WO2022120595A1

WO2022120595A1 - Super-resolution measurement system and super-resolution measurement method

Info

Publication number: WO2022120595A1
Application number: PCT/CN2020/134659
Authority: WO
Inventors: 伯恩; 宋李烟; 周胜元; 韦毅; 龙青山
Original assignee: 深圳华大智造科技股份有限公司
Priority date: 2020-12-08
Filing date: 2020-12-08
Publication date: 2022-06-16
Also published as: CN116507963A

Abstract

A super-resolution measurement system (10) and a super-resolution measurement method, used for measuring biological information of a sample (20) to be measured, and the super-resolution measurement system (10) comprising: a light source device (11), used for emitting light source light; a first microlens array (121), used for receiving and focusing the light source light to generate a reference light, the reference light being used for scanning the sample (20) to be measured, so as to cause the sample (20) to be measured to generate measurement light, and when the reference light scans the sample (20) to be tested, a focused light spot array being formed on the sample (20) to be measured; a second microlens array (151) and a filter layer (152), located on the optical path of the measurement light and used for focusing the measurement light and for filtering out stray light in the measurement light; and at least one time-delay integral camera (141,142,143, 144), the at least one time-delay integral camera (141,142,143, 144) being used for receiving the measurement light, and on the basis of the detection light, acquiring biological information of the sample (20) to be measured.

Description

Super-resolution detection system and super-resolution detection method

technical field

The invention relates to the technical field of biochemical information detection, in particular to a super-resolution detection system and a super-resolution detection method.

Background technique

Gene sequencing technology is widely used in many research fields of life sciences and medicine, including various types of genomics, the etiology of complex diseases, prenatal diagnosis, and personalized drug treatment. Gene sequencing technology refers to the technology of analyzing the sequence of four bases on DNA, including namely adenine (A), thymine (T), cytosine (C) and guanine (G).

A sequencing method is as follows: the four bases on the DNA nanospheres on the sequencing chip carry corresponding fluorophores by biochemical methods; , it will emit fluorescence of different wavelengths; by taking pictures of the sequencing chip, it is possible to detect whether the DNA nanospheres corresponding to a specific pixel point generate a fluorescent signal of a specific wavelength to identify a specific base, thereby realizing sequencing. The above-mentioned sequencing methods are limited by the optical diffraction limit, and the sample spacing on the sequencing chip can only be controlled to be more than 500 nm, and the sample density is small.

The resolution of some optical imaging systems can be reduced to sub-hundred nanometers. Such as stimulated emission depletion method (resolution up to 30-70nm), photoactivated localization microscopy (resolution up to 10-55nm), stochastic optical reconstruction microscopy (resolution up to 10-55nm), structured light Illumination microscopy (resolution up to ~80nm), spinning disk confocal microscopy based on pixel redistribution (resolution up to ~120nm), etc. However, the stimulated emission depletion method requires high excitation light intensity (hundreds of MW/cm2-GW/cm2), which is not suitable for long-read sequencing, and its point-scanning characteristics make it more suitable for rapid imaging of small-area samples . Light-activated localization microscopy and stochastic optical reconstruction microscopy can achieve high resolution, but due to their single-molecule localization characteristics, the acquisition time of each super-resolution image is basically on the order of minutes, and the sequencing speed is relatively high. slow. However, structured illumination microscopy and spinning disk confocal microscopy based on pixel redistribution can meet the requirements of large-scale, high-speed, and high-resolution imaging, and have no specific requirements for fluorescent dyes. It is also relatively low and is expected to be combined with existing sequencing technologies. Since the cost of reagents and consumables is inversely proportional to the sample density, how to increase the sample density of the sequencing chip with the help of super-resolution imaging technology and further reduce the cost of gene sequencing is an urgent problem to be solved. In the current gene sequencing method, an area array camera is used to take pictures of the sequencing chip. The area scan camera needs to take a picture of a certain object-side field of view; the stage drives the sequencing chip to move, switches to the next object-side field of view, and takes a picture again; and so on. Moving and stopping the stage is time-consuming, resulting in low sequencing throughput.

SUMMARY OF THE INVENTION

One aspect of the present application provides a super-resolution detection system for detecting biological information of a sample to be tested, and the super-resolution detection system includes:

a light source device for emitting light from the light source;

a first microlens array, used for receiving and focusing the light source light to generate reference light, the reference light is used for scanning the sample to be tested so that the sample to be tested generates detection light, the reference light scans the When a sample is to be tested, a focused spot array is formed on the sample to be tested;

a second microlens array and a filter layer, located on the optical path of the detection light, for focusing the detection light and for filtering out stray light in the detection light; and

At least one time-lapse integration camera, which is configured to receive the detection light, and obtain the biological information of the sample to be tested according to the detection light.

Another aspect of the present application provides a super-resolution detection method for detecting biological information of a sample to be tested, the super-resolution detection method is applied to a super-resolution detection system, and the super-resolution detection method includes the following steps:

Sending reference light to the sample to be tested, the reference light can form a focused spot array on the sample to be tested;

controlling the sample to be tested and the focusing spot array to generate continuous relative motion, so that the sample to be tested continues to generate detection light;

focusing the detection light and filtering out stray light in the detection light; and

The detection light after focusing and filtering out stray light is received by at least one time-lapse integrating camera, and continuous imaging is performed according to the detection light to obtain the biological information of the sample to be tested, and the detection light is in the at least one delay time. A focused spot array is formed on the target surface of the time-integrating camera.

The above-mentioned super-resolution detection system and super-resolution detection method, by setting the first microlens array, the second microlens array and the filter layer, and setting the reference light to scan the sample to be tested, the charge movement on the TDI camera makes the TDI camera and the detection The focused spot array formed by the light moves relative to each other, which can realize continuous imaging according to the detection light. Compared with the imaging method using an area array camera in the prior art, the super-resolution detection system 10 provided in this embodiment is conducive to realizing super-resolution and improving the detection speed (the speed can be increased to 5 when the area array camera is used). times), thereby reducing the detection cost.

Description of drawings

FIG. 1 is a schematic structural diagram of a super-resolution detection system, a sample to be tested, and a sequencing chip in an embodiment of the present invention.

