Automatic focusing system, gene sequencing system and automatic focusing method

Abstract

The present invention discloses an autofocus system, a gene sequencing system and an autofocus method, comprising: a light source module, used to generate a first light source signal and a second light source signal; an objective lens, used to converge the first light source signal and the second light source signal on a biochip; wherein the surface to be measured is used to reflect the first light source signal and the second light source signal to form a first reflected light signal and a second reflected light signal; a detection module, used to receive the first reflected light signal and the second reflected light signal, and generate a first target electrical signal and a second target electrical signal according to the first reflected light signal and the second reflected light signal; a control module, used to obtain a target distance according to the first target electrical signal and the second target electrical signal; and an adjustment module, used to adjust the original distance between the objective lens and the surface to be measured according to the target distance, so that the objective lens converges the first light source signal and the second light source signal on the surface to be measured. The autofocus device of the present invention can realize autofocus.

Description

Technical Field

[0001] The present invention relates to the field of optical focusing technology, and in particular to an autofocus system, a gene sequencing system and an autofocus method. Background Art

[0002] At present, the objective lens can collect the light signals emitted by the surface of the biochip to be tested, and transmit them to the camera for imaging, and detect the sample to be tested in the biochip according to the imaging results. However, in the field of microscopic imaging technology, the objective lens has the characteristics of high resolution and small depth of field. Therefore, when the relative distance between the objective lens and the biochip changes at the micron level, defocusing may occur, resulting in blurred camera imaging.

[0003] For example, in practical applications, in order to image different areas of a biochip, the biochip needs to be controlled to move so that the objective lens can collect light signals emitted from different areas. However, since different areas of the biochip surface to be tested have micrometer-level height differences, when the height of the objective lens remains unchanged, as the biochip moves, the relative distance between the objective lens and the biochip changes, resulting in defocus, which makes the camera have poor imaging effect during scanning. Summary of the invention

[0004] 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 autofocus system, a gene sequencing system and an autofocus method, which can realize the autofocus of the objective lens and keep the imaging result in the best state.

[0005] In a first aspect, the present application provides an autofocus device, comprising: a light source module, the light source module is used to generate a first light source signal and a second light source signal; an objective lens, the objective lens is used to converge the first light source signal and the second light source signal on a biochip; wherein the surface to be tested of the biochip is used to reflect the converged first light source signal to form a first reflected light signal, and the surface to be tested is also used to reflect the converged second light source signal to form a second reflected light signal; the objective lens is also used to collect the first reflected light signal and the second reflected light signal; a detection module, the detection module is used to receive The first reflected light signal and the second reflected light signal are used to generate a first target electrical signal according to the first reflected light signal and a second target electrical signal according to the second reflected light signal; a control module, the control module is connected to the detection module, and the control module is used to obtain a target distance according to the first target electrical signal and the second target electrical signal; an adjustment module, the adjustment module is respectively connected to the control module and the objective lens, and the adjustment module is used to adjust the original distance between the objective lens and the surface to be measured according to the target distance, so that the objective lens converges the first light source signal and the second light source signal on the surface to be measured.

[0006] In some embodiments, the light source module includes: a first light source, the first light source is used to emit the collimated first light source signal; a second light source, the second light source is used to emit the collimated second light source signal; wherein the frequencies of the first light source signal and the second light source signal are different.

[0007] In some embodiments, the light source module also includes: a first dichroic mirror, which is used to combine the first light source signal and the second light source signal to generate a combined beam signal; a beam expander, which is arranged behind the first dichroic mirror along the optical axis of the combined beam signal, and is used to expand the combined beam signal; the surface to be tested of the biochip is also used to reflect the expanded combined beam signal to generate a reflected combined beam signal.

[0008] In some embodiments, the detection module includes: a second dichroic mirror, the second dichroic mirror is used to perform a beam splitting operation on the reflected combined beam signal collected by the objective lens to obtain the first reflected light signal and the second reflected light signal; a first converging mirror, the first converging mirror is placed behind the second dichroic mirror along the optical axis of the first reflected light signal, and the first converging mirror is used to generate a first focusing signal according to the first reflected light signal; a first pinhole, the first pinhole is arranged behind the first converging mirror along the optical axis of the first reflected light signal, and is used to perform spatial filtering on the first focusing signal; a first detector, the first detector is used to receive the first focusing signal after spatial filtering, and generate a first target electrical signal; a second converging mirror, the second converging mirror is placed behind the second dichroic mirror along the optical axis of the second reflected light signal, and the second converging mirror is used to generate a second focusing signal according to the second reflected light signal; a second pinhole, the second pinhole is arranged behind the second lens along the optical axis of the second reflected light signal, and the second pinhole is used to perform spatial filtering on the second focusing signal; a second detector, the second detector is used to receive the second focusing signal after spatial filtering, and generate a second target electrical signal.

