Time-delayed integration scanning imaging system, method and super-resolution detection method

Abstract

The present application provides a time-delayed integration scanning imaging system, comprising: a light source module for emitting a light source laser; a light field control device for emitting stripe structured light, including a switch array, the stripe structured light being projected onto a sample to be tested to generate fluorescence, and the stripe structured light forming a stripe pattern on the sample to be tested; an imaging module, comprising an objective lens and at least one imaging channel, each imaging channel having a time-delayed integration camera, the objective lens receiving fluorescence and transmitting the fluorescence to the time-delayed integration camera, the time-delayed integration camera generating a fluorescence image based on the fluorescence, the fluorescence image being used to obtain biological information of the sample to be tested, the objective lens and the sample to be tested moving relative to each other, the scanning rate of the time-delayed integration camera being synchronized with the movement rate of the sample to be tested; and a controller for controlling the synchronization of the drive timing of the switch array and the scanning timing of the time-delayed integration camera. The present application also provides a scanning imaging method and a super-resolution detection method.

Description

Technical Field

[0001] The present application relates to the field of super-resolution detection, and in particular to a time-delayed integration scanning imaging system and a time-delayed integration scanning imaging method and a super-resolution detection method applied to the time-delayed integration scanning imaging system. Background Art

[0002] Gene sequencing technology is widely used in multiple research fields of life science and medicine. The resolution of traditional gene sequencing systems is limited by the optical diffraction limit, which also restricts the gene sequencing throughput.

[0003] One technology that improves sequencing throughput is structured illumination microscopy (SIM). This technology can increase resolution by approximately twofold, thereby boosting sequencing throughput, simply by improving illumination and algorithms. SIM imaging, based on digital micromirror devices (DMDs), enables faster acquisition of images using stripe structured light illumination, thereby increasing imaging throughput and, in turn, sequencing throughput. Another technology that improves sequencing throughput is gene sequencing technology based on time delay integration (TDI) cameras. TDI cameras combine the signals from multiple rows of linear array pixels to generate an image. The output signal's equivalent charge is the total charge in all rows. Compared to conventional linear array cameras (especially area array cameras), this reduces the unevenness of pixel response. Furthermore, TDI cameras use a line scan mode, replacing the time-consuming surface imaging mode of area array cameras (which requires the sample stage to move each time a field of view changes), resulting in higher imaging speed and throughput. Typically, the imaging speed can be increased up to 5 times by using the line scan imaging mode of a TDI camera instead of the area imaging mode.

[0004] The above two methods improve gene sequencing technology from different aspects and can both increase sequencing throughput. However, if the above two methods are combined for gene sequencing, there are the following problems: when projecting stripe structured light onto the sequencing chip, the stripe structured light in the field of view is stationary, that is, "static" stripes; when the sequencing chip and the stripe structured light produce relative motion, the sequencing chip is illuminated by light and dark stripes in sequence, and the fluorescence signal generated by it under the illumination of the stripe structured light is accumulated step by step by the TDI camera; at this time, due to the existence of the accumulation effect, the brightness of the stripe structured light at various locations is averaged, and the information of the stripe structured light is lost in the output image of the TDI camera, making super-resolution image reconstruction impossible. Summary of the Invention

[0005] On one hand, the present application provides a time-delayed integration scanning imaging system, comprising:

[0006] A light source module, used for emitting light source laser;

[0007] a light field control device, located in the optical path of the light source laser, for receiving and modulating the light source laser to emit stripe structured light, the light field control device including a switch array, the stripe structured light being projected onto the sample to be tested so that the sample to be tested generates fluorescence under the illumination of the stripe structured light, and the stripe structured light forming a stripe pattern on the sample to be tested;

[0008] an imaging module, the imaging module comprising an objective lens disposed opposite the sample to be tested and at least one imaging channel, each imaging channel having a time-delay integration camera, the objective lens receiving the fluorescence and transmitting the fluorescence to the time-delay integration camera, the time-delay integration camera generating a fluorescence image based on the fluorescence, the fluorescence image being used to obtain biological information of the sample to be tested, the objective lens and the sample to be tested moving relative to each other, the scanning rate of the time-delay integration camera being synchronized with the movement rate of the sample to be tested, and the scanning direction of the time-delay integration camera being perpendicular to the extension direction of the stripe pattern corresponding to the single stripe structured light; and

[0009] A controller is connected to the switch array and the time delay integration camera, and is used to control the synchronization of a driving timing of the switch array and a scanning timing of the time delay integration camera.

[0010] Another aspect of the present application provides a scanning imaging method using the above-mentioned time delay integration scanning imaging system, comprising:

[0011] The light source module emits light source laser;

[0012] The light source laser generates stripe structured light through a light field control device, and projects the stripe structured light onto a sample to be tested, so that the sample to be tested generates fluorescence according to the stripe structured light and forms a stripe pattern on the sample to be tested;

[0013] An objective lens receives the fluorescence and transmits the fluorescence to at least one imaging channel. A time-delayed integration camera in the imaging channel generates a fluorescence image based on the fluorescence. The objective lens and the sample to be tested move relative to each other. The scanning rate of the time-delayed integration camera is synchronized with the movement rate of the sample to be tested. The scanning direction of the time-delayed integration camera is perpendicular to the extension direction of the stripe pattern corresponding to the single stripe structured light.

