Automatic focusing system, microscopic imaging system and automatic focusing method

Abstract

The present invention discloses an autofocus system, a microscopic imaging system and an autofocus method, comprising: a first light source for emitting a first light signal; a first dichroic mirror coupled to the first light source for transmitting the first light signal; an objective lens coupled to the first dichroic mirror for converging the first light signal on a sample to be tested; wherein the sample to be tested is used to generate a reflected light signal according to the first light signal; the objective lens is also used to transmit the reflected light signal to the first dichroic mirror; a first imaging module coupled to the first dichroic mirror for receiving the reflected light signal and generating a first image signal according to the reflected light signal; a control module connected to the first imaging module for generating a target distance according to the first imaging module; and an adjustment module respectively connected to the control module and the objective lens for adjusting the original distance between the objective lens and the sample to be tested according to the target distance. The autofocus system of the present invention can improve the imaging speed and ensure good imaging quality.

Description

Technical Field

[0001] The present invention relates to the field of microscopic imaging technology, and in particular to an automatic focusing system, a microscopic imaging system and an automatic focusing method. Background Art

[0002] At present, the sample to be tested can receive the laser signal of the prism and form a fluorescence signal under the excitation of the laser signal. The objective lens collects the fluorescence signal and transmits it to the imaging system for collection to obtain the corresponding image signal. However, there are many limitations to the imaging method of the above-mentioned sample to be tested. Among them, the size and placement of the sample to be tested need to match the objective lens, and the angle of the incident laser signal of the prism needs to be readjusted each time after the sample is changed. Correspondingly, it takes a lot of time to adjust the relative distance between the objective lens and the sample to be tested so that the objective lens converges the fluorescence signal to the imaging system, thereby obtaining a clear image signal. Therefore, in the related art, the imaging speed of the imaging system scanning the sample to be tested is slow. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides an automatic focusing system, a microscopic imaging system and an automatic focusing method, which can improve the imaging speed and ensure good imaging quality.

[0004] In a first aspect, the present application provides an autofocus system, the autofocus system is used to automatically adjust the original distance between an objective lens and a sample to be tested so that the objective lens converges a first light signal to the sample to be tested, the autofocus system comprising: a first light source, the first light source is used to emit the first light signal; a first dichroic mirror, the first dichroic mirror is coupled to the first light source, the first dichroic mirror is used to transmit the first light signal; an objective lens, the objective lens is coupled to the first dichroic mirror, the objective lens is used to converge the first light signal on the sample to be tested; wherein the sample to be tested is used to reflect the first light signal to generate a reflected light signal; the objective lens The mirror is also used to collect the reflected light signal and transmit it to the first dichroic mirror; the first dichroic mirror is also used to reflect the reflected light signal; a first imaging module, the first imaging module is coupled to the first dichroic mirror, the first imaging module is used to receive the reflected light signal and generate a first image signal according to the reflected light signal; a control module, the control module is connected to the first imaging module, the control module is used to generate a target distance according to the first imaging module; an adjustment module, the adjustment module is respectively connected to the control module and the objective lens, the adjustment module is used to adjust the original distance between the objective lens and the sample to be tested according to the target distance.

[0005] In the embodiment of the present invention, the autofocus system transmits a first light signal through a first light source, and the first light signal is collected by a first dichroic mirror lens and an objective lens and then irradiated on a sample to be tested, and the sample to be tested is used to generate a reflected light signal according to the first light signal. The reflected light signal is collected and converged by the objective lens, and is transmitted to the first imaging module after being reflected by the first dichroic mirror. The first imaging module generates a corresponding first image signal with the reflected light signal, and transmits it to the control module for calculation. The control module calculates the defocus distance between the objective lens and the surface to be tested at this time according to the first image signal, and generates a corresponding target distance and transmits it to the adjustment module. The adjustment module can adjust the original distance between the objective lens and the surface to be tested according to the target distance, so that the surface to be tested is at the focal plane of the objective lens, thereby ensuring clear imaging, and the imaging speed of scanning the sample to be tested can be improved by autofocusing, and the autofocus system of the embodiment of the present invention is simple and practical.

[0006] In some embodiments, the autofocus system also includes: a first slit, the first slit is coupled to the first light source, and the first slit is used to adjust the spot size of the first light signal; a first lens, the first lens is coupled to the first slit, and the first lens is used to collimate and converge the first light signal; a second lens, the second lens is coupled to the first lens, and the second lens is used to collimate and converge the first light signal.

