CN115452716A - Light homogenizing device, gene sequencing system and control method of gene sequencing system - Google Patents

Light homogenizing device, gene sequencing system and control method of gene sequencing system Download PDF

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CN115452716A
CN115452716A CN202211004575.1A CN202211004575A CN115452716A CN 115452716 A CN115452716 A CN 115452716A CN 202211004575 A CN202211004575 A CN 202211004575A CN 115452716 A CN115452716 A CN 115452716A
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陈龙超
梁倩
王谷丰
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Shenzhen Sailu Medical Technology Co ltd
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract

The invention discloses a light homogenizing device, a gene sequencing system and a control method of the gene sequencing system, wherein the light homogenizing device comprises: a light source for generating a laser signal; the diaphragm is coupled with the light source and used for filtering an incident laser signal to form a filtering signal; the even-order aspheric surface reflector is coupled with the diaphragm and used for forming a target uniform light beam according to the filtering signal; the target uniform light beam is used for exciting a sample to be detected to generate a fluorescence signal; the shape and size of the target dodging light beam are matched with the shape and size of the camera imaging surface; wherein the camera is configured to acquire the fluorescence signal within the imaging plane. The light homogenizing device can achieve a good excitation lighting effect.

Description

Light homogenizing device, gene sequencing system and control method of gene sequencing system
Technical Field
The invention relates to the technical field of gene sequencing, in particular to a light homogenizing device, a gene sequencing system and a control method of the gene sequencing system.
Background
At present, based on the excitation characteristics of the fluorescent dye to the laser, a corresponding illumination area can be formed through the laser to excite and illuminate the fluorescent dye in the illumination area, so as to obtain a corresponding fluorescent signal. However, since the laser has the characteristic of gaussian distribution, the light intensity at the center of the illumination area is strong, while the light intensity at the edge of the illumination area is weak, and the excitation illumination effect is poor.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a light homogenizing device, a gene sequencing system and a control method of the gene sequencing system, wherein the light homogenizing device can achieve a good excitation illumination effect.
In a first aspect, the present application provides a light unifying apparatus comprising: a light source for generating a laser signal; the diaphragm is arranged behind the light source along the optical axis of the laser signal and is used for carrying out spatial filtering on the incident laser signal so as to form a filtering signal; the even-order aspheric reflector is placed behind the diaphragm along the optical axis of the filtering signal and is used for forming a target uniform light beam according to the filtering signal; the target uniform light beam is used for exciting a sample to be detected to generate a fluorescence signal; the shape and size of the target dodging light beam are matched with the shape and size of the camera imaging surface; wherein the camera is configured to acquire the fluorescence signal within the imaging plane.
In the embodiment, the dodging device performs spatial filtering on the collimated laser signal through the diaphragm, filters the edge part with weaker light intensity of the laser signal, and initially improves the uniformity of the laser signal, so as to obtain a filtered signal; the dodging device further shapes and dodges the filtering signals through the even-order aspheric surface reflector so as to obtain a target dodging light beam with the shape and size matched with the shape and size of the camera imaging surface. The target dodging light beam is uniformly distributed on the light intensity, and the photobleaching speed can be prevented from being accelerated and a sample to be detected can be prevented from being damaged under the condition of ensuring the signal to noise ratio required by camera imaging. In addition, the shape and the size of the target dodging light beam are matched with the shape and the size of the imaging surface of the camera, so that photobleaching of a non-imaging area and phototoxicity generated on DNA (a sample to be detected) can be reduced. The light homogenizing device in the embodiment can achieve a better excitation lighting effect through a simple structure.
In some embodiments, the even aspheric mirror has a surface formula that satisfies the following relationship:
Figure BDA0003808495810000021
wherein c is the curvature, k is the conic coefficient, alpha 1 Is a second order aspheric coefficient, α 2 Is a fourth order aspheric coefficient, alpha 3 Is a sixth order aspheric coefficient, alpha 3 And x and y are specific coordinate positions of the aspheric surface.
In some embodiments of the present invention, the,
Figure BDA0003808495810000022
k＝-146.5，α 1 ＝0，α 2 ＝1.848E-4，α 3 ＝-4.159E-6，α 4 ＝3.216E-8。
in some embodiments, the even aspheric mirror satisfies the following relationship: 14.6< -f < -16.1, wherein f is the focal length of the even-order aspheric mirror.
In some embodiments, the light unifying apparatus further comprises: the objective lens is arranged behind the even-order aspheric surface reflector along the optical axis of the target dodging light beam and is used for converging the target dodging light beam to form a converged light signal.
In a second aspect, the present application further provides a gene sequencing system, where the gene sequencing system is configured to test a sample to be detected to generate a detection result, and the gene sequencing system includes: a light unifying apparatus as described in any of the above embodiments; the biochip bears a sample to be detected and is used for being irradiated and excited by the target uniform light beam to generate a fluorescent signal; the fluorescence splitting module is used for splitting fluorescence signals with different wavelengths; at least one fluorescence acquisition module, wherein the fluorescence acquisition module is used for acquiring fluorescence signals subjected to the light splitting operation and generating image signals; and the processing module is connected with the fluorescence acquisition module and is used for generating a detection result according to the image signal.
In some embodiments, the gene sequencing system further comprises: a displacement stage for carrying the biochip; wherein the biochip is rectangular in shape; the shape of the target dodging light beam is an ellipse or an ellipse-like or a rectangle; the displacement table is also connected with the processing module; the processing module is further configured to control the displacement stage to move, so that the short axis of the target dodging beam moves relative to the long side of the biochip, and the short axis of the target dodging beam is parallel to the long side of the biochip, so that the camera scans the biochip; the imaging surface of the camera is a rectangular imaging surface, and the length-width ratio of the rectangular imaging surface is L: w; the ratio of the long axis to the short axis of the light spot of the target dodging light beam is L: w; wherein L, W are positive integers.
