CN115453736A - Automatic focusing system, microscopic imaging system and automatic focusing method - Google Patents
Automatic focusing system, microscopic imaging system and automatic focusing method Download PDFInfo
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Abstract
The invention discloses an automatic focusing system, a microscopic imaging system and an automatic focusing method, which comprise the following steps: a first light source for emitting a first optical signal; the first dichroic mirror is coupled with the first light source and used for transmitting the first optical signal; the objective lens is coupled with the first dichroic mirror and used for converging the first optical signal on a sample to be detected; the sample to be detected is used for generating a reflected light signal according to the first light signal; the objective lens is also used for transmitting the reflected light signal to the first dichroic mirror; the first imaging module is coupled with the first dichroic mirror and used for receiving the reflected light signals and generating first image signals according to the reflected light signals; the control module is connected with the first imaging module and used for generating a target distance according to the first imaging module; and the adjusting module is respectively connected with the control module and the objective lens and is used for adjusting the original distance between the objective lens and the sample to be measured according to the target distance. The automatic focusing system can improve the imaging speed and ensure better imaging quality.
Description
Technical Field
The invention relates to the technical field of microscopic imaging, in particular to an automatic focusing system, a microscopic imaging system and an automatic focusing method.
Background
At present, a sample to be detected can receive a laser signal of a prism and form a fluorescence signal under the excitation of the laser signal, and an objective lens collects the fluorescence signal and transmits the fluorescence signal to an imaging system for collection so as to obtain a corresponding image signal. However, there are many limitations to the above-mentioned imaging methods of the sample to be measured. The size and placement of the sample to be measured need to be matched with the objective lens, and the angle of the laser signal incident on the prism needs to be readjusted after the sample is replaced every time, and accordingly, a large amount of time is needed to be spent on adjusting the relative distance between the objective lens and the sample to be measured, so that the objective lens converges the fluorescent signal to the imaging system, and a clear image signal is obtained. Therefore, in the related art, the imaging speed of the imaging system for scanning the sample to be measured is slow.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides an automatic focusing system, a microscopic imaging system and an automatic focusing method, which can improve the imaging speed and ensure better imaging quality.
In a first aspect, the present application provides an auto-focusing system for automatically adjusting an original distance between an objective lens and a sample to be measured, so that the objective lens converges a first optical signal to the sample to be measured, the auto-focusing system comprising: a first optical source for emitting the first optical signal; a first dichroic mirror coupled to the first light source, the first dichroic mirror configured to transmit the first optical signal; the objective lens is coupled with the first dichroic mirror and is used for converging the first optical signal on a sample to be measured; the sample to be detected is used for reflecting the first optical signal to generate a reflected optical signal; the objective lens is also used for collecting the reflected light signal and transmitting the reflected light signal to the first dichroic mirror; the first dichroic mirror is further used for reflecting the reflected light signal; the first imaging module is coupled with the first dichroic mirror and used 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, the control module configured to generate a target distance according to the first imaging module; and the adjusting module is respectively connected with the control module and the objective lens and is used for adjusting the original distance between the objective lens and the sample to be measured according to the target distance.
In the embodiment of the invention, the automatic focusing system emits a first optical signal through the first light source, the first optical signal is collected through the first dichroic mirror lens and the objective lens and then irradiates on a sample to be detected, and the sample to be detected is used for generating a reflected light signal according to the first optical signal. The reflected light signals are collected and converged by the objective lens, are reflected by the first dichroic mirror and then are transmitted to the first imaging module, the first imaging module generates corresponding first image signals along with the reflected light signals and transmits the first image signals to the control module for calculation, the control module calculates the defocusing distance between the objective lens and the surface to be measured at the moment according to the first image signals, generates corresponding target distance and transmits the corresponding target distance to the adjusting module. The adjusting module can adjust the original distance between the objective lens and the surface to be measured according to the target distance, so that the surface to be measured is positioned at the focal plane of the objective lens, clear imaging is guaranteed, the imaging speed of scanning a sample to be measured can be increased through an automatic focusing mode, and the automatic focusing system is simple, convenient and practical.
In some embodiments, the autofocus system further comprises: a first slit coupled to the first light source, the first slit configured to adjust a spot size of the first optical signal; a first lens coupled to the first slit, the first lens being configured to perform collimation and convergence on the first optical signal; the second lens is coupled with the first lens and is used for collimating and converging the first optical signal.
