Sequencing photographing imaging system and sequencing photographing imaging method

Abstract

The invention discloses a sequencing photographing imaging system and a sequencing photographing imaging method. The sequencing photographing imaging system comprises an illumination module, a sample carrier, an objective lens and an imaging module. The lighting module comprises a first light source which is used for emitting exciting light. The sample carrier is used for carrying a DNA sample, and four basic groups of the DNA sample are respectively marked by different fluorescent dyes. The objective lens is used for receiving the exciting light and emitting the exciting light to a DNA sample carried on the sample carrier, and the objective lens is also used for receiving four kinds of fluorescence with different wavelengths emitted from the DNA sample. And the imaging module comprises four cameras, and the four cameras are used for respectively recording four kinds of fluorescence with different wavelengths emitted after being received by the objective lens.

Description

[0001] This application is a divisional application of a patent application with an application date of February 12, 2020, application number 202080095539.3, and invention name “Optical imaging system and biochemical substance detection system using the optical imaging system”. Technical Field

[0002] The present invention relates to the field of detection, and in particular to a sequencing photographic imaging system and a sequencing photographic imaging method. Background Art

[0003] Imaging analysis instruments used in biochemical analysis, such as gene sequencers, often use optical imaging systems based on highly sensitive scientific-grade area array cameras. Because the area of the sequencing chip to be tested is typically much larger than the single visible area of the optical imaging system's objective lens, a row-by-column scanning method is required to capture the entire sequencing chip's test area. Data splicing is then performed during the data analysis phase.

[0004] Traditional scientific-grade area array cameras typically use a platform-stepping design for row-by-row scanning. That is, the platform carrying the sequencing chip is moved to meet the needs of different photo positions. When the platform moves to a certain photo position, it needs to slow down to a standstill before taking the photo, otherwise it will produce a ghosting effect in the photo. This requires the platform to have the ability to quickly stop after high-speed movement, and then resume high-speed movement after taking the photo. However, in existing technologies, both platform acceleration and deceleration require considerable time, which has become a bottleneck for high-throughput optical sequencing.

[0005] In addition, the microscope objective lens, a key component of the optical imaging system, usually cannot achieve both a large field of view and a large numerical aperture, and is not suitable for high-throughput optical imaging systems. Summary of the Invention

[0006] In order to solve some or all of the above problems and other potential problems in the prior art, it is necessary to propose a sequencing photography imaging system and a sequencing photography imaging method.

[0007] One embodiment of the present application provides a sequencing camera imaging system, comprising an illumination module, a sample carrier, an objective lens, and an imaging module. The illumination module comprises a first light source for emitting excitation light. The sample carrier is used to carry a DNA sample, wherein the four bases of the DNA sample are respectively labeled with different fluorescent dyes. The objective lens is used to receive the excitation light and emit the excitation light onto the DNA sample carried by the sample carrier. The objective lens is also used to receive four fluorescent lights of different wavelengths emitted from the DNA sample. The imaging module comprises four cameras, each of which is used to respectively record the four fluorescent lights of different wavelengths emitted after being received by the objective lens.

[0008] Another embodiment of the present application provides a sequencing photography method, which is performed using the above-mentioned sequencing photography imaging system, and four cameras respectively record four types of fluorescence with different wavelengths emitted after being received by the objective lens.

[0009] In a first aspect, an optical imaging system is provided for photographing and imaging a sample, wherein the optical imaging system comprises an illumination module and an imaging module, wherein the illumination module is used to output excitation light, and the excitation light is used to excite the sample to generate stimulated light, and the imaging module comprises a time-delayed integral linear array camera, and the time-delayed linear array camera is used to record the stimulated light.

[0010] In a second aspect, a biochemical substance detection system is provided, wherein the biochemical substance detection system includes the above-mentioned optical imaging system.

[0011] The sequencing imaging system and the sequencing imaging method provided in the embodiments of the present invention have the following beneficial effects: 1. An industrial-grade or scientific-grade TDI camera is used to collect fluorescence, which reduces the imaging time for each sample and improves the detection throughput; 2. A filtering system composed of a spectrometer and a filter device is used, which can, on the one hand, separate fluorescence into multiple channels for imaging, and, on the other hand, prevent the interference of excitation light and other light, such as detection light, on fluorescence imaging, thereby improving the imaging quality; 3. The output end face of the light source is rectangular, or further slender rectangular, and the excitation light shaping unit is used to shape the excitation light output by the light source into an illumination spot that is compatible with the photosensitive surface of the TDI camera, which is very convenient. It maximizes the use of light source energy and improves the uniformity of the illumination spot; 4. A specially structured objective lens is used to meet the requirements of a large field of view and a large numerical aperture, while reducing distortion and flat-field achromatism; 5. A focusing system is used to adjust the distance between the objective lens and the sample carrier in real time, so that the sample carrier remains on the focal plane of the objective lens, compensating for changes in the distance between the sample carrier and the objective lens caused by changes in ambient temperature, mechanical vibration, sample bending or other reasons, thereby ensuring the quality of imaging; 6. The focusing system uses a specific method to calculate the distance between the sample carrier and the objective lens, effectively eliminating the influence of signal noise and changes in the brightness of the detection light source on the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0013] Figure 1 Schematic diagram of the optical imaging system in one embodiment of the present invention.