[0007] In a second aspect, the present application provides a microscopic imaging system, comprising: an autofocus system as described in any of the above embodiments; an illumination module, the illumination module being used to generate a first laser signal; a total reflection prism, the total reflection prism being coupled to the illumination module, the total reflection prism being used to receive the first laser signal, and generate a second laser signal based on the first laser signal; the sample to be tested generates total reflection based on the second laser signal, and generates a corresponding fluorescence signal; a second imaging module, the imaging module being used to receive the fluorescence signal collected by the objective lens, the imaging module being also used to generate a second image signal based on the fluorescence signal; wherein the second image signal is used to characterize the imaging result of the sample to be tested.

[0008] In some embodiments, the lighting module includes: a second light source, the second light source is used to generate a second light signal; a third light source, the third light source is used to generate a third light signal; a second dichroic mirror, the second dichroic mirror is used to combine the second light signal and the third light signal to generate a first laser signal; an optical path adjustment component, the optical path adjustment component is coupled to the second dichroic mirror, the optical path adjustment component is used to adjust the incident angle of the first laser signal so that the first laser signal is incident on the total reflection prism at a preset angle; a third lens, the third lens is coupled to the optical path adjustment component, the third lens is used to converge the first laser signal to the total reflection prism.

[0009] In some embodiments, the lighting module further includes: a half-wave plate, which is coupled to the second dichroic mirror; and a polarization beam splitter prism, which is coupled to the half-wave plate; wherein the half-wave plate and the polarization beam splitter prism are both used to adjust the polarization state and light intensity of the first laser signal.

[0010] In some embodiments, the second imaging module includes: a third dichroic mirror, the third dichroic mirror is coupled to the objective lens, and the third dichroic mirror is used to transmit the fluorescence signal; a filtering unit, the filtering unit is coupled to the third dichroic mirror, and the filtering unit is used to filter the fluorescence signal; a fourth lens, the fourth lens is coupled to the filtering unit, and the fourth lens is used to converge the fluorescence signal; and an image sensor, the image sensor is used to receive the converged fluorescence signal to generate the second image signal.

[0011] In some embodiments, the second imaging module further includes: a second slit, the second slit is coupled to the third dichroic mirror, and the second slit is used to adjust the spot size of the fluorescent signal; and a fifth lens, the fifth lens is coupled to the second slit, and the fifth lens is used to converge the fluorescent signal to the filtering unit.

[0012] In some embodiments, the filtering unit includes: a fourth dichroic mirror, the fourth dichroic mirror is coupled to the fifth lens, the fourth dichroic mirror is used to separate the fluorescence signal to form at least one sub-fluorescence signal; at least one filter, the filter is coupled to the fourth dichroic mirror, the filter is used to filter the sub-fluorescence signal to form a filtered signal; a fifth dichroic mirror, the fifth dichroic mirror is coupled to the filter, and the fifth dichroic mirror is used to combine the sub-fluorescence signals.

[0013] In a third aspect, the present application further provides an autofocus method, which is applied to the autofocus system described in any one of the above embodiments, and the method includes:

[0014] Acquire a first image signal of the reflected light signal;

[0015] Obtaining a target distance according to the first image signal;

[0016] The original distance between the objective lens and the sample to be measured is adjusted according to the target distance.

[0017] In some embodiments, before acquiring the first image signal of the reflected light signal, the method further includes:

[0018] Adjusting the original distance between the objective lens and the sample to be tested according to a preset distance to obtain a corresponding sample image;

[0019] Extracting information from the sample image to obtain image information;

[0020] Inputting the image information into a preset original image model for distance prediction to obtain a training distance;

[0021] Adjusting parameters of the original image model according to the training distance and the preset distance to obtain a target image model;

[0022] The obtaining the target distance according to the first image signal comprises:

[0023] Extracting information from the first image signal to obtain a target image;

[0024] The target image is input into the target image model for distance prediction to obtain the target distance.

[0025] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0027] Figure 1 A schematic diagram of the structure of an automatic focusing system according to an embodiment of the present invention;

[0028] Figure 2 is a structural schematic diagram of a microscopic imaging system according to an embodiment of the present invention;

[0029] Figure 3 A schematic diagram of a process of an automatic focusing method according to an embodiment of the present invention;

[0030] Figure 4 FIG. 4 is another flowchart of the automatic focusing method according to an embodiment of the present invention.