In a second aspect, the present application provides a microscopic imaging system comprising: an autofocus system as in any of the embodiments above; an illumination module to generate a first laser signal; the total reflection prism is coupled with the illumination module and used for receiving the first laser signal and generating a second laser signal according to the first laser signal; the sample to be detected generates total reflection according to the second laser signal and generates a corresponding fluorescent signal; a second imaging module for receiving the fluorescence signal collected by the objective lens, the imaging module further for generating a second image signal from the fluorescence signal; and the second image signal is used for representing the imaging result of the sample to be detected.
In some embodiments, the lighting module comprises: a second light source to generate a second light signal; a third light source to generate a third light signal; a second dichroic mirror for combining the second optical signal and the third optical signal to generate a first laser signal; the light path adjusting piece is coupled with the second dichroic mirror and used for adjusting the incident angle of the first laser signal so that the first laser signal is incident to the total reflection prism at a preset angle; and the third lens is coupled with the optical path adjusting piece and is used for converging the first laser signal to the total reflection prism.
In some embodiments, the lighting module further comprises: the half-wave plate is coupled with the second dichroic mirror; the polarization beam splitter prism is coupled with the half-wave plate; the half-wave plate and the polarization beam splitter prism are used for adjusting the polarization state and the light intensity of the first laser signal.
In some embodiments, the second imaging module comprises: the third dichroic mirror is coupled with the objective lens and is used for transmitting the fluorescent signal; the filtering unit is coupled with the third dichroic mirror and is used for filtering the fluorescent signal; the fourth lens is coupled with the filtering unit and is used for converging the fluorescent signal; an image sensor for receiving the converged fluorescent light signal to generate the second image signal.
In some embodiments, the second imaging module further comprises: the second slit is coupled with the third dichroic mirror and used for adjusting the size of the light spot of the fluorescent signal; a fifth lens coupled to the second slit, the fifth lens configured to converge the fluorescence signal to the filter unit.
In some embodiments, the filtering unit includes: the fourth dichroic mirror is coupled with the fifth lens and is used for carrying out color separation on the fluorescent signals to form at least one path of sub-fluorescent signals; the filter is coupled with the fourth dichroic mirror and used for filtering the sub-fluorescence signals to form filtering signals; and the fifth dichroic mirror is coupled with the filter plate and is used for combining the sub-fluorescence signals.
In a third aspect, the present application further provides an automatic focusing method applied to the automatic focusing system described in any one of the above embodiments, where the method includes:
acquiring a first image signal of the reflected light signal;
obtaining a target distance according to the first image signal;
and adjusting the original distance between the objective lens and the sample to be measured according to the target distance.
In some embodiments, prior to said acquiring the first image signal of the reflected light signal, the method further comprises:
adjusting the original distance between the objective lens and the sample to be detected according to a preset distance to obtain a corresponding sample image;
extracting information of the sample image to obtain image information;
inputting the image information to a preset original image model for distance prediction to obtain a training distance;
performing parameter adjustment on the original image model according to the training distance and the preset distance to obtain a target image model;
the obtaining of the target distance according to the first image signal includes:
extracting information of the first image signal to obtain a target image;
and inputting the target image into the target image model for distance prediction to obtain the target distance.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The invention is further described with reference to the following figures and examples, in which:
FIG. 1 is a schematic structural diagram of an auto-focusing system according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a microscope imaging system according to an embodiment of the present invention;
FIG. 3 is a schematic flow chart of an auto-focusing method according to an embodiment of the present invention;
FIG. 4 is a flowchart illustrating an auto-focusing method according to another embodiment of the present invention.
Reference numerals are as follows: the automatic focusing system 100, the first light source 110, the first dichroic mirror 120, the objective lens 130, the sample 300 to be measured, the first imaging module 140, the control module 150, the adjusting module 160, the first slit 170, the first lens 180, the second lens 190, the microscopic imaging system 200, the illumination module 210, the total reflection prism 220, the second imaging module 230, the second light source 211, the third light source 212, the second dichroic mirror 213, the optical path adjusting member 214, the third lens 215, the half-wave plate 216, the polarization splitting prism 217, the third dichroic mirror 231, the filtering unit 232, the fourth lens 233, the image sensor 234, the second slit 235, the fifth lens 236, the fourth dichroic mirror 237, the filtering plate 238, and the fifth dichroic mirror 239.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
In the description of the present invention, it should be understood that the orientation or positional relationship referred to in the description of the orientation, such as upper, lower, front, rear, left, right, etc., is based on the orientation or positional relationship shown in the drawings only for the convenience of description of the present invention and simplification of the description, and does 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 thus, should not be construed as limiting the present invention.