[0014] Figure 2 This is a schematic diagram comparing the time it takes to take a picture per line between TDI camera imaging and traditional area array camera imaging.

[0015] Figure 3 Schematic diagram of the optical structure of the objective lens in one embodiment of the present invention.

[0016] Figure 4 yes Figure 3 The full-field, full-band MTF curve of the objective lens shown.

[0017] Figure 5 yes Figure 3 Distortion curve diagram of the objective lens shown.

[0018] Figure 6 yes Figure 3 Envelope energy diagram for the objective shown.

[0019] Figure 7 The present invention is an embodiment of the tube mirror and Figure 3 Schematic diagram of the optical structure of the combination of objective lenses shown.

[0020] Figures 8A-8D yes Figure 7 Wavefront error curves of the objective lens and tube lens combination in imaging channels 1-4 are shown.

[0021] Figure 9 yes Figure 7 Distortion curve diagram of the combination of objective lens and tube lens shown.

[0022] Figure 10 Schematic diagram of the output end face of a light source in one embodiment of the present invention.

[0023] Figure 11 The excitation light shaping unit and Figure 3 Schematic diagram of the optical structure of the combination of objective lenses shown.

[0024] Figure 12 The excitation light shaping unit and the Figure 3 Schematic diagram of the optical structure of the combination of objective lenses shown.

[0025] Figure 13 Schematic diagram of the photosensitive surface of a TDI camera in one embodiment of the present invention.

[0026] Figure 14 is the image plane illumination map obtained by simulation.

[0027] Figure 15 yes Figure 1 The control principle diagram of the optical imaging system shown.

[0028] Figure 16 for Figure 1 The schematic diagram of the focusing module of the optical imaging system performing focusing is shown.

[0029] Figure 17 for Figure 1 The optical path principle diagram of the focusing module of the optical imaging system shown in FIG.

[0030] Figures 18A-18C for Figure 17 Schematic diagram of the sensor of the focus module sensing the reflected detection light spot.

[0031] Figure 19 for Figure 1 The signal spectra obtained by measuring when the optical imaging system is focused.

[0032] Figure 20 FIG. 4 is a schematic diagram of a biochemical substance detection system according to an embodiment of the present invention.

[0033] Figure 21 FIG. 4 is a schematic diagram of a biochemical substance detection system in another embodiment of the present invention.

[0034] The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings.

[0035] Description of main component symbols

[0036] Optical imaging system 1 Lighting module 11

[0037] Focus module 13 Imaging module 15

[0038] Objective lens 16 Light source 111, 1301

[0039] Excitation light shaping unit 113 Sample carrier 2

[0040] TDI camera 151, tube lens 152, 1303

[0041] Cut-off filter 153, 116 Spectrometer 154

[0042] Dichroic mirrors A, B, C, plane mirror 115

[0043] D.E

[0044] Photo shooting time t1, t2 first group of lenses 161

[0045] Second lens group 162 Third lens group 163

[0046] Fourth lens group 164 Fifth lens group 165

[0047] Sixth lens group 166 Seventh lens group 167

[0048] First lens L1 Second lens L2

[0049] Third lens L3 Fourth lens L4

[0050] Fifth lens L5 Sixth lens L6

[0051] Seventh lens L7 Eighth lens L8

[0052] Ninth lens L9 Tenth lens L10

[0053] Eleventh lens L11 Twelfth lens L12

[0054] Thirteenth lens L13 Fourteenth lens L14

[0055] Multimode fiber 1111 output end face 1112

[0056] First shaping unit 113a Second shaping unit 113b

[0057] Cylindrical lenses C1, C2, photosensitive surface 1511

[0058] C3, C4

[0059] Control device 17 Movable platform 18

[0060] Computer device 19 Detection light path 130

[0061] Sensing unit 131 Control unit 132

[0062] Sensor 1311 Reflector 1305

[0063] PD sensor 1312 sensing areas 1312a, 1312b

[0064] Curves O, P, Q, intersection point S

[0065] R

[0066] Horizontal line H DETAILED DESCRIPTION

[0067] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0068] It should be noted that when a component is referred to as being "fixed to" or "mounted on" another component, it may be directly on the other component or there may be a central component. When a component is referred to as being "disposed on" another component, it may be directly on the other component or there may be a central component. As used herein, the term "and / or" includes all and any combinations of one or more of the relevant listed items.