FIG. 2 is a schematic diagram of the optical path of the super-resolution detection system in FIG. 1 .

FIG. 3 is a schematic plan view of the first microlens array in FIG. 2 .

FIG. 4 is a schematic plan view of the laser focusing spot on the first focal plane of the first microlens array in FIG. 3 .

FIG. 5 is a schematic diagram of the laser focused spot array scanning the sequencing chip in FIG. 4 .

FIG. 6 is another schematic plan view of the laser focusing spot array in FIG. 4 .

FIG. 7 is another schematic plan view of the first microlens array in FIG. 3 .

FIG. 8 is a schematic diagram of the laser focusing spot array and its projection in FIG. 4 .

FIG. 9 is a schematic diagram of a light intensity distribution of two adjacently arranged laser focusing spots in FIG. 8 .

FIG. 10 is another schematic diagram of the light intensity distribution of the two adjacently arranged laser focusing spots in FIG. 8 .

FIG. 11 is another schematic diagram of the light intensity distribution of the two adjacently arranged laser focusing spots in FIG. 8 .

FIG. 12 is a schematic diagram of the light intensity distribution of a plurality of adjacently arranged laser focusing spots in FIG. 8 .

FIG. 13 is a schematic diagram of a strip-shaped area on the sequencing chip scanned by the laser focused spot array in FIG. 4 .

FIG. 14 is a schematic diagram of the process of receiving detection light of the TDI camera in FIG. 2 .

FIG. 15 is a schematic diagram of the process of generating an image according to the detected light by the TDI camera in FIG. 2 .

FIG. 16 is a schematic plan view of the second microlens array in FIG. 2 .

FIG. 17 is a schematic plan view of the filter layer in FIG. 2 .

FIG. 18 is a schematic flowchart of a super-resolution detection method according to an embodiment of the present invention.

Description of main component symbols

The following specific embodiments will further illustrate the present invention in conjunction with the above drawings.

Detailed ways

In order to more clearly understand the above objects, features and advantages of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments may be combined with each other in the case of no conflict.

In the following description, many specific details are set forth in order to facilitate a full understanding of the present invention, and the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention.

Referring to FIG. 1 , the super-resolution detection system 10 according to the embodiment of the present invention can be used to detect the biological information of the sample 20 to be tested. The sample to be tested 20 can be a nucleic acid sample (DNA or RNA), a protein or a cell or the like. In this embodiment, the sample to be tested 20 is a nucleic acid sample, and the biological information may be the base sequence information of the sample to be tested 20 .

The sample to be tested 20 is carried on the sequencing chip 30 . During the working process of the super-resolution detection system 10 , the reference light is emitted to the sample to be tested 20 . The relative movement of the sequencing chip 30 and the super-resolution detection system 10 is arranged to drive the sample to be tested 20 and the super-resolution detection system 10 to generate relative movement. In this embodiment, the sequencing chip 30 is placed on a loading platform, and the sequencing chip 30 and the sample to be tested 20 and the super-resolution detection system 10 are moved relative to each other by moving the loading platform. That is, in the process of moving the loading platform, the loading platform, the sequencing chip 30 and the sample to be tested 20 are kept stationary. On the basis of keeping the output direction of the reference light unchanged, by setting the sample 20 to be tested and the super-resolution detection system 10 to move relative to each other, the reference light can be projected to different areas on the sample 20 to be tested, and this process can also be referred to as "scanning". Since the field of view of the reference light on the sample to be tested 20 usually cannot completely cover the sample to be tested 20 , by setting the sample to be tested 20 and the super-resolution detection system 10 to move relative to each other, the super-resolution system 10 can realize the detection of the entire sample to be tested 20 . scanning.

In this embodiment, different bases on the sample 20 to be tested are marked with different fluorescent substances. When the reference light illuminates the sample to be tested 20, different fluorescent substances are excited to generate fluorescence of different wavelengths. The super-resolution system 10 is used to obtain the biological information of the sample to be tested 20 according to the fluorescence.

Please refer to Fig. 2. The solid arrows in Fig. 2 indicate the propagation direction of the laser light, and the dashed arrows indicate the propagation direction of the fluorescence. The super-resolution detection system 10 includes a light source device 11 . The light source device 11 includes a first laser 111 that emits first light and a second laser 112 that emits second light. In this embodiment, the first laser 111 and the second laser 112 are used to respectively emit laser light of different wavelengths, that is, the wavelengths of the first light and the second light are different. For example, one of the first laser 111 and the second laser 112 is used to emit red laser light, and the other is used to emit green laser light. The laser light emitted by the first laser 111 and the second laser 112 is collectively defined as light source light. In other embodiments, the light source device 11 includes other numbers of lasers, and each laser is used for emitting laser light of different wavelengths. The number of lasers may depend on the type of fluorescent substance on the sample 20 to be tested.

The light source device 11 also includes a fiber coupler 113 . The fiber coupler 113 is used to receive the first light and the second light emitted by the first laser 111 and the second laser 112, combine the first light and the second light, and output it to form light source light.

The super-resolution detection system 10 further includes a first microlens array 121 located on the exit path of the light source light, and the first microlens array 121 is used for receiving and focusing the light source light. Referring to FIG. 3 , the first microlens array 121 includes a plurality of first microlenses 1211 arranged in a regular quadrilateral array. Each of the first microlenses 1211 is used for focusing the received light source light respectively. In other embodiments, the plurality of first microlenses 1211 can also be arranged in other shapes, for example, the plurality of first microlenses 1211 are arranged in a regular triangle, regular hexagon, regular octagonal array, and the like.

Referring to FIG. 2 again, the focal length of each first microlens 1211 is the same, so the focal plane (first focal plane S1 ) of each first microlens 1211 is the same. Referring to FIG. 4 , the light from the light source is focused into multiple beams of light after passing through the first microlens array 121 , and each beam of light can respectively form a laser focused spot on the first focal plane S1 . Then, a plurality of laser focus spots can be formed on the first focal plane S1. Corresponding to the first microlens array 121 , the plurality of laser focusing spots are arranged in a regular quadrilateral array.