In some embodiments, the gene sequencing system further comprises: the objective lens is used for carrying out convergence operation on the target dodging light beam to form a converged light signal; the objective lens is also used for collecting the fluorescence signals; and the first dichroic mirror is used for reflecting or transmitting the target uniform light beam and the fluorescent signal respectively.
In some embodiments, the fluorescence spectroscopy module comprises: at least one dichroic mirror disposed behind the first dichroic mirror along an optical axis of the fluorescent signal.
In some embodiments, the fluorescence acquisition module comprises: the sleeve lens is arranged behind the fluorescence light splitting module along the optical axis of the fluorescence signal; the optical filter is placed behind the sleeve lens along the optical axis of the fluorescent signal; and the camera is placed behind the optical filter along the optical axis of the fluorescent signal.
In a third aspect, the present application further provides a method for controlling a gene sequencing system, which is applied to the gene sequencing system according to any one of the above embodiments, and includes: acquiring a scanning control signal; controlling a displacement table to move according to the scanning control signal so that the short axis of the target dodging light beam moves relative to the long edge of the biochip and is parallel to the long edge of the biochip; and in the moving process of the displacement table, sequentially acquiring image signals from the camera to scan the biochip so as to obtain the detection result.
In some embodiments, the imaging plane of the camera is a rectangular imaging plane, and the shape of the target dodging beam is elliptical or quasi-elliptical or rectangular; the length-width ratio of the rectangular imaging surface is L: w; the ratio of the long axis to the short axis of the light spot of the target dodging light beam is L: w; wherein L, W are positive integers.
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 a light uniformizing apparatus according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of an operation of a sample to be tested according to an embodiment of the present invention;
FIG. 3 is a long-axis intensity profile of a target dodging beam according to embodiments of the present invention;
FIG. 4 is a short axis intensity profile of a target dodging beam according to embodiments of the present invention;
FIG. 5 is a schematic structural diagram of a light uniformizing apparatus according to an embodiment of the present invention;
FIG. 6 is a schematic diagram of a framework of a gene sequencing system according to an embodiment of the present invention;
FIG. 7 is a schematic diagram of another embodiment of the present invention;
FIG. 8 is a schematic diagram of an operation of a biochip according to an embodiment of the present invention;
FIG. 9 is a schematic diagram of a gene sequencing system according to an embodiment of the present invention;
FIG. 10 is a flowchart of a control method of the gene sequencing system according to an embodiment of the present invention.
Reference numerals: the system comprises a light uniformizing device 100, a light source 110, a diaphragm 120, an even aspheric reflector 130, a sample to be detected 200, an objective lens 140, a gene sequencing system 300, a biochip 310, a fluorescence collection module 320, a fluorescence light splitting module 330, a processing module 340, a displacement table 350, a first dichroic mirror 360, a first dichroic mirror 331, a second dichroic mirror 332, a third dichroic mirror 333, a first reflector 334, a sleeve lens 321, an optical filter 322, a camera 323 and a second reflector 370.
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, when a sample to be detected is detected, a corresponding fluorescence signal can be obtained by exciting and illuminating a fluorescent dye in the sample to be detected through a laser signal. For example, in some application scenarios, a gene sequence in a sample to be detected needs to be detected, and therefore, a laser signal is irradiated on the sample to be detected, so that the sample to be detected generates a corresponding fluorescence signal under excitation of the laser signal, and the fluorescence signal is scanned and imaged by a camera, so that the gene sequence can be detected.
In the related art, based on the characteristics of gaussian distribution of laser signals, when a fluorescent dye in an illumination area is excited and illuminated by a laser signal, because the light intensity at the center of the illumination area is strong and the light intensity at the edge of the illumination area is weak, the excitation efficiency of the fluorescent dye at the center of the illumination area is high, the intensity of emitted fluorescent signals is also high, and the excitation efficiency of the fluorescent dye at the edge of the illumination area is low, and the intensity of emitted fluorescent signals is also lower. In order to improve the excitation efficiency of the fluorescent dye at the edge of the illumination area to improve the intensity of the fluorescent signal emitted by the fluorescent dye, so as to achieve the signal-to-noise ratio required by the camera for imaging the fluorescent signal, the power of the laser signal needs to be increased to make up for the disadvantage of low excitation efficiency at the edge of the illumination area. However, when the power of the laser signal is increased, the laser intensity at the central part of the illumination area is too high, which easily causes the photobleaching speed to increase, so that the error rate of the detection result of the sample to be detected is increased.
It can be understood that, in the related art, by homogenizing the laser signal using the homogenizing shaping optics, a target homogenizing beam having a relatively uniform light intensity can be obtained. However, the related dodging shaping optical device is generally a structure of the whole set of lens, and has the problems of large volume and high cost, and further dodging by using the kohler lighting module is required after dodging. Therefore, when the related art dodging shaping optical device is applied to a dodging device, the structure of the dodging device is complicated, and the optical path is long.
Therefore, the application provides a dodging device, which can realize a better excitation lighting effect, has a simple structure and is easy to realize.
Referring to fig. 1 to fig. 2, in a first aspect, the present application provides a light uniformizing apparatus 100, where the light uniformizing apparatus 100 includes: a light source 110, the light source 110 for generating a laser signal; a diaphragm 120, the diaphragm 120 being disposed behind the light source 110 along an optical axis of the laser signal, the diaphragm 120 being configured to spatially filter the incident laser signal to form a filtered signal; an even aspheric mirror 130, where the even aspheric mirror 130 is disposed behind the diaphragm 120 along the optical axis of the filtering signal, and the even aspheric mirror 130 is configured to form a target uniform light beam according to the filtering signal; wherein the target uniform light beam is used for exciting the sample 200 to be detected to generate a fluorescence signal.