[0069] See also Figure 1 FIG. 1 is a schematic diagram of an optical imaging system according to an embodiment of the present invention. The optical imaging system 1 includes an illumination module 11, a focusing module 13, and an imaging module 15. The imaging module 15 employs a TDI (time delay integration) line array camera (hereinafter referred to as a TDI camera) for imaging. Furthermore, the TDI camera is an industrial-grade or scientific-grade TDI camera. The illumination module 11, focusing module 13, and imaging module 15 share some optical path guidance components and an objective lens 16. The illumination module 11 includes a light source 111 and an excitation light shaping unit 113. The excitation light emitted from the light source 111 is shaped by the excitation light shaping unit 113 into a linear illumination spot compatible with the photosensitive surface of the TDI camera. This linear illumination spot is guided by the optical path guidance components to the objective lens 16, where it is emitted onto a sample carried on a sample carrier 2. The sample is excited by the excitation light to produce fluorescence, which then enters the imaging module 15 through the objective lens and is recorded by the TDI camera within the imaging module 15. The focusing module 13 emits detection light, which is guided by the optical path guiding components into the objective lens 16, and emitted to the sample carrier 2 through the objective lens 16. The detection light will not excite the sample to produce fluorescence, but will only be reflected back to the objective lens 16 by the sample carrier 2, and enter the focusing module 13 through the objective lens 16 and the optical path guiding components. The focusing module 13 analyzes and determines the positional relationship between the sample carrier 2 and the objective lens 16, and adjusts the position of the objective lens 16 relative to the sample carrier 2 according to the determined positional relationship between the sample carrier 2 and the objective lens 16, so that the sample carrier 2 is always located on the focal plane of the objective lens 16.

[0070] In this embodiment, the light source 111 is a laser light source, and the excitation light is laser light. The optical imaging system 1 is a sequencing imaging system used to photograph DNA samples for base sequence analysis. The DNA sample is carried on a sample carrier 2. The four bases A, T, G, and C in the DNA sample are labeled with different fluorescent dyes. The laser light emitted by the objective lens 16 excites the DNA sample to emit four fluorescent signals of different wavelengths. The imaging module 15 includes four TDI cameras 151. The arrangement of the four TDI cameras 151 provides the optical imaging system 1 with four imaging channels. A tube lens 152 and a cutoff filter 153 are positioned in front of each TDI camera 151. Each cutoff filter 153 only allows fluorescence of a single wavelength range to enter the corresponding TDI camera 151, so that the corresponding TDI camera 151 only records the fluorescence signal emitted by one of the bases A, T, G, and C. The imaging module 15 also includes multiple spectrometers 154. These spectrometers 154 reflect light of one wavelength range and transmit light of another wavelength range, thereby directing light of different wavelength ranges into different imaging channels, thereby being recorded by different TDI cameras 151. In this embodiment, the spectrometers 154 are dichroic mirrors, and the imaging module 15 includes dichroic mirrors A, B, C, and D. Dichroic mirror A is positioned in correspondence with the objective lens 16, forming a 45° angle with the optical axis of the objective lens 16. It transmits fluorescence light and reflects excitation light and detection light. Dichroic mirror B is positioned behind and parallel to dichroic mirror A, directing fluorescence of different wavelengths to dichroic mirrors C and D, respectively, through transmission and reflection. Dichroic mirrors C and D then direct the fluorescence of different wavelengths to different cutoff filters 153, through the cylindrical lens 152, and into the corresponding TDI cameras 151.

[0071] In this embodiment, the illumination module 11 and the focusing module 13 share the dichroic mirror A of the imaging module 15. Furthermore, the illumination module 11 and the focusing module 13 also share the dichroic mirror E. Dichroic mirror E directs excitation light and detection light incident at different angles to the dichroic mirror A, where they are then directed into the objective lens 16. The illumination module 11 also includes a plane mirror 115, which reflects the excitation light shaped by the excitation light shaping unit 113 back to the dichroic mirror E.

[0072] This embodiment uses a TDI camera 151. The multiple rows of pixels of the photosensitive chip of the TDI camera 151 can perform row-by-row charge transfer to perform multiple exposure imaging of the same target. By synchronizing the TDI camera 151 with the sample carrier 2, the time for taking pictures of each sample can be significantly reduced, thereby improving the detection throughput. Figure 2 The figure shows the comparison of the time taken to take a picture per line when imaging with the TDI camera 151 and the traditional area array camera. Figure 2Here, t1 represents the time it takes TDI camera 151 to capture each row, and t2 represents the time it takes a conventional area array camera to capture each row. While scanning each row of the sample, the detection platform carrying sample carrier 2 can be controlled to move at a constant speed, allowing TDI camera 151 to continuously scan the same row of the sample to capture image information for that row. This approach, compared to conventional sequencing where an area array camera scans each row, requiring the detection platform to accelerate and decelerate for each field of view, the TDI camera 151 only requires a single acceleration and deceleration for each row change. This improves the time utilization of the optical imaging system 1, thereby increasing the detection throughput of a biochemical substance detection device employing this optical imaging system 1 while also avoiding the risks associated with frequent acceleration and deceleration of the detection platform.

[0073] In this embodiment, the objective lens 16, on the one hand, evens the rectangular line light spot of the illumination module 11 and illuminates it onto the sample carrier 2, while also guiding the detection light of the focusing module 13 onto the sample carrier 2. On the other hand, the objective lens 16 collects fluorescence, and the collected fluorescence is transmitted through the tube lens 152 to obtain a DNA fluorescence image of each channel on each TDI camera 151.