Please refer to FIG. 2 again, the super-resolution detection system 10 further includes a light guide assembly. After the light emitted from the first microlens array 121 passes through the light guide assembly, it is projected to the sample to be tested 20 as a reference light. The reference light also includes multiple lights. When the sample to be tested 20 is irradiated by the reference light, laser focused spots arranged in a quadrilateral array can be formed on the surface of the sample to be tested 20 .

Generally, the area of the sequencing chip 30 is relatively large, while the area of the laser focused spot array formed by the reference light is relatively small, so it is necessary to set the sequencing chip 30 to move relative to the laser spot array, so that the sample to be tested 20 and the laser spot array move relative to each other. , so as to achieve a complete scan of the sequencing chip 30 .

Referring to FIG. 5 , in this embodiment, the sequencing chip 30 is a rectangle, and the lengths of the long side and the short side are defined as W and H respectively, and the physical size of the sequencing chip 30 is W×H. The chip to be tested 30 is divided into K+1 rectangular strip regions, wherein the area of the K strip regions is W×ΔH, and the area of one strip region is W×ΔH′. in:

H=K·ΔH+ΔH' (0≤ΔH'<ΔH) (1),

ΔH is the scanning width of the laser focused spot array.

The solid arrows in FIG. 5 indicate the trajectory and direction of the relative movement between the laser focusing spot array and the sequencing chip 30 , the asterisk “*” represents the turning point of the relative movement trajectory, and the dashed line indicates the region division of the sequencing chip 30 . The mode in which the laser focused spot array scans the entire sequencing chip 30 is a typical raster scanning mode, and the scanning trajectory is like a "snake eating snake". During the entire scanning process, by continuously controlling the displacement of the sequencing chip 30 relative to the laser focused spot array, the sequencing chip 30 can be continuously scanned without interruption, which is beneficial to improve the scanning speed. And for sequencing chips of any area and shape, the divided regions of the sequencing chip can be scanned, which is beneficial to increase the scanning area of the laser focused spot array.

The laser focusing spot array includes laser focusing spots with M rows and N columns. The light intensity of each laser focused spot has a Gaussian distribution. That is, for each laser focused spot, the central light intensity is the highest, and the light intensity gradually decreases from the center to the edge. As a result, the light intensity distribution of the entire laser focusing spot array is uneven. In this embodiment, in order to effectively improve the uniformity of the light intensity distribution of the laser focused spot array, the scanning angle of the laser focused spot array is set so that the row direction or column direction of the laser focused spot array is different from the laser focused spot array and the sequencing chip. The directions of relative movement between 30 are parallel, that is, the row direction or column direction of the laser focusing spot array and the direction of relative movement between the laser focusing spot array and the sequencing chip 30 form an included angle.

Please refer to FIG. 5 and FIG. 6 together, the horizontal direction is the scanning direction when the reference light scans the sequencing chip 30 (that is, the direction of relative movement between the sequencing chip 30 and the laser focused spot array). The horizontal direction of the laser focused spot array is Starting point, rotate counterclockwise by angle θ. During the process of scanning the sequencing chip 30 with reference light, the sequencing chip 30 is scanned along the scanning direction while maintaining this angle.

Referring to FIG. 7 , in order to make the laser focusing spot array and the scanning direction have the above-mentioned rotation angle θ, the first microlens array 121 is set to also have the rotation angle θ.

Hereinafter, the calculation process of the specific value of the above-mentioned rotation angle θ will be described.

The light intensity distribution law of each laser focused spot is expressed by the following formula:

Among them, x and y represent the horizontal and vertical coordinates, respectively, A represents the amplitude of the two-dimensional Gaussian distribution, μ _x and μ _y represent the center of the two-dimensional Gaussian distribution in the x and y directions, and σ _x and σ _y represent the two The variance of the dimensional Gaussian distribution represents the degree of dispersion of the light intensity of the focused spot.

In order to simplify the calculation process, in this embodiment, only the superposition of light intensities of two adjacent laser focused spots in a single dimension (y) is analyzed. In the y direction, the one-dimensional Gaussian light intensity distribution is expressed as:

The standard deviation σ _x and σ _y characterize the degree of dispersion of the focused spot light intensity, but they are not parameters that intuitively characterize the light intensity distribution. The standard deviation σ _x and σ _y can be converted into an intuitive laser focused spot width at half maximum according to the following formula (Full width at half maximum, FWHM):

As mentioned above, Δy represents the density of the distribution of the focused spot in the longitudinal direction (y direction). Referring to Figure 8, project all laser focus spots to the right, so that all laser focus spots are aligned in the longitudinal direction (in order to clearly display the outline of each laser focus spot, the laser focus spot is represented by a hollow circle instead of a solid circle ). Based on the orientation of FIG. 8 , the spots are numbered from top to bottom as spots 1 to 64 . There is overlap between adjacently arranged spots, and the light intensity will be superimposed on the overlapping area of the laser focused spot. Since the light intensity of each laser focused spot has a Gaussian distribution, it will cause uneven light intensity distribution.

The light intensity uniformity of the laser focused spot in the longitudinal direction depends on the ratio of Δy to FWHM _y . The ratio of Δy to FWHM _y exists in the following situations:

1)

When it is larger, the center distance of adjacent spots (such as spots 1 to 2) in the longitudinal direction is larger than the half-height width, which indicates the light intensity distribution of two discrete laser focusing spots. At this time, the two laser focusing spots will not overlap. And they do not affect each other (the light intensity distribution is shown in Figure 9, the abscissa in Figure 9 represents the position, and the ordinate represents the light intensity). In this case, when the sequencing chip 30 is moved, there will be a gap between the scanning tracks of the light spot 1 and the light spot 2, so that a part of the sequencing chip 30 will be missed.

2)

When it is small, the center distance of adjacent laser focus spots (such as spot 1-2) in the longitudinal direction is smaller than the half-height width, and the adjacent laser focus spots will have a partial overlap area, and the light intensity of adjacent laser focus spots in the overlapping area overlapping.