[0009] In some embodiments, the autofocus device further includes: a beam splitter, which is arranged behind the beam expander along the optical axis of the combined beam signal; and a third dichroic mirror, which is arranged behind the beam splitter along the optical axis of the combined beam signal.

[0010] In a second aspect, the present application also provides a gene sequencing system, comprising: an autofocus device as described in any of the above embodiments; an illumination module, the illumination module being used to generate a laser signal; wherein the objective lens is arranged behind the illumination module along the optical axis of the laser signal; a biochip, the surface to be tested of the biochip being used to carry a biological sample; wherein the biological sample is used to generate a fluorescence signal according to the laser signal, and the biochip may include multiple surfaces to be tested; an imaging module, the imaging module being used to generate an image signal according to the fluorescence signal; and a translation stage, the translation stage being used to control the movement of the biochip.

[0011] In a third aspect, the present application also provides an autofocus method, which is applied to the autofocus device described in any one of the above embodiments, and the method includes: obtaining a first target electrical signal of the first reflected light signal, and a second target electrical signal of the second reflected light signal; obtaining a target intensity ratio signal based on the first target electrical signal and the second target electrical signal; obtaining a target distance based on the intensity ratio signal; and adjusting the original distance between the objective lens and the surface to be measured based on the target distance.

[0012] In some embodiments, before obtaining the first target electrical signal of the first reflected light signal, the method further includes: adjusting the original distance between the objective lens and the biochip according to N preset distances; wherein N is a positive integer; obtaining a first standard electrical signal and a second standard electrical signal corresponding to each preset distance, and obtaining a standard intensity ratio signal according to the first standard electrical signal and the second standard electrical signal; constructing an intensity ratio curve according to the N standard intensity ratio signals; and fitting the intensity ratio curve to obtain a fitting equation.

[0013] In some embodiments, obtaining the target distance according to the intensity ratio signal includes: obtaining the target distance according to the target intensity ratio signal and the fitting equation.

[0014] 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

[0015] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0016] Figure 1 is a structural schematic diagram of an automatic focusing device according to an embodiment of the present invention;

[0017] Figure 2 A schematic diagram of the structure of a biochip according to an embodiment of the present invention;

[0018] Figure 3 is a light intensity variation curve diagram of the automatic focusing device according to an embodiment of the present invention;

[0019] Figure 4 is a ratio curve diagram of the automatic focusing device according to an embodiment of the present invention;

[0020] Figure 5 is an intensity ratio curve diagram of the auto-focusing device according to an embodiment of the present invention;

[0021] Figure 6 is another intensity ratio curve diagram of the auto-focusing device according to an embodiment of the present invention;

[0022] Figure 7 is another intensity ratio curve diagram of the auto-focusing device according to an embodiment of the present invention;

[0023] Figure 8 is a structural schematic diagram of a light source module according to an embodiment of the present invention;

[0024] Fig. 9 A schematic diagram of the structure of a detection module according to an embodiment of the present invention;

[0025] Fig.10is a structural schematic diagram of a lighting module according to an embodiment of the present invention;

[0026] Fig.11 is a structural schematic diagram of an imaging module according to an embodiment of the present invention;

[0027] Fig.12 A schematic diagram of a process of an automatic focusing method according to an embodiment of the present invention;

[0028] Fig.13 FIG. 4 is another flowchart of the automatic focusing method according to an embodiment of the present invention.

[0029] Figure numerals: autofocus device 100, light source module 110, objective lens 120, detection module 130, control module 140, adjustment module 150, first light source 111, second light source 112, first dichroic mirror 113, beam expander 114, second dichroic mirror 131, first pinhole 132, first detector 133, second pinhole 134, second detector 135, first converging mirror 136, second converging mirror 137, beam splitter 160, third dichroic mirror 170, gene sequencing system 200, illumination module 210, biochip 220, imaging module 230, fourth dichroic mirror 240, translation stage 300. DETAILED DESCRIPTION

[0030] Embodiments of the present invention are described in detail below, and examples of the embodiments 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.

[0031] 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.

[0032] 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.

[0033] 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 in combination with the specific content of the technical solution.

[0034] 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.

[0035] It should be noted that in order to enable the gene sequencing system to perform imaging detection on different positions of the biochip, the position of the biochip can be moved by the displacement stage. During the movement of the biochip, the camera (imaging module) of the gene sequencing system images different positions of the biochip, thereby realizing scanning and imaging of the entire biochip. In practical applications, different positions of the surface to be tested on the biochip have a height difference of micrometer level. Therefore, when the displacement stage moves the position of the biochip, the relative distance between the objective lens of the gene sequencing system and the surface to be tested will change at the micrometer level. Since the microscopic imaging system has the characteristics of high resolution and small depth of field, during the movement of the above-mentioned biochip, the position of the objective lens remains unchanged, and the relative distance between the surface to be tested and the objective lens changes at the micrometer level, that is, defocusing may occur, resulting in blurred imaging of the gene sequencing system.