[0031] Figure numerals: autofocus system 100, first light source 110, first dichroic mirror 120, objective lens 130, sample to be tested 300, first imaging module 140, control module 150, adjustment module 160, first slit 170, first lens 180, second lens 190, microscopic imaging system 200, illumination module 210, total reflection prism 220, second imaging module 230, second light source 211, third light source 212, second dichroic mirror 213, optical path adjustment member 214, third lens 215, half-wave plate 216, polarization beam splitting prism 217, third dichroic mirror 231, filtering unit 232, fourth lens 233, image sensor 234, second slit 235, fifth lens 236, fourth dichroic mirror 237, filter 238, fifth dichroic mirror 239. DETAILED DESCRIPTION

[0032] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0033] In the description of the present invention, it should be understood that descriptions involving orientations, such as up, down, front, back, left, right, etc., and orientations or positional relationships indicated are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0034] In the description of the present invention, "several" means more than one, "many" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, and "above", "below", "within", etc. are understood to include the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing the technical features, and cannot be understood as indicating or implying the relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0035] In the description of the present invention, unless otherwise clearly defined, terms such as setting, installing, connecting, etc. should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in the present invention based on the specific content of the technical solution.

[0036] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0037] It should be noted that the laser signal can excite the fluorescent molecules in the sample to be tested to generate corresponding fluorescent signals. The microscopic imaging system collects the fluorescent signals and generates image signals according to the fluorescent signals, thereby realizing the detection of the sample to be tested. In the related art, when the wide-field fluorescence microscope (microscopic imaging system) needs to image the sample to be tested with a certain thickness, the laser signal will excite the fluorescent molecules in the entire illumination area to generate fluorescent signals. At this time, the fluorescent signals at different levels of the sample to be tested interfere with each other, so that the longitudinal imaging resolution of the microscopic imaging system to the sample to be tested is reduced, thereby causing the quality of the image signal to be seriously reduced. For this reason, in the related art, the total internal reflection fluorescence imaging microscope forms a 100nm to 200nm range of evanescent waves on the other side of the sample through the laser signal, which can excite some fluorescent molecules in a specific area separately, thereby avoiding signal interference between different levels, reducing background noise, and improving the longitudinal imaging resolution of the microscopic imaging system, thereby breaking the resolution limit of the diffraction limit. Specifically, the above-mentioned total internal reflection fluorescence imaging microscope can be divided into two imaging types, one is objective type total internal reflection fluorescence imaging, and the other is prism type total internal reflection fluorescence imaging. Among them, prism-type total internal reflection fluorescence imaging means that the excitation and imaging are completed by the prism and the objective lens respectively, and the mutual interference between the two is small, which is relatively easy to control. However, this imaging method is relatively limited in the size and placement of the sample to be tested, and is more complicated in actual operation. Specifically, each time the sample to be tested is changed, the angle of the prism incident laser signal needs to be readjusted, and each time it takes a lot of time to focus the objective lens, which causes the microscopic imaging system to scan the sample to be tested at a slow speed.

[0038] To this end, the present application provides an autofocus system, a microscopic imaging system and an autofocus method, which can improve the imaging speed while ensuring the imaging quality, and are easy and practical to operate.

[0039] See also Figure 1In a first aspect, the present application provides an autofocus system 100, the autofocus system 100 is used to automatically adjust the original distance between an objective lens 130 and a sample to be tested 300, so that the objective lens 130 converges a first light signal to the sample to be tested 300, the autofocus system 100 comprises: a first light source 110, the first light source 110 is used to emit the first light signal; a first dichroic mirror 120, the first dichroic mirror 120 is coupled to the first light source 110, the first dichroic mirror 120 is used to transmit the first light signal; an objective lens 130, the objective lens 130 is coupled to the first dichroic mirror 120, the objective lens 130 is used to converge the first light signal on the sample to be tested 300; wherein the sample to be tested 300 is used to reflect the first light signal to generate a reflection light signal; the objective lens 130 is also used to collect the reflected light signal and transmit it to the first dichroic mirror 120; the first dichroic mirror 120 is also used to reflect the reflected light signal; a first imaging module 140, the first imaging module 140 is coupled to the first dichroic mirror 120, the first imaging module 140 is used to receive the reflected light signal, and generate a first image signal according to the reflected light signal; a control module 150, the control module 150 is connected to the first imaging module 140, the control module 150 is used to generate a target distance according to the first imaging module 140; an adjustment module 160, the adjustment module 160 is respectively connected to the control module 150 and the objective lens 130, the adjustment module 160 is used to adjust the original distance between the objective lens 130 and the sample 300 to be tested according to the target distance.