Please refer to Figure 10 to Figure 12. In Figure 10 to Figure 12, the abscissa represents the position, the ordinate represents the light intensity, the dotted line represents the independent light intensity distribution of one laser focus spot, and the solid line represents the superposition of adjacent laser focus spots in the overlapping area. after the light intensity distribution. In comparison, the uniformity of the light intensity distribution shown in Figure 10 is low (the light intensity at different positions varies greatly), while the light intensity distribution shown in Figure 11 has a high uniformity (the light intensity difference at different positions is large). smaller). Although the uniformity of the light intensity distribution shown in Figure 11 is relatively high, the overlapping area between the adjacent laser focus spots is relatively large, resulting in a high light intensity superimposed on the overlapping area. Therefore, when the overlapping portion of the

light spots

1 and 2 scans the sequencing chip 30, the sequencing chip 30 may be over-illuminated, resulting in the phenomenon of "phototoxicity".

3)

When certain conditions are met, the uniformity of light intensity distribution in the longitudinal direction of all longitudinally arranged 64 laser focusing spots can meet the requirements. The uniformity of light intensity distribution is expressed as:

The value of light intensity distribution uniformity is located in [0,1]. The closer the value of the light intensity distribution uniformity is to 1, the better the light intensity distribution uniformity, and the smaller the light intensity difference of the reference light received by different DNA nanospheres on the sequencing chip 30 .

In this embodiment, the uniformity of light intensity distribution is generally required to be greater than or equal to 85%, that is, the requirement (maximum illumination light intensity-minimum illumination light intensity)/mean value of illumination light intensity≤15%. Under this constraint, the theoretically calculated limit center distance of two adjacent laser focused spots in the longitudinal direction is 2.4775σ _y , and the uniformity of light intensity distribution at this time is 85%. Extending this restriction to the case of multiple laser focused spots, a relatively uniform light intensity distribution can be obtained (as shown in Figure 12). In this way, when the sequencing chip 30 and the laser focusing spot move relatively, the laser focusing spots 1 to 64 can achieve more uniform illumination for the sequencing chip 30 .

In this embodiment, the number of microlenses 121 in the first microlens array 121 is M×N (M is the number of rows, N is the number of columns), and the rotation angle θ should make the laser beams formed by all M×N microlenses 1211 focused The spot distribution is uniform in the longitudinal direction. Assuming that the linear distance between the centers of adjacent laser focus spots is ΔL, all M×N laser focus spots are uniformly distributed in the longitudinal direction, and the longitudinal distance of adjacent laser spots is Δy, the following calculation formula is established:

It can be seen that the rotation angle θ only depends on the number N of columns of the first microlens array 121 , and the larger the value of N is, the smaller the rotation angle θ is. Δy then depends on both ΔL and N. The larger ΔL and the smaller N, the larger Δy, and the sparser the distribution of all M×N laser focused spots in the longitudinal direction. The smaller ΔL and the larger N, the smaller Δy, and the denser the distribution of all M×N laser focused spots in the longitudinal direction.

Please refer to FIG. 8 again. In this embodiment, M=N=8, so there are totally 64 laser focusing spots (numbered 1-64), which are arranged in a square grid with 8 rows and 8 columns. In order to conveniently calculate the rotation angle θ, the laser focus spot 9 is moved horizontally to the left, and its horizontal movement trajectory intersects with the extension line of the connecting lines of the laser focus spots numbered 1 to 8, and the intersection is the 9' position. According to the above, the rotation angle θ should make the distribution of the laser focus spots formed by all M×N microlenses 1211 uniform in the longitudinal direction, so the laser focus spots 1 to 8 and 9' of the nine laser focus spots in the longitudinal direction have a uniform distribution in the longitudinal direction. The distribution is naturally uniform, so the distance between the centers of laser focus spots 1 and 9' is 8ΔL, and the distance between the centers of laser focus spots 1 and 9 is ΔL. Since the microlenses 1211 are arranged in a square grid, the angle ∠919' is a right angle. The way to solve the rotation angle θ in the right triangle ⊿919' is:

and

In this way, the specific value of the rotation angle θ can be determined. By controlling the overall rotation angle θ of the first microlens array 121 , when the reference light is irradiated on the sequencing chip 30 , the formed laser focusing spot array also has a rotation angle θ. The above process is beneficial to improve the laser focusing spot array on the sequencing chip 30 The uniformity of the light intensity distribution.

FIG. 13 shows the process of scanning a certain strip area on the sequencing chip 30 with reference light. During the process of scanning the sequencing chip 30 with the reference light, the outgoing direction of the reference light remains unchanged. The sequencing chip 30 is driven to move to the left at a constant speed, so that the laser focused spot array formed by the reference light on the sequencing chip 30 and the sequencing chip 30 move relatively. The direction of the relative movement is parallel to the long sides of the rectangular strip.

Figures (a) to (i) in FIG. 13 respectively show the relative positions of the laser focused spot array and the sequencing chip 30 at a certain moment. (When describing the orientation below, the orientation shown in FIG. 6 is used as a reference, and the longitudinal direction of the rectangular strip-shaped region in FIG. 6 is the horizontal direction).

Referring to (a) of FIG. 13 , the laser focused spot array just begins to scan the sequencing chip 30 , and no laser focused spot is projected onto the surface of the sequencing chip 30 , and no detection light is generated at this time.

Referring to Fig. 13 (b), several light spots at the lower right corner of the laser focused spot array have been projected onto the sequencing chip 30 due to the displacement of the sequencing chip 30, and the sequencing chip is scanned, resulting in several strip-shaped scanning tracks .

Referring to (c) in FIG. 13 , with the further movement of the sequencing chip 30 , the spot on the right half of the laser focused spot array has realized the scanning of the sequencing chip 30 , resulting in a strip-shaped scanning track, which is in the horizontal direction. In the vertical direction, the scanning trajectories of adjacent laser focus spots overlap slightly, thereby forming multiple relatively independent scanning areas.

Referring to (d) in FIG. 13 , the sequencing chip 30 continues to move, most of the laser focused spots have been transmitted to the surface of the sequencing chip 30 , and multiple relatively independent scanning areas have all been connected into a continuous area.