[0074] Since the spacing between the sample sites on the sample carrier 2 is on the order of hundreds of nanometers, according to the Rayleigh Criterion, the numerical aperture NA of the objective lens 16 is required to be greater than 0.8. In addition, for high-throughput imaging systems, the effective imaging field of view is a narrow, rectangular area with a large aspect ratio, so the TDI camera 151 imaging requires a large linear field of view. For example, for a sequencing throughput requirement of 6T / day, the object diameter field of view of the objective lens 16 is required to be greater than 1.5mm. Furthermore, to achieve ultra-high throughput, the objective lens 16 needs to have both a large numerical aperture and a large field of view. However, current commercial microscopes generally have low-magnification objective lenses with a large field of view but a small numerical aperture, while high-magnification objective lenses have a large numerical aperture but a small field of view. The product of the two is usually less than 0.8, making it difficult to achieve ultra-high throughput.

[0075] Therefore, the objective lens 16 provided in this embodiment generally meets the following specifications:

[0076] Numerical aperture NA>=0.7;

[0077] Effective working distance>1.2mm;

[0078] Focal length = 12.5mm;

[0079] The apochromatic aberration of 500nm~800nm wavelength light is less than 0.4um;

[0080] Field curvature <0.4um;

[0081] Object side field of view

[0082] The distortion when combined with the tube lens is <1%.

[0083] In general, the objective lens 16 provided in this embodiment is a plan achromatic objective lens with a large field of view, a large numerical aperture, and small distortion, and is suitable for a 0.25 mm cover glass and a 0.05 mm water layer.

[0084] See also Figure 3 , which is a schematic diagram of the specific optical structure of the objective lens 16 provided in this embodiment.

[0085] The objective lens 16 includes seven lens groups 161-167. The first lens group 161 to the seventh lens group 167 are arranged in sequence from the object side to the image side. The first three lens groups 161-163 have negative optical power, and the last four lens groups 164-167 have positive optical power. The first lens group 161 includes a first lens L1, which is a thick meniscus lens with positive optical power, forming a uniform lens and generating positive field curvature. The second lens group 162 includes a second lens L2, which is a meniscus lens with positive optical power and, together with the first lens L1, has a positive deflection angle. The third lens group 163 includes a third lens L3, a fourth lens L4, and a fifth lens L5. The third lens L3, the fourth lens L4, and the fifth lens L5 are cemented in sequence to form a triplet lens group, which has a positive field curvature. The fourth lens group 164 includes the sixth lens L6, a thin meniscus lens with positive optical power. The fifth lens group 165 includes the seventh lens L7 and the eighth lens L8, which are cemented together to form a doublet lens group with positive optical power. The sixth lens group 166 includes the ninth lens L9 and the tenth lens L10, which are cemented together to form a doublet lens group with negative optical power. The seventh lens group 167 includes the eleventh lens L11 and the twelfth lens L12, which are cemented together to form a negative meniscus doublet lens group with negative optical power. The sixth and seventh lens groups 166 and 167 are used to control field curvature and distortion. Among them, the thicknesses of the three types of lenses referred to in this embodiment, namely, the thick meniscus lens, the meniscus lens, and the thin meniscus lens, along the optical axis direction decrease in sequence. That is, the thickness of the thick meniscus lens along the optical axis direction is greater than the thickness of the meniscus lens in the same direction, and the thickness of the meniscus lens along the optical axis direction is greater than the thickness of the thin meniscus lens in the same direction. The "thick" and "thin" used in this embodiment are merely relative concepts used to describe the thickness of an object.

[0086] The first group of lenses 161 meet: 9.2 <f1 / fobj<9.7;

[0087] The second lens group 162 meets: 4.7 <f2 / fobj<5.5;

[0088] The third lens group 163 meets: 4.6 <f3 / fobj<5.6;

[0089] The fourth lens group 164 meets: 3.25 <f4 / fobj<3.88;

[0090] The fifth lens group 165 meets: 5.71 <f5 / fobj<6.11;

[0091] The sixth lens group 166 meets: -4.76 <f6 / fobj<-3;

[0092] The seventh lens group 167 meets: -23.9 <f7 / fobj<-19.5。

[0093] Among them, f1 is the focal length of the first lens group 161, f2 is the focal length of the second lens group 162, f3 is the focal length of the third lens group 163, f4 is the focal length of the fourth lens group 164, f5 is the focal length of the fifth lens group 165, f6 is the focal length of the sixth lens group 166, f7 is the focal length of the seventh lens group 167, and fobj is the focal length of the objective lens 16.

[0094] See also Figures 4 to 6 As shown, Figure 4 The MTF (Modulation Transfer Function) curve of the full field of view and full band of the objective lens 16 obtained by simulation is: Figure 5 is the distortion curve of the objective lens 16 obtained by simulation, Figure 6 The envelope energy diagram of the objective lens 16 obtained by simulation is shown in FIG. It can be seen from the above simulation result diagram that the objective lens 16 is close to the diffraction limit in imaging results, with a normalized field distortion of less than 1% and high resolution.

[0095] See also Figure 7 , which is a schematic diagram of the optical structure of the combination of the objective lens 16 and the tube lens 152 provided in this embodiment. In this embodiment, the tube lens 152 includes four lenses, which are cemented together to form two doublets, hereinafter referred to as the thirteenth lens L13 and the fourteenth lens L14. The focal length of the tube lens 152 is 150 mm to 250 mm. Specifically, in a specific application, the specific focal length value of the tube lens 152 is also set based on the focal length of the objective lens 16, the pixel size and number of the TDI camera 151, and the magnification and resolution required by the optical imaging system 1. The combination of the tube lens 152 and the objective lens 16 must meet imaging constraints such as distortion and field curvature. The tube lens 152 compensates for the residual aberrations of the objective lens 16.