[0036] In some application scenarios, the relative distance between the objective lens and the surface to be tested can be adjusted by the autofocus technology, so that the surface to be tested is kept on the focal plane of the objective lens, so that the imaging result during the scanning process is kept in the best state, and the best state represents that the imaging result obtained by the gene sequencing system at this time is the clearest. Specifically, in the related art, the relative distance between the surface to be tested of the biochip and the objective lens changes, that is, when defocusing occurs, the pixel position of the strongest point of the reflected light spot on the surface to be tested on the CCD or CMOS image will also change. Therefore, by detecting the position signal of the reflected light spot on the surface to be tested, the defocus distance between the objective lens and the surface to be tested can be determined at this time. The applicant has found in practice that in the process of gene sequencing, the camera of the gene sequencing system needs to take tens of thousands to hundreds of thousands of images, and needs to focus tens of thousands to hundreds of thousands of times. However, in the above-mentioned related art, CCD or CMOS, as a multi-point detector, needs to perform a large amount of calculation when collecting the position information of multiple pixels, and the response time is long, so it is easy to cause a high delay in the adjustment of the objective lens during the focus tracking (focusing) process, thereby reducing the speed of the focus tracking response.

[0037] In addition, in some application scenarios, the biochip is a multi-surface chip with a flow channel (for example, a biochip with a cover glass). Since the reflectivity of different surfaces of the biochip to light is different, the reflectivity of the upper surface of the cover glass is more than ten times stronger than the reflectivity of the surface to be tested in the flow channel. Therefore, when the microscopic imaging system needs to detect the surface to be tested in the flow channel, the reflected light spot detected by the camera on the surface to be tested will be submerged in the reflected light spot on the upper surface of the cover glass, and it is impossible to achieve tracking focus and imaging detection of the surface to be tested.

[0038] To this end, the present application provides an automatic focusing device that can detect and adjust the relative distance between the objective lens and the surface to be measured in real time, so that the surface to be measured remains at the focal plane of the objective lens, thereby achieving automatic focusing of the objective lens and keeping the imaging result in the best state.

[0039] See also Figure 1 In a first aspect, the present application provides an autofocus device 100, comprising: a light source module 110, the light source module 110 is used to generate a first light source signal and a second light source signal; an objective lens 120, the objective lens 120 is used to converge the first light source signal and the second light source signal on a biochip 220; wherein the surface to be tested of the biochip 220 is used to reflect the converged first light source signal to form a first reflected light signal, and the surface to be tested is also used to reflect the converged second light source signal to form a second reflected light signal; the objective lens 120 is also used to collect the first reflected light signal and the second reflected light signal; a detection module 130, the detection module 130 is used to receive the first reflected light signal and the second reflected light signal, and to generate a first target electrical signal according to the first reflected light signal, and to generate a second target electrical signal according to the second reflected light signal; a control module 140, which is connected to the detection module 130, and is used to obtain a target distance according to the first target electrical signal and the second target electrical signal; an adjustment module 150, which is respectively connected to the control module 140 and the objective lens 120, and is used to adjust the original distance between the objective lens 120 and the surface to be measured according to the target distance, so that the objective lens 120 converges the first light source signal and the second light source signal on the surface to be measured.

[0040] It can be understood that the autofocus device 100 in the embodiment of the present invention includes a light source module 110, a detection module 130, an objective lens 120, an adjustment module 150 and a control module 140. Among them, the light source module 110 is used to emit a first light source signal and a second light source signal, and the objective lens 120 and the biochip 220 are arranged in sequence along the emission direction of the first light source signal or the second light source signal. The surface to be measured of the biochip 220 reflects the first light source signal to generate a first reflected light signal, and reflects the second light source signal to generate a second reflected light signal. The first reflected light signal and the second reflected light signal are collected by the objective lens 120 and transmitted to the detection module 130. The detection module 130 is connected to the control module 140, and the adjustment module 150 is connected to the control module 140 and the objective lens 120 respectively.