Referring to Fig. 13 (e), the sequencing chip 30 continues to move to the left, all the laser focusing spots have been projected onto the surface of the sequencing chip 30, and have been scanned for a distance from left to right, and the scanning trajectory of each laser focusing spot is the same Scanning of the sequencing chip 30 is achieved.

Referring to (f) in FIG. 13 , the sequencing chip 30 continues to move to the left, and the laser focused spot array has gradually scanned out of the strip area.

Referring to (g) to (i) in FIG. 13 , the sequencing chip 30 continues to move to the left until the laser focusing spot on the far left scans out of the strip area. complete scan of the region.

At the next moment, continue to scan the next adjacent strip region with the scanning trajectory as shown in FIG. 5 until the entire sequencing chip 30 is scanned.

Please refer to FIG. 2 again, when the sample to be tested 20 is irradiated with the reference light, the detection light is generated. The detection light is received by the super-resolution detection system 10 . In this embodiment, when the sample to be tested 20 is irradiated with the reference light, four different wavelengths of fluorescence are generated. The above-mentioned four different wavelengths of fluorescence are used together as detection light. The detection light emitted from the sample to be tested 20 is incident into the light guide assembly.

The super-resolution detection system 10 also includes at least one Time Delay Integration (TDI) camera. The light guide module is also used to guide the detection light into the at least one TDI camera. The super-resolution detection system 10 in this embodiment includes four TDI cameras, and the four TDI cameras are

TDI cameras

141 , 142 , 143 and 144 respectively. The light guide module is used to guide the fluorescence of four different wavelengths in the detection light to different TDI cameras respectively. Each TDI camera is used for photoelectric conversion based on the received fluorescence to generate image information. The image information can finally obtain the biological information of the sample 20 to be tested after further data processing. In other embodiments, the detection light may include different amounts of fluorescence with different wavelengths, such as fluorescence with two different wavelengths, and the super-resolution detection system 10 may include two TDI cameras.

The following describes the process of TDI camera receiving detection light:

When the detection light is projected onto the target surface of each TDI camera, an array of fluorescent focusing spots is formed on the target surface of the TDI camera. Each TDI camera is used for photoelectric conversion according to the received detection light, thereby generating an electrical signal corresponding to the detection light. The working principle of each TDI camera is basically the same, and the working process of one of the TDI cameras is described below.

Please refer to FIG. 14 , the fluorescent focusing spot array is coincident with the photosensitive array of the TDI camera, and each solid line arranged vertically in FIG. 14 represents each stage of the linear photosensitive element of the TDI camera. The relative movement between the laser focused spot array and the sequencing chip 30 is realized by moving the sequencing chip 30 , and the relative movement realizes the scanning of the sequencing chip (object plane) 30 . The fluorescence focusing spot array and the target surface of the TDI camera are relatively static physically. However, since the charge of the TDI camera will be transferred step by step from left to right, it can be regarded as a relative movement between the fluorescent focusing spot array and the charge of the TDI camera, and this relative movement realizes the scanning of the target surface (image surface) of the TDI camera. The object plane corresponds to the scan of the image plane.

Figure (a) in Figure 14 corresponds to Figure (a) in Figure 13 . At the moment shown in (a) of FIG. 15 , the fluorescent focused spot array has not scanned the surface of the sequencing chip 30, and no fluorescent focused spot is projected on the target surface of the TDI camera (the open circles indicate that the target surface of the TDI camera is not The state of receiving the fluorescent focused spot).

Figure (b) in Figure 14 corresponds to Figure (b) in Figure 13. At this time, there are several laser focused spots on the right side of the laser focused spot array that are scanned onto the surface of the sequencing chip 30, so several fluorescent focused spots are also projected to the TDI accordingly. The target surface of the camera (solid circles indicate the state that the target surface of the TDI camera receives the fluorescent focused spot).

Figures (c) and (d) in Figure 14 correspond to Figures (c) and (d) in Figure 13. With the relative movement of the sequencing chip 30 and the laser focus spot array, more and more laser focus spots are scanned to On the surface of the sequencing chip 30, there are correspondingly more fluorescent focused spots projected onto the target surface of the TDI camera.

Figure (e) in Figure 14 corresponds to Figure (e) in Figure 13. When all laser focused spots are scanned onto the surface of sequencing chip 30, all corresponding fluorescent focused spots are projected onto the target surface of the TDI camera.

Figures (f) to (i) in Figure 14 correspond to Figures (f) to (i) in Figure 13 . When the laser focused spot array scans to the tail of a strip-shaped area of the sequencing chip 30, the laser focused spot array gradually Moving out of this band-shaped area, the number of fluorescent focused spots on the target surface of the TDI camera will gradually decrease until all of them disappear.

The characteristic of the TDI camera is that the charges generated by the detected light can be transferred and accumulated step by step. Please refer to Fig. 15. When all the laser focused spots are scanned onto the surface of the sequencing chip 30, the fluorescent focused spot array is projected onto the target surface of the TDI camera, and photoelectric conversion occurs to generate corresponding electrical signals. Each fluorescent focused spot corresponds to an electrical signal. Signal. The electrical signals are successively transferred to the next stage, thereby forming a strip-shaped image on the target surface of the TDI camera, and finally read out from the last stage of the multi-stage line-array photosensitive element of the TDI camera.

In (a) of FIG. 15 , the fluorescent focused spot array generates charges (electrical signals) at positions corresponding to the target surface of the TDI camera. At this time, the last stage of the linear array photosensitive element of the TDI camera only receives the electrical signal formed by a fluorescent focusing spot in the lower right corner, so the readout of the TDI camera is shown on the right side of (a) in Figure 16. There is only one Discrete spots.

In (b) of Figure 15, the fluorescent focused spot array is exposed at the original position, and photoelectric conversion occurs again, converting the fluorescent signal into an electrical signal. The charge generated in Figure 6a is transferred to the lower part of the multi-level linear array photosensitive element The first stage, the last stage of the linear array photosensitive element receives the electrical signal formed by the two light spots in the lower right corner. The readout of the TDI camera is shown in Figure 16(b), with two discrete light spots.