[0096] See also Figures 8A-8D and Figure 9 As shown, Figures 8A to 8DThey are the wavefront aberration curves of the combination of the objective lens 16 and the tube lens 152 in imaging channels 1-4 obtained by simulation, Figure 9 is the distortion curve of the combination of the objective lens 16 and the tube lens 152 obtained by simulation. Figures 8A-8D The horizontal line H in the figure represents the diffraction limit. The wavefront aberration of the combination of the objective lens 16 and the tube lens 152 in the imaging channels 1-4 is lower than the horizontal line H, which proves that the design of the combination of the objective lens 16 and the tube lens 152 has reached the limit of the required resolution. Figure 9 It can be seen that the normalized field distortion of the combination of the objective lens 16 and the tube lens 152 is less than 1%.

[0097] In this embodiment, the objective lens 16 is an infinity-corrected objective lens, which converts the fluorescence emitted from the sample into parallel light or quasi-parallel light. This makes it convenient to add a spectrometer 154 between the objective lens 16 and the tube lens 152 as needed.

[0098] See also Figure 10 Figure 1 is a schematic diagram of the output end face of light source 111 in this embodiment. In this embodiment, light source 111 is a laser light source that emits red and green laser light. The light source includes a fiber coupler (not shown) and a multimode optical fiber 1111. The red and green laser light is coupled to multimode optical fiber 1111 via the fiber coupler for output. Multimode optical fiber 1111 has an output end face 1112, which serves as the output end face of light source 111. In this embodiment, output end face 1112 is rectangular to match the long, strip-shaped photosensitive surface of TDI camera 151. Therefore, compared to the circular output end face commonly used in the prior art, the rectangular output end face of light source 111 is more compatible with TDI camera 151, thereby improving the utilization rate of the excitation light.

[0099] See also Figure 11 The figure shows an optical structure diagram of a combination of an excitation light shaping unit 113 and an objective lens 16 in one embodiment. The excitation light shaping unit 113 includes a first shaping unit 113a and a second shaping unit 113b, and the first shaping unit 113a and the second shaping unit 113b are arranged in sequence from the image side to the object side. The first shaping unit 113a is used to shape the rectangular light spot emitted by the light source 111 in the first direction, and the second shaping unit 113b is used to shape the illumination light spot emitted by the light source 111 in the second direction, wherein the first direction and the second direction are perpendicular to each other. After the illumination light spot is shaped by the first shaping unit 113a and the second shaping unit 113b, a linear light spot with an aspect ratio required for imaging scanning by the TDI camera 151 is formed. In this embodiment, the first shaping unit 113a and the second shaping unit 113b are cylindrical lenses C1 and C2 respectively, and the cylindrical lens C1 shapes the illumination light spot in the first direction. Figure 1 As shown in the X direction, cylindrical lens C2 enlarges the illumination spot in Figure 1The Y-direction shown is reduced, so that the illumination spot ultimately achieves the aspect ratio required for imaging scanning by the TDI camera 151. By properly selecting the focal lengths of the cylindrical lenses C1 and C2, the aspect ratio of the illumination spot output by the illumination module 11 can be adjusted. In this embodiment, the cylindrical lenses C1 and C2 can achieve an aspect ratio of 10:1 for the illumination spot. Combined with the rectangular output end face 1112 of the multiplying optical fiber 1111, the aspect ratio of the illumination spot output by the illumination module 11 can reach approximately 20:1. In this embodiment, the use of two cylindrical lenses C1 and C2 to shape the illumination spot simplifies the structure of the excitation light shaping unit 113, making it easier to assemble and adjust due to the reduced number of shaping components. Furthermore, this reduces illumination spot deviation caused by component and assembly tolerance variations. Furthermore, this embodiment utilizes critical illumination to strictly control the linear uniformity of the illumination, maximizing the energy of the light source 111 while providing uniform illumination for fluorescence excitation.

[0100] See also Figure 12 FIG. 1 is a schematic diagram of the optical structure of the combination of the excitation light shaping unit 113 and the objective lens 16 in another embodiment. Different from the previous embodiment, in this embodiment, the first shaping unit 113a and the second shaping unit 113b are cylindrical lenses C3 and C4 respectively. The cylindrical lens C3 shapes the illumination spot at Figure 1 As shown in the Y direction, cylindrical lens C4 reduces the illumination spot to Figure 1 The X direction is enlarged as shown, so that the illumination spot finally forms the aspect ratio required for the imaging scan of the TDI camera 151.

[0101] See also Figure 13 and Figure 14 As shown, Figure 13 Schematic diagram of the photosensitive surface of the TDI camera 151 in this embodiment. The photosensitive surface 1511 of the TDI camera 151 is a rectangular surface or, further, a long strip. Figure 14 is the image plane illumination diagram obtained by simulation. Specifically, Figure 14 This image plane illumination diagram is obtained by simulating optical imaging system 1 using non-sequential simulation software. In the simulation, the input power of light source 111 is normalized to 1W, the dichroic mirror AE is simplified to a beam splitter with a 50 / 50 reflection / transmission ratio, and the transmittance of all lenses is assumed to be 100%. The image plane illumination diagram shows that the image-side illumination uniformity is greater than 85%.