[0041] It can be understood that, according to the principle of dispersion, the positions of the light focal points of light signals of different wavelengths after passing through the objective lens 120 are different. Therefore, by detecting the positions of the light focal points of light signals of different wavelengths, the focal plane position of the objective lens 120 at this time can be determined, thereby adjusting the original distance between the objective lens 120 and the surface to be measured so that the surface to be measured is located at the focal plane of the objective lens 120. To this end, the autofocus device 100 of the embodiment of the present invention is provided with a light source module 110. As can be seen from the above content, the light source module 110 is used to generate a first light source signal and a second light source signal, wherein the frequencies (wavelengths) of the first light source signal and the second light source signal are different. The first light source signal and the second light source signal are reflected by the beam splitter 160, and then reflected by the third dichroic mirror 170 and transmitted to the objective lens 120. The objective lens 120 collects the first light source signal and the second light source signal and converges them on the biochip 220. At this time, the surface to be measured of the biochip 220 reflects the incident first light source signal to form a first reflected light signal, and reflects the second light source signal to form a second reflected light signal. The frequency of the first reflected light signal is the same as the first light source signal, and the frequency of the second reflected light signal is the same as the second light source signal. The first reflected light signal and the second reflected light signal are collected by the objective lens 120, reflected by the third dichroic mirror 170, and transmitted to the detection module 130 after being transmitted by the spectroscope 160. The detection module 130 receives the first reflected light signal and the second reflected light signal, and generates a first target electrical signal and a second target electrical signal. The first target electrical signal represents the light intensity of the first reflected light signal, and the second target electrical signal represents the light intensity of the second reflected light signal. It can be seen from the above content that the positions of the light focal points of light signals of different frequencies are different after being focused by the objective lens 120, that is, the positions of the light focal points of the first reflected light signal and the second reflected light signal are different after being collected by the objective lens 120. Therefore, the light intensity of the first reflected light signal and the light intensity of the second reflected light signal received by the detection module 130 varies with the distance between the objective lens 120 and the biochip 220, and the autofocus device 100 can judge the defocus distance at this time by the light intensity of the first reflected light signal and the light intensity of the second reflected light signal.

[0042] The embodiment of the present invention is described by taking a biochip 220 having multiple test surfaces as an example. Figure 2As shown, the thickness of the cover glass of the biochip 220 is 170um, and the thickness of the flow channel is 80um. Among them, the multiple surfaces to be tested are the upper surface of the cover glass, the upper surface of the flow channel and the lower surface of the flow channel. When the detection reagent is injected into the flow channel of the biochip 220, the biological sample can be attached to the upper surface of the flow channel and the lower surface of the flow channel. It is understandable that the gene sequencing system 200 can perform imaging detection on the biological sample on the upper surface of the flow channel. When the upper surface of the flow channel is located on the focal plane of the objective lens 120, the imaging effect of the light signal collected by the objective lens 120 is the best. When the upper surface of the flow channel is out of focus, the imaging result is blurred. To this end, the autofocus device 100 of the embodiment of the present invention can determine the defocus distance of the objective lens 120 at this time by detecting the light intensity of the first reflected light signal and the second reflected light signal reflected on the upper surface of the flow channel, thereby adjusting the original distance between the objective lens 120 and the upper surface of the flow channel so that the upper surface of the flow channel is on the focal plane of the objective lens 120. Secondly, the biological sample on the lower surface of the flow channel can also be imaged. The specific imaging method is similar to the method for imaging the biological sample on the upper surface of the flow channel, and will not be described in detail in the embodiments of the present application.

[0043] Specifically, the first light source signal and the second light source signal are collected by the objective lens 120 and irradiated on the upper surface of the flow channel, and are reflected to form a first reflected light signal and a second reflected light signal. The detection module 130 receives the first reflected light signal and the second reflected light signal, and generates a first target electrical signal according to the light intensity of the first reflected light signal, and generates a second target electrical signal according to the light intensity of the second reflected light signal. The detection module 130 transmits the first target electrical signal and the second target electrical signal to the control module 140, and the control module 140 establishes a first target electrical signal and a second target electrical signal according to the first target electrical signal and the second target electrical signal. Figure 3 The light intensity variation curve shown in FIG. Figure 3 As shown, the horizontal axis is the defocus distance (target distance) between the objective lens 120 and the surface to be measured (upper surface of the flow channel) of the biochip 220, and the vertical axis is the light intensity of the first reflected light signal and the second reflected light signal, that is, the value of the first detection electrical signal and the second target electrical signal. Figure 3 The solid line is the light intensity variation curve of the first reflected light signal. Figure 3 The dotted line is the light intensity variation curve of the second reflected light signal. Figure 3There are three groups of light intensity variation curve intervals with approximate Gaussian distribution, wherein the horizontal axis represents the positions of the three surfaces to be measured of the biochip 220, namely, the upper surface of the coverslip, the upper surface of the flow channel, and the lower surface of the flow channel. The Gaussian distribution light intensity variation curve represents the light intensity variation near the above three surfaces to be measured. Among them, the peak value of the first reflected light signal light intensity variation curve and the peak value of the second reflected light signal light intensity variation curve are respectively distributed on both sides of the focal planes of the three surfaces to be measured, and the distance between the peak values ​​is greater than 8um. It can be understood that the range between the two peak values ​​corresponding to a focal plane is the actual working range of the autofocus device 100. In addition, the control module 140 is also used to determine the working range of the autofocus device 100 according to the above. Figure 3 The light intensity variation curve is generated as follows Figure 4 Specifically, the control module 140 will Figure 3 By dividing the values ​​corresponding to the two light intensity change curves at the same defocus distance to obtain multiple intensity ratio signals, we can obtain Figure 4 Further, the control module 140 is also used to Figure 4 The value of the ratio curve is taken in a specific interval, so as to obtain Figure 5 , Figure 6 , Figure 7 The intensity ratio curve of monotonically changing is shown in FIG. Figure 5 Characterizes the relationship between the defocus distance of the objective lens 120 and the intensity ratio signal when the lower surface of the flow channel is used as the surface to be tested. Figure 6 Characterizes the relationship between the defocus distance of the objective lens 120 and the intensity ratio signal when the upper surface of the flow channel is used as the surface to be tested. Figure 7 The relationship between the defocus distance of the objective lens 120 and the intensity ratio signal when the upper surface of the cover glass is used as the surface to be tested is characterized.