In the description of the present invention, the meaning of a plurality is one or more, the meaning of a plurality is two or more, and the above, below, exceeding, etc. are understood as excluding the present numbers, and the above, below, within, etc. are understood as including the present numbers. If the first and second are described for the purpose of distinguishing technical features, they are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
In the description of the present invention, unless otherwise explicitly limited, terms such as arrangement, installation, connection and the like should be understood in a broad sense, and those skilled in the art can reasonably determine the specific meanings of the above terms in the present invention in combination with the specific contents of the technical solutions.
In the description of the present invention, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
It should be noted that the laser signal can excite the fluorescent molecules in the sample to be detected to generate a corresponding fluorescent signal, and the microscopic imaging system acquires the fluorescent signal and generates an image signal according to the fluorescent signal, thereby realizing the detection of the sample to be detected. In the related art, when a wide-field fluorescence microscope (a microscopic imaging system) needs to image a sample to be detected with a certain thickness, a laser signal excites fluorescent molecules in the whole illumination area to generate a fluorescent signal, and at the moment, the fluorescent signals of different layers of the sample to be detected generate mutual interference, so that the longitudinal imaging resolution of the sample to be detected is reduced by the microscopic imaging system, and the quality of an image signal is seriously reduced. Therefore, in the related technology, the total internal reflection fluorescence imaging microscope forms an evanescent wave in the range of 100nm to 200nm on the other side of the sample through a laser signal, and can separately excite part of fluorescent molecules in a specific area, so that signal interference among different layers is avoided, background noise is reduced, the longitudinal imaging resolution of the microscopic imaging system is improved, and the resolution limit of a diffraction limit is broken. Specifically, the total internal reflection fluorescence imaging microscope can be divided into two imaging types, one is objective total internal reflection fluorescence imaging, and the other is prism total internal reflection fluorescence imaging. The prism type total internal reflection fluorescence imaging means that excitation and imaging are respectively completed by a prism and an objective lens, mutual interference between the prism and the objective lens is small, and control is relatively easy. However, the imaging method is more complicated in practical operation because the size and the placement of the sample to be measured are limited. Specifically, the angle of the laser signal incident on the prism needs to be readjusted each time the sample to be measured is replaced, and a large amount of time is required to perform objective focusing each time, so that the speed of the microscopic imaging system scanning the sample to be measured is slow.
Therefore, the application provides an automatic focusing system, a microscopic imaging system and an automatic focusing method, which can improve the imaging speed, ensure the imaging quality and have simple and practical operation.
Referring to fig. 1, in a first aspect, the present application provides an auto-focus system 100, the auto-focus system 100 is configured to automatically adjust an original distance between an objective lens 130 and a sample 300 to be measured, so that the objective lens 130 converges a first optical signal to the sample 300 to be measured, and the auto-focus system 100 includes: a first optical source 110, the first optical source 110 being configured to emit a first optical signal; the first dichroic mirror 120, the first dichroic mirror 120 is coupled to the first light source 110, and the first dichroic mirror 120 is configured to transmit the first optical signal; the objective lens 130, the objective lens 130 is coupled to the first dichroic mirror 120, and the objective lens 130 is configured to converge the first optical signal on the sample 300 to be measured; the sample 300 to be measured is used for reflecting the first optical signal to generate a reflected optical signal; the objective lens 130 is further configured to collect the reflected light signal and transmit the reflected light signal to the first dichroic mirror 120; the first dichroic mirror 120 is also used to reflect the reflected light signal; the first imaging module 140, the first imaging module 140 is coupled to the first dichroic mirror 120, and the first imaging module 140 is configured to receive the reflected light signal and generate a first image signal according to the reflected light signal; the control module 150, the control module 150 is connected to the first imaging module 140, and the control module 150 is configured to generate a target distance according to the first imaging module 140; and the adjusting module 160, wherein the adjusting module 160 is respectively connected to the control module 150 and the objective lens 130, and the adjusting module 160 is configured to adjust an original distance between the objective lens 130 and the sample 300 to be measured according to the target distance.