It is understood that the dodging device 100 in the present embodiment includes a light source 110, a diaphragm 120 and an even aspheric mirror 130. The light source 110 is configured to generate a laser signal, and a diaphragm 120, an even aspheric mirror 130, and a sample 200 to be detected are sequentially disposed along an optical axis of the laser signal.
It will be appreciated that the light source 110 is used to generate a collimated laser signal having a spot shape that is circular. The laser signal is transmitted to the diaphragm 120. The diaphragm 120 is a device that limits a light beam in an optical system, and when a laser signal enters the diaphragm 120, the diaphragm 120 can filter the light beam of the laser signal, so as to block an edge portion with weak light intensity of the laser signal, thereby facilitating further light uniformization of the laser signal by the even aspheric mirror 130 in the following.
It is understood that the laser signal is a circular spot with a gaussian distribution, i.e., the light intensity is in a gaussian distribution in the spot area (illumination area) of the laser signal. For this purpose, the dodging device 100 of the present embodiment is provided with a corresponding even aspheric mirror 130, and the even aspheric mirror 130 is used for dodging the incident filtered signal to form a uniform illumination spot of the target dodging beam with a certain shape and size. Compared with the illumination light spot which is not subjected to the dodging operation and is only filtered by the diaphragm space, the light intensity distribution in the illumination area of the target dodging light beam is more uniform. In addition, the even-order aspheric mirror 130 is also used to reflect the target dodging beam so that the target dodging beam irradiates the sample 200 to be detected. The dodging device 100 of the present embodiment performs the dodging operation on the filtered signal through the even-order aspheric mirror 130 to obtain the target dodging beam with uniform light intensity distribution. When the target uniform light beam is used for exciting and illuminating the sample 200 to be detected, the light intensity in each illumination area is uniform, and the problem of inconsistent excitation efficiency can be avoided. Therefore, the dodging device 100 of the present embodiment can achieve a better excitation illumination effect with a simple structure.
It can be understood that the sample 200 to be detected generates a fluorescence signal under the excitation illumination of the target dodging beam, and the shape and size of the target dodging beam are the same as those of the fluorescence signal. Wherein the fluorescence signal is capable of characterizing different detection results of the sample 200 to be detected. For example, assume that the sample 200 to be detected contains different gene sequences, and that the different gene sequences can be labeled with different spectra of fluorescence. Then, when the target uniform light beam excites and illuminates the sample 200 to be detected, different gene sequences in the sample 200 to be detected can generate fluorescent signals with different spectra after being excited. Therefore, the detection result of the gene sequence of the sample 200 to be detected can be obtained through different fluorescent signals.
It is understood that, in order to obtain the detection result of the sample 200 to be detected, the fluorescence signal generated by the sample 200 to be detected can be scanned and imaged by the camera 323 to obtain the corresponding detection result. Specifically, the fluorescence signal is transmitted to an imaging surface of the camera 323, and the camera 323 can scan and image light rays in the imaging surface thereof to obtain an image signal corresponding to the fluorescence signal. In addition, the camera 323 needs to scan and image a plurality of areas of the sample 200 to be detected to obtain image signals of different areas, so as to obtain detection results of different areas. However, in the related art, the camera 323 is inefficient in scan-imaging the plurality of regions of the sample 200 to be detected. Specifically, the dodging effect of the dodging shaping optical device in the related art is isotropic, that is, after the circular light spot of the filtering signal passes through the dodging shaping optical device, the formed target dodging light beam is still a circular light spot, and then the fluorescence signal generated by the sample 200 to be detected according to the target dodging light beam is also a circular light spot. In some application scenarios, as shown in fig. 2, the imaging plane a of the camera 323 may be rectangular or have other shapes, and in order to enable the fluorescence signal to enter the imaging plane a of the camera 323 for imaging, the target dodging beam can only excite and illuminate a small area of the sample 200 to be detected, so as to obtain the fluorescence signal with a small circular spot B. At this time, as shown in fig. 2, since the imaging plane a of the camera 323 is rectangular, the fluorescence signal scanning is not performed on the remaining portion of the imaging plane a of the camera 323 except for the portion of the circular spot B where the fluorescence signal can be collected, and thus the efficiency of the scanning imaging by the camera 323 is likely to be low. In order to enable the fluorescence signal to completely cover the imaging plane a of the camera 323 so as to improve the scanning and imaging efficiency of the camera 323, as shown in fig. 2, the circular spot C of the target dodging beam needs to be larger than the imaging plane a of the camera 323 so that the fluorescence signal can completely cover the imaging plane a of the camera 323. However, the camera 323 can only scan and image the fluorescence signal of the imaged part (the part located in the imaging plane a), and the part of the fluorescence signal that exceeds the imaging plane a of the camera 323 cannot be scanned and imaged by the camera 323, i.e. no corresponding detection result is obtained, so it is also necessary to move the sample 200 to be detected, so that the exceeding part enters the imaging plane a of the camera 323, and perform excitation illumination again by using the target dodging beam to generate the corresponding fluorescence signal. It will be appreciated that the excess portion may cause photobleaching of the area after more than one pass of the excitation illumination, thereby reducing the signal-to-noise ratio of the image produced by the camera 323. And may damage the substances in the sample 200 to be detected, affecting the subsequent detection results. Therefore, in the related art, the efficiency of scan imaging the multiple regions of the sample 200 to be detected by the camera 323 is low.
It is understood that, in order to improve the scanning imaging efficiency of the camera 323, the even aspheric mirror 130 of the light equalizing device 100 in the present embodiment is used for shaping the filtered signal in addition to the light equalizing operation of the filtered signal. Specifically, the even-order aspheric mirror 130 can shape the shape of the laser signal into an ellipse or an ellipse-like shape, so that an ellipse or an ellipse-like fluorescence signal is generated after the formed target dodging light beam excites and illuminates the sample 200 to be detected in the illumination area, wherein the shape and size of the target dodging light beam are matched with the imaging surface a of the camera 323, that is, the shape and size of the light spot of the fluorescence signal are matched.