[0044] Specifically, the control module 140 performs Figure 5 By fitting the data points obtained by taking values, we can get Figure 5 The fitting curve in the figure has a curve equation of y=-57.934-1.407*ln(x+0.174), where x is the intensity ratio. According to the curve equation, the defocus distance y between the lower surface of the flow channel and the objective lens 120 can be solved. To evaluate the goodness of fit of the curve equation. Specifically, Figure 5 R of the fitted curve 2 =0.9994, indicating that the curve has a high degree of fitting.

[0045] Specifically, the control module 140 performs Figure 6 By fitting the data points obtained by taking values, we can get Figure 6The fitting curve in the figure has a curve equation of y=3.279-1.71*ln(x+0.547), where x is the intensity ratio. According to the curve equation, the defocus distance y between the lower surface of the flow channel and the objective lens 120 can be solved. To evaluate the goodness of fit of the curve equation. Specifically, Figure 6 R of the fitted curve 2 =0.9996, indicating that the curve has a high degree of fitting.

[0046] Specifically, the control module 140 performs Figure 7 By fitting the data points obtained by taking values, we can get Figure 7 The fitting curve in the figure has a curve equation of y=112.382-1.624*ln(x+0.266), where x is the intensity ratio. According to the curve equation, the defocus distance y between the lower surface of the flow channel and the objective lens 120 can be solved. To evaluate the goodness of fit of the curve equation. Specifically, Figure 7 R of the fitted curve 2 =0.9986, indicating that the curve has a high degree of fitting.

[0047] It can be understood that, from the above content, the control module 140 can establish the relationship between the intensity ratio signal and the defocus distance by constructing the above curve equation. In practical applications, the detection module 130 generates the corresponding first target electrical signal and the second target electrical signal according to the first reflected light signal and the second reflected light signal, and transmits them to the control module 140. The control module 140 generates the corresponding intensity ratio signal according to the first target electrical signal and the second target electrical signal, and determines the corresponding target distance according to the intensity ratio signal and the curve equation. The control module 140 is also used to send the target distance to the adjustment module 150, and the adjustment module 150 adjusts the original distance between the objective lens 120 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 120. At this time, the first light source signal and the second light source signal can converge on the surface to be measured.

[0048] In this embodiment, the auto-focusing device 100 generates a first light source signal and a second light source signal through the light source module 110. The first light source signal and the second light source signal are collected by the objective lens 120 and irradiated on the surface to be measured. The surface to be measured reflects to generate a first reflected light signal and a second reflected light signal. The detection module 130 generates a corresponding first target electrical signal and a second target electrical signal according to the first reflected light signal and the second reflected light signal and transmits them to the control module 140. The control module 140 can generate a corresponding target distance according to the first target electrical signal and the second target electrical signal. The adjustment module 150 adjusts the original distance between the objective lens 120 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 120, that is, the first light source signal and the second light source signal can converge on the surface to be measured. The auto-focusing device 100 of this embodiment can detect and adjust the original distance between the objective lens 120 and the surface to be measured in real time, so that the surface to be measured is kept at the focal plane of the objective lens 120, thereby realizing automatic focusing of the objective lens 120 and keeping the imaging result in the best state.

[0049] See also Figure 8 In some embodiments, the light source module 110 includes: a first light source 111, the first light source 111 is used to emit a collimated first light source signal; a second light source 112, the second light source 112 is used to emit a collimated second light source signal; wherein the frequencies of the first light source signal and the second light source signal are different.

[0050] It can be understood that, from the above content, the frequencies of the first light source signal and the second light source signal are different. Therefore, the autofocus device 100 in the embodiment of the present invention is provided with a corresponding first light source 111 and a second light source 112, so as to generate two light source signals of different frequencies. In practical applications, the gene sequencing system 200 uses a laser signal of a certain wavelength band to excite the biological sample to generate a corresponding fluorescence signal. In order to avoid affecting the fluorescence signal, the first light source 111 and the second light source 112 in the autofocus device 100 can be set to a light signal with a wavelength different from that of the laser signal and the fluorescence signal. Specifically, in the embodiment of the present invention, the wavelength of the first light source signal emitted by the first light source 111 can be 405nm, and the wavelength of the second light source signal emitted by the second light source 112 can be 980nm. In addition, corresponding collimators are also provided in the first light source 111 and the second light source 112, and the collimators are used to collimate the first light source signal or the second light source signal so that the first light source signal and the second light source signal are collimated beams when emitted.