It can be understood that, when the sample 300 to be detected is located on the focal plane of the objective lens 130, the objective lens 130 can collect the fluorescence signal generated by the sample 300 to be detected and accurately focus the fluorescence signal to the corresponding imaging module (the second imaging module 230) to generate a clear second image signal, where the second image signal represents the detection result of the sample 300 to be detected. As can be seen from the above, when the prism-type total internal reflection fluorescence imaging method is used to excite the sample 300 to be detected, the laser signal must irradiate the sample 300 to be detected at a specific incident angle, so that the sample 300 to be detected is inconvenient to move after being replaced, otherwise, the incident angle of the laser signal needs to be readjusted, thereby affecting the detection efficiency. However, in practical applications, the sample 300 to be measured often needs to be replaced many times for detection, and after the sample 300 to be measured is replaced, the sample 300 to be measured may have an out-of-focus distance from the focal plane of the objective lens 130, which easily causes imaging blur. Therefore, in the embodiment of the present invention, the autofocus system 100 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 implementing autofocus.
Specifically, the auto-focusing system 100 includes a first light source 110, a first dichroic mirror 120, and an objective lens 130, which are coupled in sequence, wherein the first dichroic mirror 120 is further configured to be coupled with a first imaging module 140, and a control module 150 is respectively connected with the first imaging module 140 and the objective lens 130.
It is understood that the first light source 110 is used for generating a first light signal, and the first light signal may be an infrared laser signal with a wavelength of 830nm in the embodiment of the present invention. The first light source 110 emits a first optical signal to the first dichroic mirror 120. The first dichroic mirror 120 is capable of transmitting optical signals with specific wavelengths and reflecting optical signals with other wavelengths. In an embodiment of the present invention, the first dichroic mirror 120 is capable of transmitting the first optical signal to emit the first optical signal to the objective lens 130. The objective lens 130 is used for collecting the first optical signal and irradiating the first optical signal onto the sample 300 to be measured. The sample 300 to be measured can reflect the first optical signal to generate a reflected optical signal, the reflected optical signal can be collected by the objective 130 and transmitted to the first dichroic mirror 120, the first dichroic mirror 120 reflects the reflected optical signal to transmit the reflected optical signal to the first imaging module 140, and in some application scenarios, the first imaging module 140 may be a camera. It can be understood that when the original distance between the surface to be measured and the objective lens 130 is different, the reflected light signal collected by the objective lens 130 is imaged by the first imaging module 140 with different results, i.e. the first imaging module 140 generates different first image signals according to the reflected light signal. Therefore, the auto-focusing system 100 can determine the defocus distance between the objective lens 130 and the surface to be measured by processing the first image signal, thereby implementing auto-focusing.
Specifically, the auto-focusing apparatus according to the embodiment of the present invention is provided with a corresponding control module 150, and the control module 150 is configured to receive the first image signal generated by the first imaging module 140. The control module 150 is further configured to calculate a target distance (defocus distance) from the first image signal, and send the target distance to the adjustment module 160. The adjusting module 160 is configured to adjust a 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 maintained at the focal plane of the objective lens 130, thereby ensuring that the microscopic imaging system 200 can perform clear imaging.
In the embodiment of the present invention, the auto-focusing system 100 emits a first optical signal through the first light source 110, the first optical signal is collected through the first dichroic mirror 120 and the objective lens 130 and then irradiates on the sample 300 to be measured, and the sample 300 to be measured is configured to generate a reflected light signal according to the first optical signal. The reflected light signal is collected and converged by the objective lens 130, reflected by the first dichroic mirror 120 and transmitted to the first imaging module 140, the first imaging module 140 generates a corresponding first image signal with the reflected light signal, and transmits the first image signal to the control module 150 for calculation, and the control module 150 calculates the defocus distance between the objective lens 130 and the surface to be measured at that time according to the first image signal, generates a corresponding target distance, and transmits the target distance to the adjusting module 160. The adjusting module 160 can adjust the original 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 located at the focal plane of the objective lens 130, thereby ensuring clear imaging, and the imaging speed of scanning the sample 300 to be measured can be increased by the automatic focusing mode, and the automatic focusing system 100 of the embodiment of the invention is simple, convenient and practical.
Referring again to fig. 1, in some embodiments, the auto-focusing system 100 further includes: a first slit 170, the first slit 170 being coupled to the first light source 110, the first slit 170 being used for adjusting a spot size of the first light signal; the first lens 180, the first lens 180 is coupled to the first slit 170, and the first lens 180 is configured to perform collimation and convergence on the first optical signal; and a second lens 190, the second lens 190 being coupled to the first lens 180, the second lens 190 being used for collimating and converging the first optical signal.