Please refer to Figures (c) to (e) of Figure 15. With the step-by-step transfer of charges (electrical signals), the last-stage linear photosensitive element of the TDI camera gradually receives the Nth (N=8) row of fluorescent focusing spots. The generated charges, at this time, are M (M=8) discrete light spot signals in total.

Please refer to (f) of FIG. 15 , the charges continue to be transferred step by step, and the charges generated by the fluorescent focusing spot in the N-1 (N-1 = 7) column are transferred step by step to the last linear photosensitive element and read out. At this time, a total of M discrete fluorescent focusing spots are read out. At this time, the discrete fluorescent focused spot is composed of two lines of spot signals, so compared with the spots in (a) to (d) in FIG. 15 , it is longer in the longitudinal direction.

Please refer to (g) of FIG. 15 , the charges generated by the fluorescent focusing spot in the N-2 (N-2=6) column are transferred to the last linear photosensitive element step by step and read out.

Please refer to (h) of Figure 15. When the charges generated by the fluorescent focusing spot in the first column are transferred to the last-stage linear photosensitive element step by step and read out, the discrete fluorescent focusing spots can be connected together. The images that can be read out are continuous.

The laser focused spot array starts to scan from the position shown in (a) in FIG. 13 and ends at the position shown in (i) in FIG. 13 , completing the scanning of a single strip area of the sequencing chip 30 .

The TDI camera continued to read the image during the above scanning process, and the obtained image is shown in Fig. 15(i). Due to the rotation angle θ of the first microlens array 121 , the images formed by each fluorescent focused spot in (i) of FIG. 15 are staggered. In order to make the obtained image consistent with the real sequencing chip 30 , it is necessary to perform a simple alignment operation on the image generated by the TDI camera, and the subsequent image is shown in (j) of FIG. 15 .

The laser focusing spot array and the sequencing chip 30 move relative to each other, and the charge transfer of the TDI camera is equivalent to making the fluorescence focusing spot and the TDI camera move relative to each other. Then the relative movement speed between the laser focusing spot array and the sequencing chip 30 is the same as that of the TDI camera. The frame rate of satisfies the following relationship:

The maximum frame rate of the TDI camera is expressed as f _max Hz, and the maximum speed of the relative motion between the laser focusing spot array and the sequencing chip 30 is expressed as υ _max mm/s. In this embodiment, taking the object surface 1 mm as an example for calculation, when the relative movement between the laser focusing spot array and the sequencing chip 30 is the above-mentioned maximum speed, the time required for moving 1 mm is

Assuming that the magnification of the TDI camera is Mag, and the width of the pixels of the TDI camera along the charge transfer direction is expressed as w mm, the number of series of linear photosensitive elements occupied by the 1mm object surface imaged to the target surface of the TDI camera is:

The time required for each stage of charge transfer is

In order to enable the information of the object surface to be collected in time by the TDI camera, the minimum required charge transfer frame rate is:

Therefore, the relationship between the maximum frame rate of the TDI camera and the maximum speed of the relative movement between the laser focusing spot array and the sequencing chip 30 is:

Please refer to FIG. 2 again. In this embodiment, the super-resolution detection system 10 further includes a second microlens array 151 and a filter layer 152 . The second microlens array 151 is used for focusing the detection light, and the filter layer 152 is used for filtering out stray light in the detection light, so as to enhance the contrast of the images generated by the time-

lapse integrator cameras

141 , 142 , 143 and 144 .

Referring to FIG. 16 , in this embodiment, the second microlens array 151 includes a plurality of second microlenses 1511 . Each microlens 1511 is used to focus the received detection light respectively. Corresponding to the reference light, the detection light also includes a plurality of beams. The detection light can form a plurality of fluorescent focused spots arranged in an array on the second microlens array 151 . The plurality of fluorescent focusing spots are in one-to-one correspondence with the plurality of second microlenses 1511 , that is, the plurality of light beams in the detection light are in one-to-one correspondence with the plurality of second microlenses 1511 . Each second microlens 1511 is used for focusing a corresponding beam of light.

In other embodiments, the plurality of second microlenses 1511 can also be arranged in other shapes, for example, the plurality of second microlenses 1511 are arranged in a regular triangle, regular hexagon, regular octagonal array, and the like. The structure of the second microlens array 151 can be kept the same as that of the first microlens array 121 .

The focal length of each second microlens 1511 is the same, and thus the focal plane (second focal plane S2 ) of each second microlens 1511 is the same. After the detection light passes through the second microlens array 151, a plurality of fluorescent focusing light spots can be formed on the second focal plane S2. A plurality of fluorescent focused spots are arranged in a regular quadrilateral array.

In this embodiment, the focal length of the second microlens 1511 is one-half of the focal length of the first microlens 1211 , so the multiple beams formed by focusing in the detection light are respectively focused again.

Referring to FIG. 17 , in this embodiment, the filter layer 152 includes a non-transparent plate-like structure 1521 , and the plate-like structure 1521 is provided with a plurality of circular holes 1522 of the same size. The number of the circular holes 1522 is the same as the number of the second microlenses 1511 in the second microlens array 151 . The plurality of circular holes 1522 are in one-to-one correspondence with the plurality of microlenses 1511 .

Please refer to FIG. 2 and FIG. 17 together, each circular hole 1522 is located at the focal point of its corresponding microlens 1521 , that is, the plane where each circular hole 1522 is located is the same as the second focal plane S2 .

The detection light emitted from each second microlens 1511 is at least partially incident to the only one corresponding circular hole 1522 and exits from the circular hole 1522 . Since the circular hole 1522 is opened on the opaque plate-like structure 1521, the detection light accurately incident to the circular hole 1522 can pass through the filter layer 152, and the rest of the detection light is blocked by the opaque plate-like structure 1521 and cannot be transmitted from the light-tight plate structure 1521. The filter layer 152 exits. The filter layer 152 in this embodiment is beneficial to filter out stray light at the focus of each second microlens 1511 .

As shown in FIG. 2 , in this embodiment, the detection light first passes through the second microlens array 151 , and then passes through the filter layer 152 . In a modified embodiment, the detection light can also be set to pass through the filter layer 152 first, and then pass through the second microlens array 151 . That is, in this modified embodiment, each second microlens 1511 is used for focusing the detection light emitted from the corresponding circular hole 1522 .