[0040] It is understandable that when the sample 300 to be tested is on the focal plane of the objective lens 130, the objective lens 130 can collect the fluorescent signal generated by the sample 300 to be tested and accurately focus it on the corresponding imaging module (the second imaging module 230) to generate a clear second image signal, wherein the second image signal represents the detection result of the sample 300 to be tested. As can be seen from the above content, when the sample 300 to be tested is excited by the prism-type total internal reflection fluorescence imaging method, since the laser signal must irradiate the sample 300 to be tested at a specific incident angle, it is not convenient to move the sample 300 to be tested after replacement, otherwise the incident angle of the laser signal needs to be readjusted, thereby affecting the efficiency of the detection. However, in actual applications, it is often necessary to replace the sample 300 to be tested multiple times for detection, and after replacing the sample 300 to be tested, the sample 300 to be tested may be out of focus from the focal plane of the objective lens 130, which easily leads to blurred imaging. To this end, the automatic focusing system 100 in the embodiment of the present invention can automatically adjust the original distance between the objective lens 130 and the surface to be measured, so that the surface to be measured is always kept on the focal plane of the objective lens 130, thereby achieving automatic focusing.

[0041] Specifically, the autofocus system 100 includes a first light source 110, a first dichroic mirror 120, and an objective lens 130 that are coupled in sequence, wherein the first dichroic mirror 120 is also used to be coupled with a first imaging module 140, and a control module 150 is respectively connected to the first imaging module 140 and the objective lens 130.

[0042] It can be understood that the first light source 110 is used to generate a first light signal, and in the embodiment of the present invention, the first light signal can be an infrared laser signal with a wavelength of 830nm. The first light source 110 transmits the first light signal to the first dichroic mirror 120. Among them, the first dichroic mirror 120 can transmit light signals of a specific wavelength and reflect light signals of other wavelengths. In the embodiment of the present invention, the first dichroic mirror 120 can transmit the first light signal to transmit the first light signal to the objective lens 130. The objective lens 130 is used to collect the first light signal and irradiate the first light signal onto the sample to be tested 300. The sample to be tested 300 can reflect the first light signal to generate a reflected light signal, and the reflected light signal can be collected by the objective lens 130 and transmitted to the first dichroic mirror 120. The first dichroic mirror 120 reflects the reflected light signal, thereby transmitting the reflected light signal to the first imaging module 140. In some application scenarios, the first imaging module 140 can be a camera. It is understandable that when the original distance between the surface to be measured and the objective lens 130 is different, the image formation result of the reflected light signal collected by the objective lens 130 in the first imaging module 140 is different, that is, the first imaging module 140 generates different first image signals according to the reflected light signal. Therefore, the autofocus system 100 can determine the defocus distance between the objective lens 130 and the surface to be measured at this time by processing the first image signal, thereby achieving autofocus.

[0043] Specifically, the autofocus device of the embodiment of the present invention is provided with a corresponding control module 150, and the control module 150 is used to receive the first image signal generated by the first imaging module 140. The control module 150 is also used to calculate the target distance (defocus distance) according to the first image signal, and send the target distance to the adjustment module 160. The adjustment module 160 is used to adjust the relative distance between the objective lens 130 and the surface to be measured according to the target distance, so that the surface to be measured is kept at the focal plane of the objective lens 130, thereby ensuring clear imaging of the microscopic imaging system 200.

[0044] In the embodiment of the present invention, the autofocus system 100 emits a first light signal through the first light source 110, and the first light signal is collected by the first dichroic mirror 120 lens and the objective lens 130 and then irradiated on the sample to be tested 300, and the sample to be tested 300 is used to generate a reflected light signal according to the first light signal. The reflected light signal is collected and converged by the objective lens 130, and is transmitted to the first imaging module 140 after being reflected by the first dichroic mirror 120. The first imaging module 140 generates a corresponding first image signal with the reflected light signal, and transmits it to the control module 150 for calculation. The control module 150 calculates the defocus distance between the objective lens 130 and the surface to be tested at this time according to the first image signal, and generates a corresponding target distance and transmits it to the adjustment module 160. The adjustment module 160 can adjust the original distance between the objective lens 130 and the surface to be tested according to the target distance, so that the surface to be tested is at the focal plane of the objective lens 130, thereby ensuring clear imaging, and the imaging speed of scanning the sample to be tested 300 can be improved by the automatic focusing method, and the autofocus system 100 of the embodiment of the present invention is simple and practical.

[0045] Please refer again Figure 1 In some embodiments, the autofocus system 100 further includes: a first slit 170, the first slit 170 is coupled to the first light source 110, and the first slit 170 is used to adjust the spot size of the first light signal; a first lens 180, the first lens 180 is coupled to the first slit 170, and the first lens 180 is used to collimate and converge the first light signal; a second lens 190, the second lens 190 is coupled to the first lens 180, and the second lens 190 is used to collimate and converge the first light signal.