In a specific embodiment, as shown in fig. 2, when the imaging plane a of the camera 323 is a rectangle with an aspect ratio L: W of 2:1, the laser signal is filtered by the diaphragm 120 to form a filtered signal and is incident on the reflection surface of the even aspheric mirror 130, and the even aspheric mirror 130 performs the dodging and shaping operations on the filtered signal to form the target dodging beam. After being reflected from the even-order aspheric mirror 130, the target uniform light beam is converged and then diverged, and finally irradiates on the sample 200 to be detected for excitation illumination. The illumination area formed by the target dodging beam on the sample 200 to be detected is an elliptical light spot D, the ratio of the long axis to the short axis of the elliptical light spot D is 2:1, at this time, the shape and size of the target dodging beam are matched with the shape and size of the imaging plane a of the camera 323, that is, the shape and size of the fluorescence signal are matched with the shape and size of the imaging plane a of the camera 323. When the target dodging light beam irradiates on the sample 200 to be detected, since the illumination area formed by the target dodging light beam is elliptical, a plurality of areas of the sample 200 to be detected can be simultaneously excited and illuminated, and since the imaging surface a of the camera 323 is matched with the target dodging light beam, the camera 323 can scan and image all generated fluorescence signals, so that the scanning and imaging efficiency of the camera 323 is effectively improved, and the damage caused by excessive excitation illumination on the same area of the sample 200 to be detected for multiple times is avoided.
It will be appreciated that the size of the shape of the imaging plane a of the camera 323 matches the size of the shape of the target dodging beam spot D when the aspect ratio of the imaging plane a of the camera 323 is equal to the ratio of the major and minor axes of the target dodging beam spot D. Similarly, when the imaging surface a of the camera 323 has other shapes, reasonable parameter setting can be performed on the even aspheric mirror 130 to obtain the target dodging beam spot D with the shape and size matched with the imaging surface, so as to improve the detection efficiency of the sample 200 to be detected.
It can be understood that, in the embodiment, since the dodging device 100 performs dodging and shaping operations on the laser signal through the reflection surface of the even-order aspheric mirror 130, only the reflection surface of the even-order aspheric mirror 130 needs to be designed and manufactured correspondingly, and no additional mirror needs to be added, so that the size of the dodging device 100 can be reduced to a certain extent, and the structure is simplified.
In this embodiment, the light equalizing device 100 filters the collimated laser signal through the diaphragm 120 to adjust the light intensity of the laser signal and filter the edge portion with weaker light intensity of the laser signal, so as to obtain a filtered signal; the dodging device 100 further shapes and dodges the filtered signal through the even aspheric mirror 130 to obtain a target dodging beam with a shape and size matching the shape and size of the imaging surface of the camera 323. The target dodging light beam is uniformly distributed on the light intensity, and the photo-bleaching speed can be accelerated and the sample to be detected 200 can be prevented from being damaged under the condition that the signal to noise ratio required by the imaging of the camera 323 is ensured. In addition, the shape and the size of the target dodging beam are matched with the shape and the size of the imaging surface of the camera 323, so that the scanning imaging efficiency of the camera 323 can be effectively improved. In the embodiment, the light uniformizing device 100 can achieve a better excitation illumination effect through a simple structure.
Referring to fig. 1, 3 and 4, in some embodiments, the surface form formula of the even aspheric mirror 130 satisfies the following relationship:
Figure BDA0003808495810000081
wherein c is the curvature, k is the conic coefficient, alpha 1 Is a second order aspheric coefficient, α 2 Is a fourth order aspheric coefficient, alpha 3 Is a sixth order aspheric coefficient, alpha 3 And x and y are specific coordinate positions of the aspheric surface.
It can be understood that when the even-order aspheric mirror 130 satisfies the above formula, the filtered signal can obtain the target dodging beam with the elliptical light spot after being reflected by the even-order aspheric mirror 130. In addition, by setting specific face parameters: curvature, cone coefficients, second-order aspheric coefficients, fourth-order aspheric coefficients, sixth-order aspheric coefficients and eighth-order aspheric coefficients, and specific coordinate positions of aspheric surfaces are determined, so that target uniform light beams with different shapes and sizes can be obtained, namely the major axis and the minor axis of the elliptic light spots are different. From the above, the target dodging beam with the elliptical light spot is matched with the rectangular imaging surface of the camera 323, so that the scanning and imaging efficiency of the camera 323 can be improved. In addition, in the embodiment, the even-order aspheric mirror 130 forms a target dodging beam with a good dodging effect, so that a better excitation illumination effect can be achieved.
Specifically, fig. 3 is a light intensity distribution diagram in the long axis direction of the light spots of the target dodging light beam, and fig. 4 is a light intensity distribution diagram in the short axis direction of the light spots of the target dodging light beam. In the embodiment, the length of the major axis of the target dodging beam spot is 1.6mm, and the length of the minor axis is 0.8 mm. Specifically, the light intensity minimum value in the illumination area is divided by the light intensity maximum value, thereby obtaining illumination uniformity. The uniformity in the major axis direction was 81%, and the uniformity in the minor axis direction was 85%. It can be understood that when the illumination uniformity reaches more than 75%, the extraction efficiency of the imaging algorithm for the effective information of the center and the edge of the illumination area corresponding to the photographing position of the camera 323 is nearly consistent. Therefore, the light uniformizing device 100 of the present embodiment can well meet the actual use requirement.