[0102] In other embodiments, the first shaping unit 113a and the second shaping unit 113b may also be optical wedges, micro lenses, or diffractive optical elements, respectively.

[0103] Please refer to Figure 1As shown, in this embodiment, a cutoff filter 153 is placed before each tube lens 152. A cutoff filter 116 is also placed between the light source 111 and the excitation light shaping unit 113 to filter stray light outside the excitation wavelength. These cutoff filters 153 and 116, along with the dichroic mirror AE, form a filtering system that separates the four types of fluorescence light into four channels for imaging and collection by four TDI cameras 151. This filtering system not only separates the fluorescence light into individual channels for imaging, but also filters the excitation light and detection light from entering the TDI cameras 151. In this embodiment, the cutoff value of the cutoff filter 153 is 8.

[0104] See also Figure 15 As shown in the figure, it is a control principle diagram of the optical imaging system 1 in this embodiment. The optical imaging system 1 also includes a control device 17. The control device 17 is used to control the moving speed of the detection platform (in this embodiment, the movable platform 17) to match and synchronize with the frequency of the TDI camera 151. At the same time, the control device 17 is also used to control the opening of the light source 111 to synchronize with the photo taking of the TDI camera 151. Specifically, in this embodiment, in order to achieve the above purpose, the control device 17 is communicated with the movable platform 18 on which the sample carrier 2 is placed. When the movable platform 18 reaches the specified position, the control device 17 simultaneously triggers the light source 111 and the TDI camera 151 to synchronize the sample, the light source 111 and the TDI camera 151. At the same time, the frequency of the TDI camera 151 automatically matches the moving speed of the movable platform 18, so that the generated image is not deformed. Please also combine Figure 1 As shown, the movable platform 18 can be controlled to move in the X direction shown and in the Y direction perpendicular to the X direction, and can be controlled to rotate within the plane formed by the X and Y directions, so that the movable platform 18 can be moved or positioned to a predetermined position. The objective lens 16 can be controlled to move in the Z direction shown, which is perpendicular to both the X and Y directions.

[0105] In this embodiment, the focusing module 13 is used to detect the positional relationship between the sample carrier 2 and the objective lens 16 by emitting detection light, and based on the detection result, controls the movement of the objective lens 16 in the Z direction to adjust the distance between the objective lens 16 and the sample carrier 2, thereby ensuring that the sample carrier 2 is always in the focal plane of the objective lens 16. Therefore, the focusing module 13 can compensate for changes in the distance between the objective lens 16 and the sample carrier 2 caused by changes in ambient temperature, mechanical vibration, bending of the sample carrier 2, surface undulations of the sample carrier 2, and changes in the thickness of the sample carrier 2.

[0106] See also Figure 16FIG2 is a schematic diagram illustrating the principle of focusing performed by the focusing module 13 in this embodiment. In this embodiment, the detection light emitted by the focusing module 13 passes through the objective lens 16 and is irradiated onto the sample carrier 2. The detection light is then reflected back to the objective lens 16 by the sample carrier 2, and then returns to the focusing module 13 along the original optical path through the objective lens 16. The focusing module 13 then determines whether the sample carrier 2 is within the focal plane of the objective lens 16 based on the returned detection light. If the sample carrier 2 is not within the focal plane of the objective lens 16, the focusing module 13 sends a signal to the drive unit 161 that drives the movement of the objective lens 16. The drive unit 161 controls the movement of the objective lens 16 to move the objective lens 16 toward or away from the sample carrier 2, so that the sample carrier 2 is once again within the focal plane of the objective lens 16.

[0107] Specifically, in this embodiment, the imaging data of each TDI camera 151 is transmitted to a computer device 19. The computer device 19 comprehensively evaluates the imaging quality of all TDI cameras 151 according to preset rules, and determines the standard position of the objective lens 16 (i.e., the position of the objective lens 16 in the Z direction when the sample carrier 2 is located on the focal plane of the objective lens 16) based on the principle of optimal quality, thereby obtaining a standard value representing the standard position of the objective lens 16. The standard value is provided to the focusing module 13. The focusing module 13 includes a detection optical path 130, a sensor unit 131, and a control unit 132. The sensor unit 131 senses the returned detection light and outputs an electrical signal. The control unit 132 obtains a detection value based on the electrical signal output by the sensor unit 131, compares the detection value with the standard value, and obtains a control signal for controlling the driving unit 161, thereby controlling the driving unit 161 to drive the objective lens 16 toward or away from the sample carrier 2.