[0046] It is understandable that in order to adjust the spot size of the first light signal so that the first light signal can be gathered within the imaging range of the objective lens 130, the autofocus system 100 in the embodiment of the present invention is provided with a corresponding first slit 170. The first slit 170 is arranged in the light emitting direction of the first light source 110. When the first light source 110 emits the first light signal to the first slit 170, the first slit 170 can adjust the spot size of the first light signal. Preferably, the aspect ratio of the first slit 170 can be 3:1. The first slit 170 with other aspect ratios can also be selected according to actual needs, which is not limited in the embodiment of the present invention. In addition, the first slit 170 is also used to collimate the light beam of the first light signal.

[0047] It can be understood that, in order to collimate and focus the first light signal so that the first light signal can be better focused on the objective lens 130, the autofocus system 100 of the embodiment of the present invention is also provided with a corresponding first lens 180 and a second lens 190. Among them, the first lens 180 is arranged in the light emitting direction of the first slit 170, and the second lens 190 is arranged in the light emitting direction of the first lens 180. The first light signal is transmitted to the first lens 180 and the second lens 190 after being adjusted by the first slit 170. The first lens 180 and the second lens 190 can collimate and focus the first light signal so that the first light signal is focused on the objective lens 130.

[0048] See also Figure 2 In a second aspect, the present application provides a microscopic imaging system 200, comprising: an autofocus system 100 as in any of the above embodiments; an illumination module 210, the illumination module 210 is used to generate a first laser signal; a total reflection prism 220, the total reflection prism 220 is coupled to the illumination module 210, the total reflection prism 220 is used to receive the first laser signal, and generate a second laser signal according to the first laser signal; the sample 300 to be tested generates total reflection according to the second laser signal, and generates a corresponding fluorescence signal; a second imaging module 230, the imaging module is used to receive the fluorescence signal collected by the objective lens 130, and the imaging module is also used to generate a second image signal according to the fluorescence signal; wherein the second image signal is used to characterize the imaging result of the sample 300 to be tested.

[0049] It is understandable that, from the above content, the microscopic imaging system 200 can be used to perform imaging detection on the sample 300. Specifically, the microscopic imaging system 200 in the embodiment of the present invention includes: an illumination module 210, a total reflection prism 220, a second imaging module 230 and an autofocus system 100.

[0050] Specifically, the lighting module 210 is used to emit a first laser signal, and the first laser signal is incident on the total reflection prism 220 at a specific first angle. After the first laser signal passes through the total reflection prism 220, a second laser signal is generated that is emitted at a specific second angle. The second laser signal can be completely reflected by the surface of the sample 300 to be tested, thereby forming a vector field. Preferably, in the embodiment of the present invention, the first angle can be 30 degrees, and the second angle can be 70 degrees. The light intensity of the vector field decreases exponentially toward the other side of the incident surface in a direction perpendicular to the sample 300 to be tested, and forms a vector wave with a wavelength of 100nm to 200nm. The vector wave can excite the sample 300 to be tested within the range to generate a corresponding fluorescence signal. In addition, the objective lens 130 can collect the fluorescence signal generated by the sample 300 to be tested, and transmit it to the second imaging module 230. The second imaging module 230 generates a second image signal based on the fluorescence signal, and the second image signal is used to characterize the imaging result of the sample 300 to be tested, that is, the detection result.

[0051] It is understandable that, from the above content, when the sample 300 to be tested is replaced, the sample 300 to be tested may not be on the focal plane of the objective lens 130, that is, the objective lens 130 is out of focus, and the imaging of the second imaging module 230 is blurred. Therefore, in the embodiment of the present invention, the microscopic imaging system 200 can adjust the original distance between the objective lens 130 and the sample 300 to be tested in real time through the automatic focusing system 100 to avoid the defocusing of the objective lens 130, so as to make the imaging of the second imaging module 230 clear. In addition, during the process of scanning the sample 300 to be tested by the microscopic imaging system 200, the automatic focusing system 100 can perform automatic focusing, thereby improving the imaging speed of the scanning.

[0052] Please refer again Figure 2 In some embodiments, the lighting module 210 includes: a second light source 211, the second light source 211 is used to generate a second light signal; a third light source 212, the third light source 212 is used to generate a third light signal; a second dichroic mirror 213, the second dichroic mirror 213 is used to combine the second light signal and the third light signal to generate a first laser signal; an optical path adjustment component 214, the optical path adjustment component 214 is coupled to the second dichroic mirror 213, and the optical path adjustment component 214 is used to adjust the incident angle of the first laser signal so that the first laser signal is incident on the total reflection prism 220 at a preset angle; a third lens 215, the third lens 215 is coupled to the optical path adjustment component 214, and the third lens 215 is used to converge the first laser signal to the total reflection prism 220.