Referring to fig. 1 and 2, in some embodiments,
Figure BDA0003808495810000082
k＝-146.5，α 1 ＝0，α 2 ＝1.848E-4，α 3 ＝-4.159E-6，α 4 ＝3.216E-8。
it is understood that the present embodiment is described by taking the aspect ratio of the imaging plane of the camera 323 as 2:1 as an example. When the surface type parameters of the surface type formula of the even-order aspheric mirror 130 are set as above, the filtered signal is shaped and homogenized by the even-order aspheric mirror 130 to form an elliptical light spot with the length-to-minor axis ratio of 2:1, and the fluorescence signal is also an elliptical light spot D with the length-to-minor axis ratio of 2:1, and the size of the elliptical light spot is matched with the size of the imaging surface A of the camera 323. At this time, as shown in fig. 2, the spot D of the fluorescence signal can completely enter the imaging plane a of the camera 323. As can be seen from the above, the camera 323 can scan and image multiple regions of the sample 200 to be detected simultaneously under the condition of ensuring good excitation illumination effect.
In some embodiments, the even aspheric mirror 130 satisfies the following relationship: 14.6 woven-over-f woven-over 16.1, wherein f is the focal length of the even-order aspherical mirror 130.
It can be understood that the focal length f of the even-order aspheric mirror 130 satisfies the above relationship, so that the even-order aspheric mirror 130 can achieve better shaping and dodging effects and generate the target dodging beam with the ratio of the long axis to the short axis of 2:1. Similarly, when the surface type parameter of the even-order aspheric mirror 130 is changed, the focal length f is also changed accordingly, and this embodiment is not described herein.
Referring to fig. 5, in some embodiments, the light uniformizing apparatus 100 further includes: the objective lens 140 is disposed behind the even-order aspheric mirror 130 along the optical axis of the target dodging beam, and the objective lens 140 is configured to perform a converging operation on the target dodging beam to form a converged optical signal.
It can be understood that the filtered signal is reflected by the even aspheric mirror 130 to form a target dodging beam, and the target dodging beam is converged and then diverged, and finally irradiates on the sample 200 to be detected. Since the light spot of the target dodging light beam is diverged during the divergence process, energy loss is caused. For this purpose, the dodging device 100 of the present embodiment is provided with a corresponding objective lens 140, the objective lens 140 is used for converging the target dodging light beam to obtain a converged light signal, and the converged light signal can be focused on the sample 200 to be detected. Therefore, the converged light signal is the focused target dodging beam, and therefore, the shape of the converged light signal is still the same as that of the target dodging beam.
It can be understood that the energy of the target dodging light beam which is not converged by the objective lens 140 is relatively divergent, and the energy of the converged light signal obtained after converging by the objective lens 140 is more concentrated than that of the target dodging light beam. Therefore, loss of energy can be avoided. In addition, in some application scenarios, it may be necessary to change the size of the imaging plane of the camera 323, and as can be seen from the above, in order to ensure the scanning imaging efficiency of the camera 323, the size of the fluorescence imaging area should be changed to follow the size of the imaging plane of the camera 323. For this reason, in the light uniformizing apparatus 100 of the present embodiment, by replacing different objective lenses 140, different converging operations can be performed on the target light uniformizing light beams to generate converging light signals with different sizes, and the size of the fluorescence imaging area can be changed accordingly. Therefore, the size of the fluorescence signal can be flexibly adjusted by setting the objective lens 140 to accommodate the cameras 323 having different imaging planes. In addition, the objective lens 140 in this embodiment is also used to collect the fluorescence signal generated by the sample 200 to be detected, so as to transmit to the camera 323 for scanning and imaging.
Referring to fig. 6, in a second aspect, the present application further provides a gene sequencing system 300, where the gene sequencing system 300 is configured to test a sample 200 to be tested to generate a test result, and the gene sequencing system 300 includes: the light evening device 100 according to any one of the above embodiments; the biochip 310 is used for carrying a sample 200 to be detected, and the biochip 310 is used for being irradiated and excited by the target dodging light beam to generate a fluorescent signal; at least one fluorescence splitting module 330, wherein the fluorescence splitting module 330 is used for splitting fluorescence signals with different wavelengths; at least one fluorescence collection module 320, wherein the fluorescence collection module 320 is used for collecting the fluorescence signal subjected to the light splitting operation and generating an image signal; a processing module 340, wherein the processing module 340 is connected to the fluorescence collecting module 320, and the processing module 340 is configured to generate a detection result according to the image signal.
It is understood that the gene sequencing system 300 in this embodiment includes the light uniformizing apparatus 100, the biochip 310, the fluorescence collecting module 320, the camera 323, and the processing module 340. The biochip 310 is configured to generate a corresponding fluorescence signal from a target uniform light beam, and a fluorescence splitting module 330 and a fluorescence collecting module 320 are sequentially disposed along an optical axis of the fluorescence signal, and the fluorescence collecting module 320 is further configured to be connected to the processing module 340.
Specifically, the dodging device 100 is configured to generate a target dodging beam and irradiate the target dodging beam on the biochip 310, and the biochip 310 can generate a corresponding fluorescence signal according to the target dodging beam. As can be seen from the above, the fluorescence signal can be imaged to obtain a corresponding image signal, and the detection result of the biochip 310 can be obtained according to the image signal. In order to perform efficient scanning imaging on the fluorescence signal and ensure the accuracy of the detection result, the gene sequencing system 300 in this embodiment is provided with a fluorescence spectroscopy module 330 and a fluorescence acquisition module 320. The fluorescence splitting module 330 is used for splitting the fluorescence signal to transmit the fluorescence signal to different fluorescence collecting modules 320, and the fluorescence collecting modules 320 are used for efficiently collecting the fluorescence signal to form a collected signal, and meanwhile, the fluorescence signal is prevented from being interfered by the outside to influence the detection result.
It can be understood that, since the image signal generated by the fluorescence acquisition module 320 can only represent the image information of the fluorescence signal, the information in the image signal needs to be processed to obtain the corresponding detection result. For this purpose, the gene sequencing system 300 in this embodiment further provides a corresponding processing module 340, and the processing module 340 is configured to analyze and process information in the image signal to obtain a detection result.