[0051] Please refer again Figure 8In some embodiments, the light source module 110 further includes: a first dichroic mirror 113, the first dichroic mirror 113 is used to combine the first light source signal and the second light source signal to generate a combined beam signal; a beam expander 114, the beam expander 114 is arranged behind the first dichroic mirror 113 along the optical axis of the combined beam signal, and the beam expander 114 is used to expand the combined beam signal; the surface to be tested of the biochip 220 is also used to reflect the expanded combined beam signal to generate a reflected combined beam signal.

[0052] It is understandable that in some application scenarios, the light source module 110 can integrate the first light source signal and the second light source signal into a light beam with the same directivity and emit it. To this end, in the embodiment of the present invention, the light source module 110 is provided with a corresponding first dichroic mirror 113, and the first dichroic mirror 113 is used to transmit or reflect the first light source signal, and to reflect or transmit the second light source signal, so that the first light source signal and the second light source signal are integrated into a combined beam signal.

[0053] It is understandable that in order to allow the combined beam signal to fill the objective lens 120, the autofocus device 100 in the embodiment of the present invention is provided with a corresponding beam expander 114, which can expand the combined beam signal to make the depth of field of the objective lens 120 smaller and the sensitivity of the autofocus device 100 higher. The combined beam signal after expansion is irradiated on the surface to be measured of the biochip 220, and is reflected by the surface to be measured to generate a corresponding reflected combined beam signal. The detection module 130 can receive the reflected combined beam signal and generate a corresponding target distance according to the above technical content, and the adjustment module 150 adjusts the original distance between the objective lens 120 and the surface to be measured according to the target distance.

[0054] See also Fig. 9In some embodiments, the detection module 130 includes: a second dichroic mirror 131, the second dichroic mirror 131 is used to perform a beam splitting operation on the reflected combined beam signal collected by the objective lens 120 to obtain a first reflected light signal and a second reflected light signal; a first converging mirror 136, the first converging mirror 136 is placed behind the second dichroic mirror 131 along the optical axis of the first reflected light signal, and the first converging mirror 136 is used to generate a first focusing signal according to the first reflected light signal; a first pinhole 132, the first pinhole 132 is arranged behind the first converging mirror 136 along the optical axis of the first reflected light signal, and is used to perform spatial filtering on the first focusing signal; a first detection The first detector 133 is used to receive the first focusing signal after spatial filtering and generate a first target electrical signal; the second converging mirror 137 is placed behind the second dichroic mirror 131 along the optical axis of the second reflected light signal, and the second converging mirror 137 is used to generate a second focusing signal according to the second reflected light signal; the second pinhole 134 is set behind the second lens along the optical axis of the second reflected light signal, and the second pinhole is used to perform spatial filtering on the second focusing signal; the second detector 135 is used to receive the second focusing signal after spatial filtering and generate a second target electrical signal.

[0055] It can be understood that after the reflected combined beam signal is reflected by the third dichroic mirror 170, it is incident on the detection module 130 through the beam splitter 160. In the embodiment of the present invention, the detection module 130 is provided with a corresponding second dichroic mirror 131, and the second dichroic mirror 131 can split the reflected combined beam signal to obtain a first reflected light signal and a second reflected light signal. Specifically, the first reflected light signal passes through the first converging mirror 136 to obtain a first focused signal, and the first focused signal passes through the first pinhole 132 and is spatially filtered before being received by the first detector 133, and the first detector 133 generates a first target electrical signal according to the light intensity of the first focused signal. The second reflected light signal passes through the second converging mirror 137 to obtain a second focused signal, and the second focused signal passes through the second pinhole 134 and is spatially filtered before being received by the second detector 135, and the second detector 135 generates a second target electrical signal according to the light intensity of the second focused signal. The control module 140 generates a corresponding target distance according to the first target electrical signal and the second target electrical signal, and the adjustment module 150 adjusts the original distance between the objective lens 120 and the surface to be measured according to the target distance.

[0056] Please refer again Figure 1 In some embodiments, the autofocus device 100 further includes: a beam splitter 160, which is disposed behind the beam expander along the optical axis of the combined beam signal; and a third dichroic mirror 170, which is disposed behind the beam splitter 160 along the optical axis of the combined beam signal.

[0057] It can be understood that the third dichroic mirror 170 is used to reflect the first light source signal, the second light source signal and the combined beam signal so as to transmit them to the objective lens 120 for convergence. The third dichroic mirror 170 is also used to reflect the first reflected light signal, the second reflected light signal and the reflected combined beam signal to the beam splitter 160. The beam splitter 160 is a half-reflecting half-mirror. When the first reflected light signal, the second reflected light signal and the reflected combined beam signal are incident, the reflection and transmission intensity of the beam splitter 160 is 1:1, and the transmitted light signal is transmitted to the detection module 130 for collection.