[0053] It can be understood that, from the above content, the lighting module 210 is used to generate a first laser signal incident on the total reflection prism 220 at a specific first angle. Specifically, the lighting module 210 is provided with a corresponding second light source 211, a third light source 212, an optical path adjustment module 160 and a third lens 215. Among them, the second dichroic mirror 213 is coupled to the second light source 211 and the third light source 212 respectively, and the optical path adjustment member 214 is coupled to the second dichroic mirror 213 and the fourth lens 233 respectively, and the fourth lens 233 is also used to be coupled to the total reflection prism 220.

[0054] Specifically, the first light source 110 is used to generate a second light signal, and the third light source 212 is used to generate a third light signal. Preferably, the wavelength of the second light signal can be 532nm, and the wavelength of the third light signal can be 638nm. The second dichroic mirror 213 is used to combine the second light signal and the third light signal into a first laser signal, and reflect it to the optical path adjustment component 214. The optical path adjustment component 214 is used to adjust the incident angle of the first laser signal so that the first laser signal is incident on the total reflection prism 220 at a specific first angle, thereby causing total reflection on the surface of the sample 300 to be tested. The third lens 215 is used to converge the adjusted first laser signal on the surface of the total reflection prism 220.

[0055] Please refer again Figure 2 In some embodiments, the lighting module 210 further includes: a half-wave plate 216, which is coupled to the second dichroic mirror 213; a polarization beam splitter prism 217, which is coupled to the half-wave plate 216; wherein the half-wave plate 216 and the polarization beam splitter prism 217 are both used to adjust the polarization state and light intensity of the first laser signal.

[0056] It can be understood that both the half-wave plate 216 and the polarization beam splitter prism 217 are used to adjust the polarization state and light intensity of the first laser signal to meet different requirements in practical applications.

[0057] Please refer again Figure 2 In some embodiments, the second imaging module 230 includes: a third color dichroic mirror 231, which is coupled to the objective lens 130 and is used to transmit the fluorescent signal; a filtering unit 232, which is coupled to the third color dichroic mirror 231 and is used to filter the fluorescent signal; a fourth lens 233, which is coupled to the filtering unit 232 and is used to converge the fluorescent signal; and an image sensor 234, which is used to receive the converged fluorescent signal to generate a second image signal.

[0058] It can be understood that, from the above content, the third dichroic mirror 231 can reflect the first light signal generated by the autofocus system 100 and the reflected light signal generated by the sample to be tested 300, and can transmit the fluorescent signal so that the fluorescent signal is emitted to the image sensor 234 for imaging, thereby avoiding interference between different light signals.

[0059] It is understandable that the light signal incident to the second imaging module 230 may include other interference signals, such as a second laser signal for excitation. In order to avoid the interference signal from causing adverse effects on the imaging of the image sensor 234, a corresponding filtering unit 232 is provided in the embodiment of the present invention, and the filtering unit 232 can filter the incident fluorescent signal, thereby filtering out the interference signal and noise, and improving the imaging quality of the image sensor 234. The filtered fluorescent signal is emitted to the fourth lens 233, and the fourth lens 233 is used to converge the fluorescent signal on the imaging surface of the image sensor 234, and the image sensor 234 generates a corresponding second image signal according to the fluorescent signal.

[0060] Please refer again Figure 2 In some embodiments, the second imaging module 230 further includes: a second slit 235, the second slit 235 is coupled to the third dichroic mirror 231, and the second slit 235 is used to adjust the spot size of the fluorescent signal; a fifth lens 236, the fifth lens 236 is coupled to the second slit 235, and the fifth lens 236 is used to converge the fluorescent signal to the filtering unit 232.

[0061] It is understandable that the second slit 235 can filter the incident fluorescent signal to prevent the second laser signal from entering, thereby improving the signal-to-noise ratio of the image sensor 234. At the same time, the second slit 235 is also used to adjust the spot size of the fluorescent signal. In addition, the second imaging module 230 is also provided with a fifth lens 236, which is used to converge the fluorescent signal to the filtering unit 232.

[0062] Please refer again Figure 2 In some embodiments, the filtering unit 232 includes: a fourth dichroic mirror 237, which is coupled to the fifth lens 236, and is used to separate the fluorescence signal to form at least one sub-fluorescence signal; at least one filter 238, which is coupled to the fourth dichroic mirror 237, and is used to filter the sub-fluorescence signal to form a filtered signal; and a fifth dichroic mirror 239, which is coupled to the filter 238, and is used to combine the sub-fluorescence signals.