In a specific embodiment, the gene sequencing system 300 is used to detect gene sequences on a biochip 310. Since different bases in the gene sequence can generate fluorescence signals with different spectra under the excitation illumination of the target dodging light beam, the obtained image signals include different spectral components after the fluorescence signals with different spectra are scanned and imaged by the camera 323. The processing module 340 can obtain a specific gene sequence, i.e. a detection result, by analyzing the spectral components.
It can be seen that the contents of the above-mentioned embodiment of the light uniformizing apparatus 100 are all applicable to the embodiment of the gene sequencing system 300, the functions implemented by the embodiment of the gene sequencing system 300 are the same as those of the above-mentioned embodiment of the light uniformizing apparatus 100, and the advantageous effects achieved by the embodiment of the light uniformizing apparatus 100 are also the same as those achieved by the above-mentioned embodiment of the light uniformizing apparatus 100.
Referring to fig. 7 and 8, in some embodiments, the gene sequencing system 300 further comprises: a displacement stage 350, the displacement stage 350 being configured to carry the biochip 310; wherein the biochip 310 is rectangular in shape; the shape of the target dodging light beam is an ellipse or an ellipse-like or a rectangle; the displacement table 350 is also connected with the processing module 340; wherein the processing module 340 is further configured to control the displacement stage 350 to move, so that the short axis of the target dodging beam moves relative to the long side of the biochip 310, and the short axis of the target dodging beam is parallel to the long side of the biochip 310, so that the camera 323 scans the biochip 310; the imaging surface of the camera 323 is a rectangular imaging surface, and the length-width ratio of the rectangular imaging surface is L: w; the ratio of the long axis to the short axis of the light spot of the target dodging light beam is L: w; wherein L, W are positive integers.
It can be understood from the above description that the camera 323 can only image the fluorescence signal in the imaging plane a, therefore, in order to scan and image different areas of the biochip 310, the gene sequencing system 300 in this embodiment is provided with a corresponding displacement stage 350, the displacement stage 350 is used to carry the biochip 310, and the displacement stage 350 is also used to connect with the processing module 340. The translation stage 350 can be moved under the control of the processing module 340 to move the biochip 310 so that the target dodging beam excites and illuminates different areas of the biochip 310.
In one embodiment, as shown in FIG. 8, the biochip 310 is rectangular, the imaging plane A of the camera 323 is a rectangular imaging plane, and the ratio of the length to the width of the rectangular imaging plane A is L: w, the ratio of the long axis to the short axis of a light spot D of the target dodging beam is L: w. When the displacement stage 350 is at the initial position, the spot D of the target dodging beam excites and illuminates the X1 region of the biochip 310 and generates a corresponding fluorescence signal, the camera 323 generates an image signal according to the above working principle, and the processing module 340 receives the image signal, thereby generating a corresponding detection result X1. When the processing module 340 obtains the detection result, the processing module 340 is further configured to generate a first control signal, and the displacement stage 350 moves according to the first control signal, so that the spot D of the target dodging beam irradiates the X2 area of the biochip 310, and obtains the detection result X2 according to the above steps. The displacement stage 350 continues to move, so that the camera 323 can scan and image the areas of the biochip 310, such as X3, X4..
It can be understood that when the length-width ratio of the rectangular imaging plane a of the camera 323 is 2:1, the ratio of the long axis to the short axis of a light spot D of the target dodging beam is 2:1, compared to the circular laser signal in the related art, the target dodging beam of the present embodiment can excite and illuminate a larger area in the biochip 310, and scan and image through the matched camera 323. Therefore, the gene sequencing system 300 of the present embodiment has high scanning and detecting efficiency, and can further improve the output of gene sequencing data in unit time.
Referring to fig. 9, in some embodiments, the gene sequencing system 300 further comprises: the objective lens 140, the objective lens 140 is configured to perform a converging operation on the target dodging beam to form a converging optical signal; the objective lens 140 is also used for collecting the fluorescence signal; the first dichroic mirror 360, the first dichroic mirror 360 is configured to reflect or transmit the target uniform light beam and the fluorescent signal, respectively.
It can be understood that, in order to distinguish the optical paths of the target dodging light beam and the fluorescence signal so as to collect the fluorescence signal to image the corresponding fluorescence collection module 320, in this embodiment, the gene sequencing system 300 is provided with a corresponding first dichroic mirror 360, and the first dichroic mirror 360 is used for splitting the target dodging light beam and the fluorescence signal.
Specifically, as shown in fig. 9, when the target uniform light beam is incident on the surface of the first dichroic mirror 360, the first dichroic mirror 360 reflects the target uniform light beam so that the target uniform light beam irradiates the biochip 310, or the target uniform light beam is focused on the biochip 310 through the objective lens 140. When the fluorescent signal is incident to the surface of the first dichroic mirror 360, the first dichroic mirror 360 transmits the fluorescent signal so that the fluorescent signal is incident to the fluorescent light collecting module 320. Similarly, the first dichroic mirror 360 can also transmit the target dodging light beam and reflect the fluorescence signal to split the target dodging light beam and the fluorescence signal.
Referring again to fig. 9, in some embodiments, the fluorescence spectroscopy module includes: at least one dichroic mirror placed behind the first dichroic mirror 360 along the optical axis of the fluorescence signal.
It will be appreciated that, since the fluorescent signal typically contains a plurality of different spectral components, in some application scenarios, the gene sequencing system 300 may only need to detect certain spectral components (bands) therein. For this reason, in the present embodiment, the gene sequencing system 300 is provided with a corresponding dichroic mirror, and the dichroic mirror can transmit or reflect the fluorescent signal of the required wavelength band, and reflect or transmit the fluorescent signal of other wavelength bands, so as to collect the fluorescent signal of the specific wavelength band and provide it to the fluorescent collection module 320 for scanning imaging, thereby avoiding interference of the fluorescent signal of other wavelength bands.