It is understood that, in order to adjust the spot size of the first optical signal so that the first optical signal can be focused within the imaging range of the objective lens 130, the auto-focusing system 100 of the embodiment of the present invention is provided with the corresponding first slit 170. The first slit 170 is disposed in the light outgoing direction of the first light source 110, and when the first light source 110 emits the first optical signal to the first slit 170, the first slit 170 can adjust the size of a light spot of the first optical signal, preferably, the aspect ratio of the first slit 170 may be 3. In addition, the first slit 170 is also used for collimating the beam of the first optical signal.
It is understood that, in order to perform the collimating focusing on the first optical signal, so as to enable the first optical signal to be better focused on the objective lens 130, the auto-focusing system 100 according to the embodiment of the present invention further provides a corresponding first lens 180 and a corresponding second lens 190. The first lens 180 is disposed in the light emitting direction of the first slit 170, and the second lens 190 is disposed in the light emitting direction of the first lens 180. The first optical signal is adjusted by the first slit 170 and then transmitted to the first lens 180 and the second lens 190, and the first lens 180 and the second lens 190 can perform collimation and focusing on the first optical signal, so that the first optical signal is focused on the objective lens 130.
Referring to fig. 2, in a second aspect, the present application provides a microscopic imaging system 200, comprising: the autofocus system 100 of any of the embodiments described above; an illumination module 210, the illumination module 210 being configured to generate a first laser signal; the total reflection prism 220, the total reflection prism 220 is coupled with the illumination module 210, and the total reflection prism 220 is configured to receive the first laser signal and generate a second laser signal according to the first laser signal; the sample 300 to be detected generates total reflection according to the second laser signal and generates a corresponding fluorescent signal; a second imaging module 230, configured to receive the fluorescence signal collected by the objective lens 130, and generate a second image signal according to the fluorescence signal; the second image signal is used for representing the imaging result of the sample 300 to be measured.
It is understood that, as can be seen from the above, the microscopic imaging system 200 can be used for imaging and detecting the sample 300 to be detected. 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 the auto-focusing system 100.
Specifically, the illumination module 210 is configured to emit a first laser signal, the first laser signal enters the total reflection prism 220 at a specific first angle, the first laser signal passes through the total reflection prism 220 to generate a second laser signal emitted at a specific second angle, and the second laser signal can be completely reflected by the surface of the sample 300 to be detected, so as to form an evanescent field. Preferably, the first angle may be 30 degrees and the second angle may be 70 degrees in the embodiment of the present invention. The light intensity of the evanescent field decreases in an exponential function in a direction perpendicular to the sample 300 to be measured toward the other side of the incident surface, and an evanescent wave with a wavelength of 100nm to 200nm is formed, and the evanescent wave can excite the sample 300 to be measured in the range to generate a corresponding fluorescence signal. In addition, the objective lens 130 can collect fluorescence signals generated by the sample 300 to be measured and transmit the fluorescence signals to the second imaging module 230. The second imaging module 230 generates a second image signal according to the fluorescence signal, and the second image signal is used for characterizing an imaging result, i.e., a detection result, of the sample 300 to be detected.
It can be understood from the above description that when the sample 300 is replaced, the sample 300 may not be in the focal plane of the objective 130, i.e. the objective 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 130 and the sample 300 to be measured in real time through the auto-focusing system 100, so as to prevent the objective 130 from being out of focus, thereby enabling the imaging of the second imaging module 230 to be clear. In addition, during the scanning of the sample 300 to be measured by the microscopic imaging system 200, the automatic focusing system 100 can perform automatic focusing, thereby increasing the imaging speed of the scanning.
Referring again to fig. 2, in some embodiments, the lighting module 210 includes: a second light source 211, the second light source 211 being configured to generate a second light signal; a third light source 212, the third light source 212 being configured to generate a third light signal; the second dichroic mirror 213, the second dichroic mirror 213 is configured to combine the second optical signal and the third optical signal to generate a first laser signal; the optical path adjusting component 214, the optical path adjusting component 214 is coupled with the second dichroic mirror 213, and the optical path adjusting component 214 is configured to adjust an incident angle of the first laser signal, so that the first laser signal is incident to the total reflection prism 220 at a preset angle; and a third lens 215, wherein the third lens 215 is coupled to the optical path adjusting member 214, and the third lens 215 is used for converging the first laser signal to the total reflection prism 220.