[0108] Please also see Figure 17 and Figures 18A-18C As shown, Figure 17 FIG. 4 is a principle diagram of the optical path for the focusing module 13 to perform focusing in this embodiment. Figures 18A-18C This is a schematic diagram of the sensing unit 131 of the focusing module 13 in this embodiment sensing whether the sample carrier 2 is in the focal plane of the objective lens 16. The detection light path includes a light source 1301 and a tube lens 1303. The sensing unit 131 includes a sensor 1311. The detection light emitted by the light source 1301 passes through the tube lens 1303 and the objective lens 16 (in this embodiment, it enters the tube lens 1303 after being reflected by the reflector 1305), irradiates the sample carrier 2 and is reflected by the sample carrier 2. The reflected detection light passes through the objective lens 16 and the tube lens 1303 and irradiates the sensor 1311. In this embodiment, the sensor 1311 adopts a photoelectric sensor, specifically a PD (Photodiode) sensor 1312. Two sensing areas 1312a and 1312b are provided on the PD sensor 1312. The position where the detection light irradiates the sensing areas 1312a and 1312b can determine the position between the sample carrier 2 and the objective lens 16. Figure 18AAs shown, the detection light spot G is biased towards the sensing area 1312a, which means that the sample carrier 2 is above the focal plane of the objective lens 16; Figure 18B As shown, the area of the detection light spot G falling on the sensing region 1312a is the same as the area falling on the sensing region 1312b or the difference between the two is within a preset range, which means that the sample carrier 2 is on the focal plane of the objective lens 16; Figure 18C As shown, the spot of the detection light is biased toward sensing area 1312b, indicating that the sample carrier 2 is below the focal plane of the objective lens 16. Sensing area 1312a converts the received light signal into an electrical signal PD1 and outputs it, while sensing area 1312b converts the received light signal into an electrical signal PD2 and outputs it. The control unit 132 determines the defocus direction and defocus amount corresponding to the sample carrier 2 based on the two received electrical signals PD1 and PD2, and then controls the drive unit 161 to drive the objective lens 16 toward or away from the sample carrier 2 by a corresponding distance.

[0109] In this embodiment, the control signal DIV for controlling the driving unit 161 is generated by dividing the difference between the electrical signals PD1 and PD2 by their sum. This effectively filters out signal noise and prevents the influence of brightness fluctuations of the light source 1301 on focusing accuracy. The calculation formula for generating the control signal DIV is as follows:

[0110] DIV=α(DIFF / SUM+β)

[0111] Wherein, α is the signal amplification factor, the difference signal DIFF=PD1−PD2, the sum signal SUM=PD1+PD2, and β is the preset voltage offset.

[0112] See also Figure 19 , which is a schematic diagram of the changes in the signals obtained during focusing. The horizontal axis represents the relative distance between the objective lens 16 and the sample carrier 2 in the Z axis, in mm. Curve O represents the difference signal DIFF, curve Q represents the sum signal SUM, curve P represents the control signal DIV, and curve R represents the relative coordinate position between the drive unit 161 and the sample carrier 2 in the Z direction. Figure 19 The intersection point S of the middle curve R and the curve P represents the focal plane of the objective lens 16 , and the value (DIV value) of the curve P at the intersection point S is the standard value representing the standard position of the objective lens 16 .

[0113] See also Figure 20 FIG. 5 is a schematic diagram of a biochemical substance detection system according to an embodiment of the present invention. The biochemical substance detection system 5 includes an optical imaging system 51 , which may be the optical imaging system 1 described in the above embodiment.

[0114] See also Figure 21FIG. 6 is a schematic diagram of a biochemical substance detection system according to another embodiment of the present invention. The biochemical substance detection system 6 includes an optical imaging system 61 and a detection platform 62. The optical imaging system 61 can be the optical imaging system 1 described in the above embodiment. The detection platform 62 is used to carry a sample carrier. The detection platform 62 is a movable platform. The control device 611 of the optical imaging system 61 is used to control and coordinate the optical imaging system 61 and the detection platform 62. For example, the control device 611 is used to control the movement speed of the detection platform 62 to match and synchronize the frequency of the TDI camera 613 of the optical imaging system 61. The control device 63 is also used to control the activation of the light source 615 of the optical imaging system 61 to synchronize the image capture of the TDI camera 613. When the detection platform 62 reaches a designated position, the control device 611 simultaneously triggers the light source 615 and the TDI camera 613, synchronizing the sample (not shown) mounted on the detection platform 62, the light source 615, and the TDI camera 613. Furthermore, the frequency of the TDI camera 613 automatically matches the movement speed of the detection platform 62 to ensure that the generated image is not distorted.

[0115] In summary, the optical imaging system and biochemical substance detection system provided by the above embodiments have the following advantages: 1. an industrial-grade or scientific-grade TDI camera is used to collect fluorescence, thereby reducing the photographing time for each sample and improving the detection throughput; 2. a filtering system is formed by a spectrometer and a filter, which can, on the one hand, separate fluorescence into multiple channels for imaging, and, on the other hand, prevent the interference of excitation light and other light, such as detection light, on fluorescence imaging, thereby improving the imaging quality; 3. the output end face of the light source is rectangular, or further elongated rectangular, and the excitation light shaping unit is used to shape the excitation light output by the light source into an illumination spot that is compatible with the photosensitive surface of the TDI camera, thereby maximizing the utilization of the optical filter. It not only uses the energy of the light source, but also improves the uniformity of the illumination spot; 4. It adopts a specially structured objective lens, which makes the objective lens meet the requirements of large field of view and large numerical aperture at the same time, while reducing distortion and flat-field achromatism; 5. It adopts a focusing system, which adjusts the distance between the objective lens and the sample carrier in real time, so that the sample carrier remains on the focal plane of the objective lens, compensating for the distance change between the sample carrier and the objective lens caused by ambient temperature changes, mechanical vibrations, sample bending or other reasons, and ensuring the quality of imaging; 6. The focusing system uses a specific method to calculate the distance between the sample carrier and the objective lens, which effectively eliminates the influence of signal noise and changes in the brightness of the detection light source on the detection results.