[0063] It is understandable that, since different fluorescent molecules can generate fluorescent signals of different wavelengths under the excitation of the second laser signal, the fluorescent signal generated by the sample to be tested 300 has multiple wavelengths. In practical applications, the fluorescent signals of one or more specific wavelengths can be collected and detected. To this end, the filter unit 232 of the embodiment of the present invention is provided with a corresponding fourth dichroic mirror 237, which can transmit or reflect the fluorescent signal of a specific wavelength, thereby separating the fluorescent signal into different sub-fluorescent signals, and a corresponding filter 238 is provided on the optical path of each sub-fluorescent signal to filter out interference signals. In order to be able to perform imaging detection on multiple sub-fluorescent signals through a single image sensor 234, the second imaging module 230 is also provided with a fifth dichroic mirror 239, which can combine the multiple sub-fluorescent signals into one fluorescent signal, and focus the fourth lens 233 to the image sensor 234. Preferably, the image sensor 234 can be an EMCCD camera.

[0064] It is understandable that a plurality of reflectors are further provided in the embodiment of the present invention, and the reflectors can reflect different light signals to reduce the overall volume of the microscopic imaging system 200 and improve the integration.

[0065] See also Figure 3 In a third aspect, the present application further provides an autofocus method, which is applied to the autofocus system of any of the above embodiments, and the method includes:

[0066] Step S101, obtaining a first image signal of a reflected light signal;

[0067] Step S102, obtaining a target distance according to the first image signal;

[0068] Step S103: adjusting the original distance between the objective lens and the sample to be measured according to the target distance.

[0069] It can be understood that, from the above content, when the original distance between the surface to be measured and the objective lens is different, the image formation result of the reflected light signal collected by the objective lens in the first imaging module is different, that is, the first imaging module generates different first image signals according to the reflected light signal. Therefore, the first image signal can be processed to obtain the corresponding target distance, and the target distance is used to characterize the defocus distance between the objective lens and the surface to be measured. The objective lens can be adjusted according to the target distance to keep the surface to be measured at the focal plane of the objective lens, thereby achieving automatic focusing.

[0070] Please refer again Figure 3 In some embodiments, before step S101, obtaining the first image signal of the reflected light signal, the method further includes:

[0071] Step S201, adjusting the original distance between the objective lens and the sample to be measured according to a preset distance to obtain a corresponding sample image;

[0072] Step S202: extract information from the sample image to obtain image information;

[0073] Step S203: input the image information into a preset original image model for distance prediction to obtain a training distance;

[0074] Step S204: adjusting the parameters of the original image model according to the training distance and the preset distance to obtain a target image model;

[0075] Obtaining the target distance according to the first image signal includes:

[0076] Step S205: extract information from the first image signal to obtain a target image;

[0077] Step S206: input the target image into the target image model for distance prediction to obtain the target distance.

[0078] It is understandable that, from the above content, the sample image (first image signal) is related to the defocus distance of the objective lens. Therefore, in the embodiment of the present invention, by calculating the sample images at different preset distances, the corresponding target image model can be obtained.

[0079] Specifically, the control module receives sample images corresponding to different preset distances, and performs Fourier transform processing on each sample image to extract the high-frequency information part to obtain multiple image information. The control module is also used to input multiple image information into a preset original image module for distance prediction to obtain a training distance, and compare the training distance with the preset distance, and adjust the parameters of the original image model according to the comparison result to finally obtain the target image model. It can be understood that the target image model obtained through the above training process can predict the optimal target distance according to the first image signal, thereby controlling the objective lens to reach the optimal focal plane to obtain a clear imaging result.

[0080] It is understandable that, when processing the sample image or the first image signal, preprocessing may be performed first, such as denoising, regularization, etc., to improve the accuracy of subsequent calculations.

[0081] In some application scenarios, when the adjustment module moves the objective lens according to the target distance, jitter may occur, thereby blurring the image. To this end, in an embodiment of the present invention, the control module is also used to set a movement threshold, and judge whether the objective lens has jitter according to the movement threshold. When jitter exists, the adjustment module stops moving the objective lens, otherwise it continues to move. In addition, the control module can also be set with a fast reset function to control the objective lens to move quickly to the initial position.

[0082] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An automatic focusing system, characterized in that: The automatic focusing system is used to automatically adjust the original distance between the objective lens and the sample to be tested so that the objective lens converges the first light signal to the sample to be tested. The automatic focusing system includes: a first light source, the first light source being used to emit the first light signal; a first dichroic mirror, the first dichroic mirror is coupled to the first light source, and the first dichroic mirror is used to transmit the first light signal; an objective lens, the objective lens being coupled to the first dichroic mirror, and the objective lens being used to converge the first light signal onto a sample to be tested; wherein the sample to be tested is used to reflect the first light signal to generate a reflected light signal; the objective lens is also used to collect the reflected light signal and transmit it to the first dichroic mirror; the first dichroic mirror is also used to reflect the reflected light signal; a first imaging module, the first imaging module is coupled to the first dichroic mirror, and the first imaging module is used to receive the reflected light signal and generate a first image signal according to the reflected light signal; A control module, the control module is connected to the first imaging module, and the control module is used to generate a target distance according to the first imaging module; An adjustment module, wherein the adjustment module is connected to the control module and the objective lens respectively, and the adjustment module is used to adjust the original distance between the objective lens and the sample to be tested according to the target distance.