[0058] See also Figures 9 to 11 In a second aspect, the present application further provides a gene sequencing system 200, comprising: an autofocus device 100 as in any of the above embodiments; an illumination module 210, the illumination module 210 is used to generate a laser signal; wherein the objective lens 120 is arranged behind the illumination module 210 along the optical axis of the laser signal; a biochip 220, the surface to be tested of the biochip 220 is used to carry a biological sample, and the biological sample is used to generate a fluorescence signal according to the laser signal; wherein the biochip 220 may include a plurality of surfaces to be tested; an imaging module 230, the imaging module 230 is used to generate an image signal according to the fluorescence signal; and a displacement stage 300, the displacement stage 300 is used to control the movement of the biochip 220.

[0059] It is understandable that the gene sequencing system 200 is used to sequence the DNA in the biological sample. Specifically, the lighting module 210 is used to generate a laser signal, wherein the wavelength of the laser signal can be a single wavelength, a dual wavelength or a band. The laser signal is used to be transmitted through the fourth dichroic mirror 240 and converged by the objective lens 120 to irradiate the surface to be tested, so as to excite the biological sample on the surface to be tested to generate a corresponding fluorescence signal. In order to obtain a better excitation effect, the lighting module 210 in the embodiment of the present invention can be provided with a corresponding beam shaping unit, a homogenizing unit, a filtering unit, etc.

[0060] It is understandable that in order to sequence the DNA in the biological sample, the gene sequencing system 200 is also provided with a corresponding imaging module 230. Specifically, the fluorescence signal is collected by the objective lens 120, and after passing through the third dichroic mirror 170, it is reflected to the imaging module 230 by the fourth dichroic mirror 240. The imaging module 230 can be provided with a corresponding color filter, a focusing lens and a camera, so as to extract the fluorescence signal of the required wavelength band for imaging and generate a corresponding image signal.

[0061] It can be seen that the contents of the above-mentioned autofocus device embodiments are all applicable to the embodiments of the present gene sequencing system. The functions specifically implemented by the embodiments of the present gene sequencing system are the same as those of the above-mentioned autofocus device embodiments, and the beneficial effects achieved are also the same as those achieved by the above-mentioned autofocus device embodiments.

[0062] See also Fig.12 In a third aspect, the present application further provides an autofocus method, which is applied to the autofocus device of any of the above embodiments, and the method comprises:

[0063] Step S101, acquiring a first target electrical signal of a first focus signal and a second target electrical signal of a second focus signal;

[0064] Step S102, obtaining a target intensity ratio signal according to the first target electrical signal and the second target electrical signal;

[0065] Step S103, obtaining the target distance according to the intensity ratio signal;

[0066] Step S104: adjusting the original distance between the objective lens and the surface to be measured according to the target distance.

[0067] It can be understood that from the above content, by obtaining the target intensity ratio signal of the first target electrical signal and the second target electrical signal, and based on the fitting curve equation, the defocus distance between the objective lens and the surface to be measured at this time can be determined, thereby determining the target distance, and adjusting 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 always on the focal plane of the objective lens.

[0068] See also Fig.13 In some embodiments, before step S101, the auto-focus method further includes:

[0069] Step S201, adjusting the distance between the objective lens and the biochip according to N preset distances;

[0070] Step S202: acquiring a first standard electrical signal and a second standard electrical signal corresponding to each preset distance, and obtaining a standard intensity ratio signal according to the first standard electrical signal and the second standard electrical signal;

[0071] Step S203, constructing an intensity ratio curve according to N standard intensity ratio signals;

[0072] Step S204: Fitting the intensity ratio curve to obtain a fitting equation.

[0073] See also Fig.13 In some embodiments, step S103 includes sub-steps:

[0074] The target distance is obtained according to the target intensity ratio signal and the fitting equation.

[0075] It can be understood that, from the above content, the autofocus device can first construct a corresponding intensity ratio curve according to a plurality of preset distances, and obtain a fitting equation by fitting the intensity ratio curve. In practical applications, when there is a defocus distance between the objective lens and the surface to be measured, the autofocus device obtains an intensity ratio signal according to the first target electrical signal and the second target electrical signal, and calculates the target distance at this time, i.e., the defocus distance, according to the fitting equation, so that the adjustment module can adjust the original distance between the objective lens and the surface to be measured according to the target distance, and finally achieve autofocus.

[0076] It can be seen that the contents of the above-mentioned automatic focusing device embodiments are all applicable to the embodiments of the present automatic focusing method, the functions specifically implemented by the embodiments of the present automatic focusing method are the same as those of the above-mentioned automatic focusing device embodiments, and the beneficial effects achieved are also the same as those achieved by the above-mentioned automatic focusing device embodiments.