[0116] It should be noted that the above description only describes one or two specific embodiments. However, those skilled in the art will readily appreciate that further embodiments can be derived from the above specific embodiments. For example, the first and second shaping units of the excitation light shaping unit are not limited to a single lens, but can be composed of multiple lenses. The lenses constituting the first and second shaping units are not limited to cylindrical lenses or aspherical lenses, but can also be spherical lenses or Fresnel lenses. Furthermore, the number of TDI cameras and corresponding imaging channels is not limited to four, but can also be one or more. The bandpass and cutoff bands of the filter system can also be adjusted according to specific circumstances. The design of the tube lens is not limited to a two-piece doublet lens structure, but can be modified to accommodate focal length or imaging requirements, such as using multiple lenses or a single lens.

[0117] It can be understood that the optical imaging system provided in the embodiments of the present invention can be applied not only to biochemical substance detection systems, but also to any other device that uses excitation light to excite fluorescence and image fluorescence to achieve rapid imaging of the device.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A sequencing imaging system, characterized in that: include: An illumination module, the illumination module comprising a first light source, the first light source being configured to emit excitation light; A sample carrier, wherein the sample carrier is used to carry a DNA sample, wherein the four bases of the DNA sample are respectively labeled with different fluorescent dyes; an objective lens, the objective lens being used to receive the excitation light and emit the excitation light to the DNA sample carried on the sample carrier, and the objective lens being further used to receive four fluorescent lights of different wavelengths emitted from the DNA sample; An imaging module includes four cameras, and the four cameras are used to respectively record four types of fluorescence with different wavelengths emitted after being received by the objective lens.

2. The sequencing imaging system according to claim 1, wherein: The imaging module includes a first dichroic mirror, a second dichroic mirror, a third dichroic mirror and a fourth dichroic mirror. The first dichroic mirror is used to reflect the excitation light emitted by the first light source to the objective lens and transmit the fluorescence of different wavelengths emitted after being received by the objective lens to the second dichroic mirror. The second dichroic mirror is used to direct the incident fluorescence of different wavelengths to the third dichroic mirror and the fourth dichroic mirror respectively. The third dichroic mirror is used to direct the incident fluorescence of different wavelengths to two of the four cameras respectively. The fourth dichroic mirror is used to direct the incident fluorescence of different wavelengths to the other two of the four cameras respectively.

3. The sequencing imaging system according to claim 1, wherein: The optical imaging system includes a filter disposed between each camera and the objective lens, and the filter is used to prevent light other than a specific wavelength from entering the corresponding camera.

4. The sequencing imaging system according to claim 1, wherein: The optical imaging system includes a focusing module, which is used to detect the positional relationship between the sample carrier carrying the DNA sample and the objective lens, and adjust the relative position between the sample carrier and the objective lens according to the sensed positional relationship so that the sample carrier is located on the focal plane of the objective lens.

5. The sequencing imaging system according to claim 4, wherein: The focusing module includes a second light source for outputting detection light to illuminate the sample carrier and a sensing unit for sensing the detection light reflected back from the sample carrier. The sensing unit outputs an electrical signal reflecting the positional relationship between the sample carrier and the objective lens based on the reflected detection light.

6. The sequencing imaging system according to claim 5, wherein: The sequencing camera imaging system includes a fifth dichroic mirror, which is used to guide the excitation light from the first light source and the detection light emitted by the second light source to the objective lens, and the fifth dichroic mirror is also used to guide the reflected detection light to the sensing unit.

7. The sequencing imaging system according to claim 1, wherein: The camera is a time-delay integration line array camera.

8. The sequencing imaging system according to claim 7, wherein: The illumination module further includes an excitation light shaping unit, wherein the excitation light shaping unit is configured to amplify the spot of the excitation light in a first direction and reduce the spot of the excitation light in a second direction perpendicular to the first direction, so that the spot shape of the excitation light is adapted to the shape of the photosensitive surface of the time-delayed integrated line array camera; alternatively, the excitation light shaping unit includes a first shaping unit and a second shaping unit, wherein the first shaping unit is configured to amplify the spot of the excitation light in the first direction, and the second shaping unit is configured to reduce the spot of the excitation light in a second direction perpendicular to the first direction; alternatively, the excitation light shaping unit includes two cylindrical lenses, wherein one of the two cylindrical lenses is configured to amplify the spot of the excitation light in the first direction, and the other is configured to reduce the spot of the excitation light in the second direction perpendicular to the first direction.

9. The sequencing imaging system according to claim 1, wherein: The sequencing photography and imaging system includes a control device, which is used to control the movement speed of the sample carrier to match and synchronize with the frequency of the camera, and to control the activation of the first light source to synchronize with the photography of the camera.

10. A sequencing camera imaging method, performed using the sequencing camera imaging system according to any one of claims 1 to 9, characterized in that: The four cameras respectively record four types of fluorescence emitted from the DNA sample.

Images