It can be understood from the above description that the illumination module 210 is used to generate the first laser signal incident on the total reflection prism 220 at a specific first angle. Specifically, the illumination module 210 is provided therein with a corresponding second light source 211, a third light source 212, the optical path adjusting module 160, and a third lens 215. The second dichroic mirror 213 is coupled with the second light source 211 and the third light source 212, the optical path adjusting component 214 is coupled with the second dichroic mirror 213 and the fourth lens 233, and the fourth lens 233 is further configured to be coupled with the total reflection prism 220.
Specifically, the first light source 110 is configured to generate a second light signal, and the third light source 212 is configured to generate a third light signal, wherein preferably, the wavelength of the second light signal may be 532nm, and the wavelength of the third light signal may be 638nm. The second dichroic mirror 213 is configured to combine the second optical signal and the third optical signal into a first laser signal, and reflect the first laser signal to the optical path adjusting component 214. The optical path adjusting element 214 is configured to adjust an incident angle of the first laser signal, so that the first laser signal is incident to the total reflection prism 220 at a specific first angle, and total reflection is generated on the surface of the sample 300 to be measured. The third lens 215 is used to condense the adjusted first laser signal on the surface of the total reflection prism 220.
Referring again to fig. 2, in some embodiments, the lighting module 210 further includes: the half-wave plate 216 is coupled with the second dichroic mirror 213; the polarization beam splitter prism 217, the polarization beam splitter prism 217 is coupled with the half-wave plate 216; the half-wave plate 216 and the polarization beam splitter 217 are used for adjusting the polarization state and the light intensity of the first laser signal.
It will be appreciated that the half-wave plate 216 and the polarization beam splitter prism 217 are used to adjust the polarization state and the light intensity of the first laser signal to meet different requirements in practical applications.
Referring again to fig. 2, in some embodiments, the second imaging module 230 includes: the third dichroic mirror 231, the third dichroic mirror 231 is coupled to the objective lens 130, and the third dichroic mirror 231 is used for transmitting the fluorescence signal; the filtering unit 232, the filtering unit 232 is coupled to the third dichroic mirror 231, and the filtering unit 232 is configured to filter the fluorescent signal; a fourth lens 233, the fourth lens 233 is coupled to the filter unit 232, and the fourth lens 233 is configured to converge the fluorescence signal; an image sensor 234, the image sensor 234 being configured to receive the converged fluorescent light signal to generate a second image signal.
It can be understood from the above description that the third dichroic mirror 231 can reflect the first optical signal generated by the automatic focusing system 100 and the reflected optical signal generated by the sample 300 to be measured, 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 optical signals.
It is understood that the optical 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 adverse effects of the interference signal on the imaging of the image sensor 234, the embodiment of the present invention provides the corresponding filtering unit 232, and the filtering unit 232 can filter the incident fluorescence signal, so as to filter the interference signal and noise, and improve the imaging quality of the image sensor 234. The filtered fluorescence signal is emitted to the fourth lens 233, the fourth lens 233 is used for converging the fluorescence signal on the imaging surface of the image sensor 234, and the image sensor 234 generates a corresponding second image signal according to the fluorescence signal.
Referring again to fig. 2, in some embodiments, the second imaging module 230 further includes: a second slit 235, wherein the second slit 235 is coupled to the third dichroic mirror 231, and the second slit 235 is used for adjusting the size of the light spot of the fluorescent signal; and a fifth lens 236, wherein the fifth lens 236 is coupled to the second slit 235, and the fifth lens 236 is used for converging the fluorescence signal to the filtering unit 232.
It is understood that the second slit 235 can filter the incident fluorescence signal to prevent the second laser signal from entering, thereby improving the signal-to-noise ratio of the image sensor 234, and at the same time, the second slit 235 is also used for adjusting the spot size of the fluorescence signal. In addition, the second imaging module 230 is further provided with a fifth lens 236, and the fifth lens 236 is used for converging the fluorescence signal to the filtering unit 232.
Referring to fig. 2 again, in some embodiments, the filtering unit 232 includes: the fourth dichroic mirror 237, the fourth dichroic mirror 237 is coupled to the fifth lens 236, and the fourth dichroic mirror 237 is configured to color-separate the fluorescent signal to form at least one sub-fluorescent signal; at least one filter 238, the filter 238 is coupled to the fourth dichroic mirror 237, the filter 238 is configured to filter the sub-fluorescence signal to form a filtered signal; the fifth dichroic mirror 239, the fifth dichroic mirror 239 and the filter 238 are coupled, and the fifth dichroic mirror 239 is used for combining sub-fluorescence signals.