[0077] 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 device, characterized in that: include: A light source module, wherein the light source module is used to generate a first light source signal and a second light source signal; An objective lens, the objective lens is used to converge the first light source signal and the second light source signal on the biochip; wherein the surface to be tested of the biochip is used to reflect the converged first light source signal to form a first reflected light signal, and the surface to be tested is also used to reflect the converged second light source signal to form a second reflected light signal; the objective lens is also used to collect the first reflected light signal and the second reflected light signal; a detection module, the detection module being used to receive the first reflected light signal and the second reflected light signal, and to generate a first target electrical signal according to the first reflected light signal, and to generate a second target electrical signal according to the second reflected light signal; A control module, the control module is connected to the detection module, the control module is used to obtain a target intensity ratio signal according to the first target electrical signal and the second target electrical signal, and calculate the target distance according to the target intensity ratio signal and a fitting equation; 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 surface to be measured according to the target distance, so that the objective lens converges the first light source signal and the second light source signal on the surface to be measured.

2. The automatic focusing device according to claim 1, characterized in that: The light source module comprises: a first light source, the first light source being used to emit a collimated first light source signal; A second light source, the second light source is used to emit a collimated second light source signal; wherein the frequencies of the first light source signal and the second light source signal are different.

3. The automatic focusing device according to claim 2, characterized in that: The light source module further includes: a first dichroic mirror, wherein the first dichroic mirror is used to combine the first light source signal and the second light source signal to generate a combined beam signal; A beam expander is arranged behind the first dichroic mirror along the optical axis of the combined beam signal, and is used to expand the combined beam signal; the surface to be tested of the biochip is also used to reflect the expanded combined beam signal to generate a reflected combined beam signal.

4. The automatic focusing device according to claim 3, characterized in that: The detection module comprises: a second dichroic mirror, the second dichroic mirror being used for performing a beam splitting operation on the reflected combined beam signal collected by the objective lens to obtain the first reflected light signal and the second reflected light signal; a first converging mirror, the first converging mirror being placed behind the second dichroic mirror along the optical axis of the first reflected light signal, the first converging mirror being used to generate a first focusing signal according to the first reflected light signal; a first pinhole, the first pinhole being arranged behind the first converging mirror along the optical axis of the first reflected light signal and being used for spatially filtering the first focused signal; a first detector, the first detector being used to receive the first focused signal after spatial filtering and generate a first target electrical signal; a second converging mirror, the second converging mirror being disposed behind the second dichroic mirror along the optical axis of the second reflected light signal, the second converging mirror being used to generate a second focusing signal according to the second reflected light signal; a second pinhole, the second pinhole being arranged behind the second converging mirror along the optical axis of the second reflected light signal, the second pinhole being used for spatial filtering the second focused signal; A second detector is used to receive the second focused signal after spatial filtering and generate a second target electrical signal.

5. The automatic focusing device according to any one of claims 3 to 4, characterized in that: Also includes: A beam splitter, the beam splitter is arranged behind the beam expander along the optical axis of the combined beam signal; A third dichroic mirror is arranged behind the beam splitter along the optical axis of the combined beam signal.

6. A gene sequencing system, characterized in that: include: The automatic focusing device according to any one of claims 1 to 5; An illumination module, the illumination module being used to generate a laser signal; wherein the objective lens is arranged behind the illumination module along the optical axis of the laser signal; A biochip, wherein the surface to be tested of the biochip is used to carry a biological sample; wherein the biological sample is used to generate a fluorescent signal according to the laser signal, and the biochip comprises a plurality of surfaces to be tested; An imaging module, wherein the imaging module is used to generate an image signal according to the fluorescent signal; A translation stage is used to control the movement of the biochip.

7. An autofocus method, applied to the autofocus device as claimed in claim 4, characterized in that: The method comprises: Acquire a first target electrical signal of the first focus signal and a second target electrical signal of the second focus signal; Obtaining a target intensity ratio signal according to the first target electrical signal and the second target electrical signal; Obtaining a target distance according to the intensity ratio signal; The original distance between the objective lens and the surface to be measured is adjusted according to the target distance.

8. The automatic focusing method according to claim 7, characterized in that: Before acquiring the first target electrical signal of the first focusing signal, the method further includes: Adjusting the original distance between the objective lens and the biochip according to N preset distances; wherein N is a positive integer; Acquire a first standard electrical signal and a second standard electrical signal corresponding to each preset distance, and obtain a standard intensity ratio signal according to the first standard electrical signal and the second standard electrical signal; Constructing an intensity ratio curve according to the N standard intensity ratio signals; The intensity ratio curve is fitted to obtain a fitting equation.

9. The automatic focusing method according to claim 8, characterized in that: The step of obtaining the target distance according to the intensity ratio signal comprises: The target distance is obtained according to the target intensity ratio signal and the fitting equation.

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