Referring again to fig. 9, in some embodiments, the fluorescence acquisition module 320 includes: a sleeve lens 321, wherein the sleeve lens 321 is disposed behind the fluorescence splitting module 330 along an optical axis of the fluorescence signal; an optical filter 322, wherein the optical filter 322 is disposed behind the sleeve lens 321 along an optical axis of the fluorescent signal; a camera 323, the camera 323 being placed behind the filter 322 along an optical axis of the fluorescence signal.
Specifically, the fluorescent signal is transmitted through the dichroic mirror and then transmitted to the sleeve lens 321 for convergence, and then the filter 322 filters the residual interference signal in the fluorescent signal, such as the target dodging beam, and finally transmitted to the camera 323 for scanning and imaging.
It can be understood that, the gene sequencing can detect four bases in the gene, and the four bases can generate fluorescence signals with different spectra (wave bands) under the excitation illumination of the target uniform light beam, and in order to perform the comprehensive gene sequencing, the present embodiment is provided with four corresponding fluorescence collecting modules 320 to form four channels to detect the four bases in the gene respectively. Wherein, the four basic groups are A, T, G, C respectively, and the wave bands for generating fluorescence signals are f1, f2, f3 and f4 respectively.
Specifically, when the fluorescence signal passes through the first dichroic mirror 331, the fluorescence signal with the specific frequency f1 is transmitted by the first dichroic mirror 331, enters the corresponding fluorescence collection module 320, is transmitted to the sleeve lens 321 for convergence, is filtered by the filter 322 for removing the residual interference signal in the fluorescence signal, and is finally transmitted to the camera 323 for scanning and imaging, so as to obtain the detection result of the base a in the biochip 310. The first dichroic mirror 331 is further configured to reflect the fluorescence signals of the remaining frequencies so as to transmit the fluorescence signals to the second dichroic mirror 332, and the second dichroic mirror 332 may reflect the fluorescence signals of the f2 specific frequency in the fluorescence signals so as to enable the fluorescence signals of the f2 specific frequency to enter the corresponding fluorescence acquisition module 320 for scanning imaging, so as to obtain a detection result about the base T in the biochip 310. The second dichroic mirror 332 is further configured to transmit the fluorescence signals of the remaining frequencies to the third dichroic mirror 333, and the third dichroic mirror 333 is capable of reflecting the fluorescence signals of the f3 specific frequency therein, so that the fluorescence signals of the f3 frequency enter the corresponding fluorescence acquisition module 320 for scanning imaging to obtain a detection result about the base G in the biochip 310. The third dichroic mirror 333 also serves to transmit the fluorescence signals of the remaining frequencies to be transmitted to the first reflecting mirror 334. The first mirror 334 can reflect the incident fluorescence signal, so that the fluorescence signal with f4 frequency is reflected into the corresponding fluorescence collection module 320 for scanning imaging to obtain the detection result of the base C in the biochip 310. Further, the sleeve lens 321 can receive the fluorescent signal emitted from the first dichroic mirror 360 via the second reflecting mirror 370. The second reflecting mirror 370 can reflect the fluorescent signal to transmit the reflected fluorescent signal to the first dichroic mirror 331, thereby improving the integration of the gene sequencing system 300 and reducing the distribution volume thereof.
It can be understood that, according to actual requirements, the dichroic mirrors in the fluorescence collection module 320 and the fluorescence splitting module 330330 may be added or reduced to respectively detect fluorescence signals of different wavelength bands, which is not limited herein.
Referring to fig. 10 and 8, in a third aspect, the present application further provides a control method of a gene sequencing system 300, which is applied to the gene sequencing system 300 according to any of the above embodiments, and includes: s101, acquiring a scanning control signal;
step S102, controlling the displacement table 350 to move according to the scanning control signal;
step S103, sequentially acquiring image signals from the camera 323 in the moving process of the displacement table 350;
it can be understood that, according to the scanning control signal, the displacement stage 350 is controlled to move, so that the short axis of the target dodging beam moves relative to the long side of the biochip 310, and the short axis of the target dodging beam and the long side of the biochip 310 are parallel to each other; sequentially acquiring image signals from the camera 323 during the movement of the displacement stage 350; so as to perform a scanning operation on the biochip 310, thereby obtaining the detection result.
It can be understood from the above description that, when the processing module 340 generates the detection result according to the image signal, the processing module 340 is further configured to generate a first control signal, i.e. a scanning control signal, to control the displacement stage 350 to move. Wherein, the displacement stage 350 can move according to a preset time period and a moving distance. The camera 323 can scan and image the moved biochip 310 within a preset time period to obtain a corresponding detection result. The displacement stage 350 moves a preset moving distance, so that the light spot of the target dodging beam can be irradiated on different areas of the biochip 310. Specifically, as shown in fig. 8, the long axis of the target dodging beam spot is perpendicular to the long side of the biochip 310, and the target dodging beam moves along the long side of the biochip 310, so that the camera 323 performs scanning operation imaging on the X1 region, the X2 region, and the like on the biochip 310, thereby obtaining the detection result.
It can be understood that, since the long side of the chip corresponds to the short side of the target fluorescence signal, i.e., the long side of the chip corresponds to the short side of the camera 323, the single moving distance of the translation stage 350 can be shortened compared to the scanning mode in which the long side of the chip corresponds to the short side of the camera 323, thereby improving the scanning efficiency of the camera 323.
Therefore, the contents of the embodiment of the gene sequencing system 300 are all applicable to the embodiment of the control method, the functions implemented by the embodiment of the control method are the same as those of the embodiment of the gene sequencing system 300, and the beneficial effects achieved by the embodiment of the gene sequencing system 300 are also the same as those achieved by the embodiment of the gene sequencing system 300.