It will be appreciated that the fluorescence signal generated by the sample 300 to be tested has a plurality of wavelengths, since different fluorescent molecules can generate fluorescence signals of different wavelengths under the excitation of the second laser signal. In practical application, the fluorescent signals of specific one or more wavelengths can be collected and detected. For this purpose, the filtering unit 232 according to the embodiment of the present invention is provided with a corresponding fourth dichroic mirror 237, the fourth dichroic mirror 237 can transmit or reflect the fluorescence signal with a specific wavelength, so as to color-separate the fluorescence signal into different sub-fluorescence signals, and a corresponding filtering sheet 238 is disposed on the optical path of each sub-fluorescence to filter the interference signal. In order to perform imaging detection on multiple paths of sub-fluorescent signals through the single image sensor 234, the second imaging module 230 is further provided with a fifth dichroic mirror 239, and the fifth dichroic mirror 239 can combine the multiple paths of sub-fluorescent signals into one path of fluorescent signal and focus the fluorescent signal to the image sensor 234 through the fourth lens 233. Preferably, the image sensor 234 may be an EMCCD camera.
It can be understood that, in the embodiment of the present invention, a plurality of mirrors are further provided, and the mirrors can reflect different optical signals, so as to reduce the overall size of the microscopic imaging system 200 and improve the integration level.
Referring to fig. 3, in a third aspect, the present application further provides an auto-focusing method applied to the auto-focusing system of any one of the above embodiments, the method includes:
step S101, acquiring a first image signal of a reflected light signal;
step S102, obtaining a target distance according to the first image signal;
and S103, adjusting the original distance between the objective lens and the sample to be measured according to the target distance.
It can be understood from the above that, when the original distance between the surface to be measured and the objective lens is different, the result of imaging the reflected light signal collected by the objective lens at 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 is processed to obtain a corresponding target distance, the target distance is used for representing the defocusing distance between the objective lens and the surface to be measured, the objective lens is adjusted according to the target distance, the surface to be measured can be kept at the focal plane of the objective lens, and automatic focusing is achieved.
Referring to fig. 3 again, in some embodiments, before the step S101 of acquiring the first image signal of the reflected light signal, the method further includes:
step S201, adjusting the original distance between an objective lens and a sample to be detected according to a preset distance to obtain a corresponding sample image;
step S202, extracting information of a sample image to obtain image information;
step S203, inputting image information to a preset original image model for distance prediction to obtain a training distance;
step S204, adjusting parameters of the original image model according to the training distance and the preset distance to obtain a target image model;
obtaining a target distance from the first image signal, comprising:
s205, extracting information of the first image signal to obtain a target image;
and S206, inputting the target image into the target image model for distance prediction to obtain the target distance.
It is understood from the above that the sample image (first image signal) is related to the defocus distance of the objective lens. Therefore, in the embodiment of the invention, the corresponding target image model can be obtained by calculating the sample images at different preset distances.
Specifically, the control module receives corresponding sample images at different preset distances, performs Fourier transform processing on each sample image, and extracts a high-frequency information part to obtain a plurality of image information. The control module is further used for inputting the image information into a preset original image module for distance prediction to obtain a training distance, comparing the training distance with the preset distance, and performing parameter adjustment on the original image model according to a comparison result to finally obtain a target image model. It can be understood that the target image model obtained through the training process can predict the optimal target distance according to the first image signal, so as to control the objective lens to reach the optimal focal plane, thereby obtaining a clear imaging result.
It is understood that, when the sample image or the first image signal is processed, pre-processing, such as denoising, regularization, etc., may be performed first to improve the accuracy of the subsequent calculation.
In some application scenarios, when the adjusting module moves the objective lens according to the target distance, jitter may occur, thereby blurring the image. Therefore, in the embodiment of the present invention, the control module is further configured to set a moving threshold, and determine whether the objective lens shakes according to the moving threshold, and when shaking does exist, the adjusting module stops moving the objective lens, otherwise, the adjusting module continues moving the objective lens. In addition, the control module can also be provided with a quick reset function so as to control the objective lens to move to the initial position quickly.