2. The automatic focusing system according to claim 1, characterized in that: Also includes: a first slit, wherein the first slit is coupled to the first light source, and the first slit is used to adjust the spot size of the first light signal; A first lens, the first lens is coupled to the first slit, and the first lens is used to collimate and converge the first optical signal; A second lens is coupled to the first lens, and the second lens is used for collimating and converging the first optical signal.

3. A microscopic imaging system, characterized in that: include: The automatic focusing system according to any one of claims 1 to 2; An illumination module, wherein the illumination module is used to generate a first laser signal; A total reflection prism, the total reflection prism is coupled to the lighting module, the total reflection prism is used to receive the first laser signal, and generate a second laser signal according to the first laser signal; the sample to be tested generates total reflection according to the second laser signal, and generates a corresponding fluorescence signal; A second imaging module, the imaging module is used to receive the fluorescent signal collected by the objective lens, and the imaging module is also used to generate a second image signal according to the fluorescent signal; wherein the second image signal is used to characterize the imaging result of the sample to be tested.

4. The microscopic imaging system according to claim 3, characterized in that: The lighting module comprises: a second light source, the second light source being used to generate a second light signal; a third light source, the third light source being used to generate a third light signal; a second dichroic mirror, the second dichroic mirror being used to combine the second optical signal and the third optical signal to generate a first laser signal; an optical path adjusting member, the optical path adjusting member being coupled to the second dichroic mirror, and the optical path adjusting member being used for adjusting an incident angle of the first laser signal so that the first laser signal is incident on the total reflection prism at a preset angle; A third lens is coupled to the optical path adjustment component and is used to converge the first laser signal to the total reflection prism.

5. The microscopic imaging system according to claim 4, characterized in that: The lighting module further comprises: A half-wave plate, wherein the half-wave plate is coupled to the second dichroic mirror; A polarization beam splitter prism, wherein the polarization beam splitter prism is coupled to the half-wave plate; Wherein, the half-wave plate and the polarization beam splitter prism are both used to adjust the polarization state and light intensity of the first laser signal.

6. The microscopic imaging system according to claim 3, characterized in that: The second imaging module comprises: a third dichroic mirror, the third dichroic mirror being coupled to the objective lens and configured to transmit the fluorescent signal; A filtering unit, the filtering unit is coupled to the third dichroic mirror, and the filtering unit is used to filter the fluorescence signal; a fourth lens, the fourth lens being coupled to the filtering unit and configured to converge the fluorescence signal; An image sensor is used to receive the converged fluorescent signal to generate the second image signal.

7. The microscopic imaging system according to claim 6, characterized in that: The second imaging module further includes: a second slit, the second slit is coupled to the third dichroic mirror, and the second slit is used to adjust the spot size of the fluorescent signal; A fifth lens is coupled to the second slit, and is used to converge the fluorescence signal to the filtering unit.

8. The microscopic imaging system according to claim 7, characterized in that: The filtering unit comprises: a fourth dichroic mirror, the fourth dichroic mirror being coupled to the fifth lens, and the fourth dichroic mirror being used for performing color separation on the fluorescent signal to form at least one sub-fluorescent signal; at least one filter, the filter being coupled to the fourth dichroic mirror, and the filter being used to filter the sub-fluorescence signal to form a filtered signal; A fifth dichroic mirror is coupled to the filter, and is used for combining the sub-fluorescence signals.

9. An autofocus method, applied to the autofocus system according to any one of claims 1 to 2, characterized in that: The method comprises: Acquire a first image signal of the reflected light signal; Obtaining a target distance according to the first image signal; The original distance between the objective lens and the sample to be measured is adjusted according to the target distance.

10. The automatic focusing method according to claim 9, characterized in that: Before acquiring the first image signal of the reflected light signal, the method further includes: Adjusting the original distance between the objective lens and the sample to be tested according to a preset distance to obtain a corresponding sample image; Extracting information from the sample image to obtain image information; Inputting the image information into a preset original image model for distance prediction to obtain a training distance; Adjusting parameters of the original image model according to the training distance and the preset distance to obtain a target image model; The obtaining the target distance according to the first image signal comprises: Extracting information from the first image signal to obtain a target image; The target image is input into the target image model for distance prediction to obtain the target distance.

Images