In some embodiments, the imaging plane of the camera 323 is a rectangular imaging plane, and the shape of the target dodging beam is elliptical or quasi-elliptical or rectangular; the length-width ratio of the rectangular imaging surface is L: w; the ratio of the long axis to the short axis of the light spot of the target dodging light beam is L: w; wherein L, W are positive integers.
In one particular embodiment, the imaging plane of the camera 323 is a rectangular imaging plane and the shape of the target dodging beam is elliptical or elliptical-like or rectangular. From the above, when the ratio of the length to the width of the rectangular imaging surface and the ratio of the length to the short axis of the spot of the target dodging beam are both L: and W, the shape and size of the imaging surface of the characterization camera 323 are matched with the shape and size of the target dodging beam. In this case, the efficiency of scanning the biochip 310 by the camera 323 is higher, and the amount of output sequencing data per unit time can be increased.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above 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 (12)

1. A light unifying apparatus, characterized in that the light unifying apparatus comprises:
a light source for generating a laser signal;
the diaphragm is arranged behind the light source along the optical axis of the laser signal and is used for carrying out spatial filtering on the incident laser signal so as to form a filtering signal;
the even-order aspheric mirror is placed behind the diaphragm along the optical axis of the filtering signal and is used for forming a target uniform light beam according to the filtering signal; the target uniform light beam is used for exciting a sample to be detected to generate a fluorescence signal;
the shape and size of the target dodging light beam are matched with the shape and size of the camera imaging surface; wherein the camera is configured to acquire the fluorescence signal within the imaging plane.
2. The light unifying apparatus according to claim 1, wherein the even aspheric mirror has a surface type formula satisfying the following relationship:
Figure FDA0003808495800000011
wherein c is the curvature, k is the conic coefficient, alpha 1 Is a second order aspheric coefficient, α 2 Is a fourth order aspheric coefficient, alpha 3 Is a sixth order aspheric coefficient, alpha 3 And x and y are specific coordinate positions of the aspheric surface.
3. The light homogenizing device of claim 2,
Figure FDA0003808495800000012
k＝-146.5，α 1 ＝0，α 2 ＝1.848E-4，α 3 ＝-4.159E-6，α 4 ＝3.216E-8。
4. a light distributing device as claimed in claim 3, wherein said even aspheric mirror satisfies the following relation:
14.6 woven-over-f woven-over-16.1, wherein f is the focal length of the even-order aspheric mirror.
5. The light unifying apparatus according to claim 1, further comprising:
the objective lens is arranged behind the even-order aspheric surface reflector along the optical axis of the target dodging light beam and is used for converging the target dodging light beam to form a converged light signal.
6. A gene sequencing system, wherein the gene sequencing system is configured to test a test sample to generate a test result, and the gene sequencing system comprises:
the light unifying apparatus as claimed in any one of claims 1 to 5;
the biochip bears a sample to be detected and is used for being irradiated and excited by the target uniform light beam to generate a fluorescence signal;
the fluorescence splitting module is used for splitting fluorescence signals with different wavelengths;
at least one fluorescence acquisition module, wherein the fluorescence acquisition module is used for acquiring fluorescence signals subjected to the light splitting operation and generating image signals;
and the processing module is connected with the fluorescence acquisition module and is used for generating a detection result according to the image signal.
7. The gene sequencing system of claim 6, further comprising:
a displacement stage for carrying the biochip; wherein the biochip is rectangular in shape; the shape of the target dodging light beam is an ellipse or an ellipse-like or a rectangle; the displacement table is also connected with the processing module;
the processing module is further configured to control the displacement stage to move, so that the short axis of the target dodging beam moves relative to the long side of the biochip, and the short axis of the target dodging beam is parallel to the long side of the biochip, so that the camera scans the biochip;
the imaging surface of the camera is a rectangular imaging surface, and the length-width ratio of the rectangular imaging surface is L: w; the ratio of the long axis to the short axis of the light spot of the target dodging light beam is L: w; wherein L, W are positive integers.
8. The gene sequencing system of claim 6, further comprising:
the objective lens is used for converging the target dodging light beam to form a converged light signal; the objective lens is also used for collecting the fluorescence signals;
and the first dichroic mirror is used for respectively reflecting or transmitting the target uniform light beam and the fluorescent signal.
9. The gene sequencing system of claim 7, wherein the fluorescence spectroscopy module comprises:
at least one dichroic mirror disposed behind the first dichroic mirror along an optical axis of the fluorescence signal.
10. The gene sequencing system of claim 6, wherein the fluorescence acquisition module comprises:
the sleeve lens is arranged behind the fluorescence light splitting module along the optical axis of the fluorescence signal;
the optical filter is placed behind the sleeve lens along the optical axis of the fluorescent signal;
a camera disposed behind the optical filter along an optical axis of the fluorescence signal.
11. The method for controlling a gene sequencing system, which is applied to the gene sequencing system according to any one of claims 6 to 10, comprising:
acquiring a scanning control signal;
controlling a displacement table to move according to the scanning control signal so that the short axis of the target dodging light beam moves relative to the long edge of the biochip and is parallel to the long edge of the biochip;
and in the moving process of the displacement table, sequentially acquiring image signals from the camera to scan the biochip so as to obtain the detection result.
12. The method of claim 11, wherein the imaging plane of the camera is a rectangular imaging plane, and the shape of the target dodging beam is an ellipse or an ellipse-like or a rectangle; the length-width ratio of the rectangular imaging surface is L: w; the ratio of the long axis to the short axis of the light spot of the target dodging light beam is L: w; wherein L, W are positive integers.
CN202211004575.1A 2022-08-22 2022-08-22 Light homogenizing device, gene sequencing system and control method of gene sequencing system Pending CN115452716A (en)

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