The embodiments of the present invention have been described in detail with reference to the drawings, but the present invention is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (10)
1. An auto-focus system, the auto-focus system configured to automatically adjust an original distance between an objective lens and a sample to be measured, so that the objective lens converges a first optical signal to the sample to be measured, the auto-focus system comprising:
a first optical source for emitting the first optical signal;
the first dichroic mirror is coupled with the first light source and is used for transmitting the first optical signal;
the objective lens is coupled with the first dichroic mirror and is used for converging the first optical signal on a sample to be measured; the sample to be detected is used for reflecting the first optical signal to generate a reflected optical signal; the objective lens is also used for collecting the reflected light signal and transmitting the reflected light signal to the first dichroic mirror; the first dichroic mirror is further used for reflecting the reflected light signal;
the first imaging module is coupled with the first dichroic mirror and used for receiving the reflected light signal and generating a first image signal according to the reflected light signal;
the control module is connected with the first imaging module and is used for generating a target distance according to the first imaging module;
and the adjusting module is respectively connected with the control module and the objective lens and is used for adjusting the original distance between the objective lens and the sample to be measured according to the target distance.
2. The autofocus system of claim 1, further comprising:
a first slit coupled to the first light source, the first slit configured to adjust a spot size of the first optical signal;
a first lens coupled to the first slit, the first lens being configured to perform collimation and convergence on the first optical signal;
the second lens is coupled with the first lens and is used for collimating and converging the first optical signal.
3. A microscopic imaging system, comprising:
the autofocus system of any of claims 1 to 2;
an illumination module to generate a first laser signal;
the total reflection prism is coupled with the illumination module and used for receiving the first laser signal and generating a second laser signal according to the first laser signal; the sample to be detected generates total reflection according to the second laser signal and generates a corresponding fluorescent signal;
a second imaging module, the imaging module being configured to receive the fluorescence signal collected by the objective lens, the imaging module being further configured to generate a second image signal according to the fluorescence signal; and the second image signal is used for representing the imaging result of the sample to be detected.
4. The microscopy imaging system of claim 3, wherein the illumination module comprises:
a second light source for generating a second light signal;
a third light source to generate a third light signal;
a second dichroic mirror for combining the second optical signal and the third optical signal to generate a first laser signal;
the light path adjusting piece is coupled with the second dichroic mirror and used for adjusting the incident angle of the first laser signal so as to enable the first laser signal to be incident to the total reflection prism at a preset angle;
and the third lens is coupled with the optical path adjusting piece and is used for converging the first laser signal to the total reflection prism.
5. The microscopy imaging system of claim 4, wherein the illumination module further comprises:
the half-wave plate is coupled with the second dichroic mirror;
the polarization beam splitter prism is coupled with the half-wave plate;
the half-wave plate and the polarization beam splitter prism are used for adjusting the polarization state and the light intensity of the first laser signal.
6. The microscopy imaging system of claim 3, wherein the second imaging module comprises:
the third dichroic mirror is coupled with the objective lens and is used for transmitting the fluorescent signal;
the filtering unit is coupled with the third dichroic mirror and is used for filtering the fluorescent signal;
the fourth lens is coupled with the filtering unit and is used for converging the fluorescent signal;
an image sensor for receiving the converged fluorescent light signal to generate the second image signal.
7. The microscopy imaging system of claim 6, wherein the second imaging module further comprises:
the second slit is coupled with the third dichroic mirror and used for adjusting the size of the light spot of the fluorescent signal;
a fifth lens coupled to the second slit, the fifth lens configured to converge the fluorescence signal to the filter unit.
8. The microscopy imaging system of claim 7, wherein the filtering unit comprises:
the fourth dichroic mirror is coupled with the fifth lens and is used for carrying out color separation on the fluorescent signals so as to form at least one path of sub-fluorescent signals;
the filter plate is coupled with the fourth dichroic mirror and used for filtering the sub-fluorescence signals to form filtering signals;
and the fifth dichroic mirror is coupled with the filter plate and is used for combining the sub-fluorescence signals.
9. Auto-focusing method applied to the auto-focusing system according to any one of claims 1 to 2, characterized in that the method comprises:
acquiring a first image signal of the reflected light signal;
obtaining a target distance according to the first image signal;
and adjusting the original distance between the objective lens and the sample to be measured according to the target distance.
10. The auto-focusing method of claim 9, wherein prior to said acquiring the first image signal of the reflected light signal, the method further comprises:
adjusting the original distance between the objective lens and the sample to be detected according to a preset distance to obtain a corresponding sample image;
extracting information of 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 of the target distance according to the first image signal includes:
extracting information of the first image signal to obtain a target image;
and inputting the target image into the target image model for distance prediction to obtain the target distance.
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