Compared with the modified embodiment, the diameter of the circular hole 1522 of the filter layer 152 in this embodiment is smaller.

By focusing the detection light again and filtering out the stray light at the focus of each second microlens 1511 , the contrast of the images generated by the time-

lapse integration cameras

141 , 142 , 143 and 144 can be improved.

As described above, to improve the light intensity distribution uniformity of the laser focused spot array formed by the reference light on the sequencing chip 30 , the entire laser focused spot array is set to have a rotation angle θ relative to the scanning direction. Correspondingly, as shown in FIGS. 16 and 17 , both the second microlens array 151 and the filter layer 152 are also arranged to be rotated by an angle θ.

Please refer to FIG. 2 again, the light guide module includes a plurality of optical elements for guiding light (eg, light source light, reference light, detection light, etc.).

In this embodiment, the light guide module includes a lens 1311 , a lens 1312 and a lens 1313 arranged in sequence between the light source device 11 and the first microlens array 121 . The lens 1311 is used for collimating the light source light to emit parallel light source light. The

lenses

1312 and 1313 are used to jointly perform beam expansion processing on the light source light emitted by the lens 1311, so as to expand the diameter of the light source light. By adjusting the ratio between the distances of the

lenses

1312 and 1313, the magnification of the light diameter of the light source can be adjusted.

The light guide module further includes a lens 1314 , a dichroic mirror 1315 , a reflector 1316 , a lens 1317 , a lens 1318 , a lens 1319 and a lens 1320 . The lens 1315 is used to collimate the multiple beams of light emitted by the microlens array 121 respectively, and the collimated multiple beams pass through the dichroic mirror 1315, the mirror 1316, the lens 1317, the lens 1318 and the lens 1319 in sequence, and are used as a reference Light is projected onto the sample 20 to be tested. When the sample to be tested 20 is irradiated with the reference light, fluorescence is generated, and the fluorescence is incident on the second microlens array 151 as the detection light through the lens 1319 , the lens 1318 , the lens 1317 , the mirror 1316 , the dichroic mirror 1315 and the lens 1320 in sequence. Dichroic mirror 1315 is used to transmit laser light and reflect fluorescent light. The mirror 1316 is used to reflect the received light. Lens 1314 and lens 1318 are used to collimate the received light. The light exiting from the lens 1317 may form an array of laser focused spots arranged in an array at its focal plane. The lens 1319 is used to focus the received light and project it to the sample 20 to be tested. The focal position of the lens 1320 coincides with the focal position of the microlens array 131 . The focal plane of the lens 1320 can form an array of fluorescent focusing spots arranged in a square.

The light guide module further includes a lens 1321 , an emission mirror 1322 and

dichroic mirrors

1323 , 1324 and 1325 . The lens 1321 is used to collimate the received beam. The mirror 1322 is used to reflect the received light beam, and the

dichroic mirrors

1323, 1324 and 1325 are respectively used to split the received light beam according to the wavelength, that is, the

dichroic mirrors

1323, 1324 and 1325 respectively allow only specific wavelengths (or wavelength bands) of light pass through, thereby directing four wavelengths of fluorescence to

TDI cameras

141, 142, 143, and 144, respectively.

The light guide module further includes four filters 1326 and four lenses 1327 respectively corresponding to the four TDI cameras. The detection light (fluorescence) of the four wavelengths is filtered by a filter 1326 and then focused by a lens 1327 on the target surface of a TDI camera.

In the optical path shown in FIG. 2 , the focal planes of lens 1311 and lens 1312 are conjugate focal planes, the focal planes of

lenses

1314 , 1317 , 1318 , 1319 , 1320 and 1321 are conjugate focal planes, and the focal planes of four lenses 1327 is the conjugate focal plane. The focal planes that are conjugate to each other are equivalent focal planes.

The focal lengths of the

lenses

1314, 1317, 1320, and 1321 can be set to be the same, so that when the focal lengths of the lens 1319 (objective lens) and the lens 1327 are both determined, the focal length of the lens 1318 can be changed to adjust the beam diameter of the reference light. Adjust the imaging magnification of the TDI camera.

In other embodiments, the light guide module may have various types and numbers of optical elements. The specific structure of the light guide module is not limited. The specific structure of the light guide module is configured according to the specific construction method of the light path. The description of the specific structure of the light guide module in this application is only an example.

In this embodiment, the super-resolution detection system 10 further includes a necessary control device (not shown in the figure) to realize the control function. For example, the control device can be used to control the first laser 111 and the second laser 112 to emit laser light, to control the movement of the sequencing chip 30 and so on. The control device can be, for example, a computer, a control chip, or the like.

This embodiment also provides a super-resolution detection method, which is applied to the above-mentioned super-resolution detection system. Referring to FIG. 18 , the super-resolution detection method provided by this embodiment includes the following steps:

Step S1, emitting reference light to the sample to be tested, and the reference light can form a focused spot array on the sample to be tested;

Step S2, controlling the sample to be tested and the focused spot array to generate continuous relative motion, so that the sample to be tested continues to generate detection light;

Step S3, focusing the detection light and filtering out stray light in the detection light;

Step S4, receiving the detection light after focusing and filtering out the stray light with at least one time-lapse integrator, and performing continuous imaging according to the detection light, so as to obtain the biological information of the sample to be tested, and the detection light is in the A focused light spot array is formed on the target surface of at least one time-lapse integrator camera.

The above method steps are consistent with the working process of the aforementioned super-resolution detection system 10 , and will not be repeated here. Specifically: for step S1, please refer to the aforementioned description of the light source device 11 and the first microlens array 121; for step S2, please refer to the aforementioned description of the scanning process in FIG. 5 and FIG. 13; for step S3, please refer to the aforementioned description of the second microlens array 151 and the description of the filter layer 152; for step S4, please refer to the foregoing description of the working process of the TDI camera.

