling the control parameter of the acousto-optic modulator in the light source module by the external host computer to make the light source module output the monochromatic collimated illumination beam with the set wavelength comprises:

the external upper computer compares the wavelength value of the first illumination beam measured by the measuring module with the wavelength of the monochromatic collimated illumination beam output by the light source module;

and the external upper computer feeds back and regulates control parameters of the acousto-optic modulator in the light source module according to the comparison result, so that the light source module outputs monochromatic collimated illumination beams with set wavelengths.

21. The measurement method according to claim 14, further comprising, after the collecting the sequence of microscopic images of the sample to be measured:

and carrying out data processing on the microscopic image sequence of the sample to be detected by using a database matching method based on sample model data.

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