In the super-resolution detection system 10 and the super-resolution detection method provided by this embodiment, in the first aspect, the first microlens array 121 , the second microlens array 151 and the filter layer 152 are arranged, and the sequencing chip and the laser focusing spot array are arranged. The relative motion is generated, so that the laser focusing spot can continuously scan the sequencing chip (see the scanning method shown in Figure 5); further, through the charge movement on the TDI camera, the fluorescent focusing spot array formed by the TDI camera and the detection light is formed Relative motion occurs, enabling continuous imaging based on detection light. Compared with the method of using area scan camera imaging in the prior art, the super-resolution detection system 10 based on the line scanning mode of the TDI camera provided in this embodiment is conducive to realizing super-resolution and improving the detection speed (the speed can be increased to 5 times when using an area scan camera), thereby reducing the inspection cost.

Second, since the laser focused spot continuously scans the sequencing chip, when the area of the sequencing chip is large, the sequencing chip can also be continuously scanned by partition, which is beneficial to improve the sequencing throughput of the super-resolution detection system 10 .

In the third aspect, through the second microlens array 151 , the fluorescent focusing light spot can be further focused, which is beneficial to improve the detection accuracy of the super-resolution detection system 10 . By arranging the filter layer 152 , stray light can be filtered out, which is beneficial to improve the contrast of the images generated by the

TDI cameras

141 , 142 , 143 and 144 .

In the fourth aspect, by setting the rotation angle θ of the first microlens array 121, the second microlens array 151 and the filter layer 152, it is beneficial to make the light intensity distribution of the laser focusing spot more uniform, thereby helping to obtain more accurate detection results. .

In the fifth aspect, by adjusting the beam size of the reference light, it is beneficial to better match the field of view (FOV) size of the objective lens (lens 1319 ), so that the objective lens FOV can be fully utilized. By adjusting the imaging magnification, it is beneficial to match the size of the target surface of each TDI camera, so as to make full use of every pixel on the target surface of the TDI camera.

Those of ordinary skill in the art should realize that the above embodiments are only used to illustrate the present invention, rather than being used to limit the present invention, as long as the above embodiments are suitable for the scope of the spirit of the present invention Variations and variations fall within the scope of the claimed invention.

Claims

A super-resolution detection system for detecting biological information of a sample to be tested, characterized in that the super-resolution detection system comprises:

a light source device for emitting light from the light source;

a first microlens array, used for receiving and focusing the light source light to generate reference light, the reference light is used for scanning the sample to be tested so that the sample to be tested generates detection light, the reference light scans the When the sample is to be tested, a focused spot array is formed on the sample to be tested;

a second microlens array and a filter layer, located on the optical path of the detection light, for focusing the detection light and for filtering out stray light in the detection light; and

At least one time-lapse integration camera, which is used to receive the detection light, and obtain the biological information of the sample to be tested according to the detection light.
The super-resolution detection system according to claim 1, wherein when the reference light is projected on the sample to be tested, the sample to be tested and the reference light continue to move relative to each other until the reference light completes scanning the sample to be tested.
The super-resolution detection system according to claim 2, wherein the detection light forms a focused spot array on the at least one time-lapse integrator camera, and the at least one time-lapse integrator camera is used to measure the focused spot according to the The array generates electric charge;

The focused light spot array and the electric charge are continuously moved relative to each other, and the at least one time-lapse integral camera is used for continuous imaging according to the electric charge, so as to obtain the biological information of the sample to be tested.
The super-resolution detection system according to claim 1, wherein the maximum frame frequency of the at least one time-lapse integrator camera is defined as f max Hz, and the relative relationship between the focused spot array formed by the reference light and the sample to be tested is defined. The maximum speed of movement is υ max mm/s, the width of the pixel along the charge transfer direction of the at least one time-lapse camera is defined as w, and the magnification of the at least one time-lapse camera is defined as Mag, then:
The super-resolution detection system according to claim 1, wherein the focused spot array formed by the reference light comprises multiple rows and multiple columns, and the row direction or column direction of the focused spot array is the same as that of the focused spot array and The directions of relative movement between the samples to be tested form an included angle θ.
The super-resolution detection system according to claim 5, wherein the focused light spot array formed by the reference light comprises N columns, and the distance between the centers of the adjacently arranged focused light spots is defined as ΔL, and the defined The distance of the light spot in the column direction is Δy, then:
The super-resolution detection system according to claim 1, wherein the first microlens array comprises a plurality of first microlenses arranged in an array, and the focal planes of each first microlens are the same, and the focal planes of the first microlenses are the same. The reference light emitted from the first microlens can form a plurality of focused light spots arranged in an array on the focal plane.
The super-resolution detection system according to claim 1, wherein the second microlens array comprises a plurality of second microlenses arranged in an array, and the focal planes of each second microlens are the same. The detection light emitted from the second microlenses may form a plurality of focused light spots arranged in an array on the focal plane of the plurality of second microlenses.
The super-resolution detection system according to claim 8, wherein the filter layer is provided with a plurality of holes, and each hole corresponds to a second microlens;

Each hole is used for filtering out stray light in the light emitted by the corresponding second microlens, or each second microlens is used for focusing the detection light emitted by the corresponding hole.
The super-resolution detection system according to claim 9, wherein each hole is used to filter out stray light in the light emitted by the corresponding second microlens, and each hole is located in the plurality of second microlenses. on the focal plane of the lens.
The super-resolution detection system according to claim 1, wherein the light source light is laser light, and the detection light is fluorescence;

The sample to be tested is a nucleic acid sample, and the biological information is base sequence information of the sample to be tested.
A super-resolution detection method for detecting biological information of a sample to be tested, the super-resolution detection method being applied to a super-resolution detection system, and characterized in that the super-resolution detection method comprises the following steps:

Sending reference light to the sample to be tested, the reference light can form a focused spot array on the sample to be tested;

controlling the sample to be tested and the focused spot array to generate continuous relative motion, so that the sample to be tested continues to generate detection light;

focusing the detection light and filtering out stray light in the detection light;

The detection light after focusing and filtering out stray light is received by at least one time-lapse integrating camera, and continuous imaging is performed according to the detection light to obtain the biological information of the sample to be tested, and the detection light is in the at least one delay time. A focused spot array is formed on the target surface of the time-integrating camera.