[0014] Controlling the driving timing of the light field control device to be synchronized with the scanning timing of the time delay integration camera;

[0015] During the synchronous scanning of the time-delayed integration camera relative to the movement of the sample to be tested, the objective lens continuously receives the fluorescence, the time-delayed integration camera obtains a fluorescence image based on the fluorescence, and obtains biological information of the sample to be tested based on the fluorescence image.

[0016] Another aspect of the present application provides a super-resolution detection method using the above-mentioned time-delayed integration scanning imaging system, comprising:

[0017] Starting step: the light source module emits a light source laser; the light source laser generates a stripe structured light through the switch array, and the stripe structured light is projected onto the sample to be tested, so that the sample to be tested generates fluorescence according to the stripe structured light and forms a stripe pattern on the sample to be tested;

[0018] Adjusting the initial deflection state of the switch array, and repeating the starting step to generate the stripe structured light with different phases, and performing the image acquisition step under the stripe structured light with different phases to acquire a fluorescence image; and

[0019] Performing super-resolution reconstruction based on the fluorescence image to obtain a super-resolution image to obtain biological information of the sample to be tested; wherein

[0020] The image acquisition step includes: an objective lens receives the fluorescence and transmits the fluorescence to at least one imaging channel, a time-delayed integration camera in the imaging channel generates a fluorescence image based on the fluorescence, the objective lens and the sample to be tested move relative to each other, the scanning rate of the time-delayed integration camera is synchronized with the movement rate of the sample to be tested, and the scanning direction is perpendicular to the extension direction of the stripe pattern corresponding to the single stripe structured light; controlling the driving timing of the switch array to be synchronized with the scanning timing of the time-delayed integration camera; during the period when the time-delayed integration camera performs synchronous scanning relative to the movement of the sample to be tested, the objective lens continuously receives the fluorescence, and the time-delayed integration camera obtains the fluorescence image based on the fluorescence.

[0021] The above-mentioned time-delayed integral scanning imaging system enables relative movement between the objective lens and the sample to be measured and synchronizes the scanning rate of the time-delayed integral camera with the movement rate of the sample to be measured, so that the information of the stripe structured light in the image output by the imaging module is fully retained without being accumulated and averaged by the imaging module. This is conducive to solving the technical problem of "loss of stripe structured light information and inability to perform super-resolution image reconstruction." BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic structural diagram of the time-delayed integration scanning imaging system, the sample to be tested, the sequencing chip and the platform according to an embodiment of the present application.

[0023] Figure 2 for Figure 1 Schematic diagram of the optical path of the time-delayed integration scanning imaging system.

[0024] Figure 3 for Figure 2 Schematic diagram of the planar structure of the medium light field control device.

[0025] Figure 4 for Figure 3 Schematic diagram of the deflection state of the micromirror.

[0026] Figure 5 For the embodiment of this application Figure 1 Schematic diagram of the flow of the scanning imaging method of the time delay integration scanning imaging system.

[0027] Figure 6 For the embodiment of this application Figure 1 Schematic diagram of the process flow of the super-resolution detection method in the time-delayed integration scanning imaging system.

[0028] Figure 7 Schematic diagram of the formation process of stripe structured light with phase 0.

[0029] Figure 8 Schematic diagram of the formation process of stripe structured light with a phase of π / 2.

[0030] Figure 9 Schematic diagram of the formation process of stripe structured light with a phase of π.

[0031] Figure 10 for Figure 2 Schematic diagram of the planar structure of the sequencing chip.

[0032] Figure 11 Schematic diagram of the scanning process of the sequencing chip.

[0033] Figure 12 for Figure 2 Schematic diagram of a working state of the time delay integration scanning imaging system.

[0034] Figure 13 for Figure 2 Schematic diagram of another working state of the time delay integration scanning imaging system.

[0035] Figure 14 for Figure 2 Schematic diagram of another working state of the time delay integration scanning imaging system.

[0036] Figure 15 for Figure 2 Schematic diagram of another working state of the time delay integration scanning imaging system.

[0037] Figure 16 for Figure 2 Schematic diagram of another working state of the time delay integration scanning imaging system.

[0038] Figure 17 for Figure 2 Schematic diagram of another working state of the time delay integration scanning imaging system.

[0039] Figure 18 for Figure 2 Schematic diagram of another working state of the time delay integration scanning imaging system.

[0040] Figure 19 for Figure 2 Schematic diagram of another working state of the time delay integration scanning imaging system.

[0041] Figure 20 for Figure 2 Schematic diagram of another working state of the time delay integration scanning imaging system.

[0042] Description of main component symbols

[0043] Time delay integration scanning imaging system 10

[0044] Laser 111

[0045] Multimode fiber 112

[0046] Light field control device 12

[0047] Micromirror 121

[0048] Filter 131

[0049] TDI Camera 132

[0050] Beam expansion and homogenization lens 151

[0051] Total internal reflection mirror 152

[0052] Focusing lens 153

[0053] Dichroic mirror 154

[0054] Sample to be tested 20

[0055] Sequencing chip 30

[0056] Platform 40

[0057] Steps S11, S12, S13, S14, S15, S21, S22, S23, S24

[0058] The following specific implementation methods will further illustrate the present application in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0059] See also Figure 1 The time-delayed integration scanning imaging system 10 of the embodiment of the present application can be used to detect biological information of a sample 20 to be tested. The sample 20 to be tested can be a nucleic acid sample (DNA or RNA), a protein, or a cell. In this embodiment, the sample 20 to be tested is a nucleic acid sample, and the biological information can be the base sequence information of the sample 20 to be tested.

[0060] The sample 20 to be tested is carried on a sequencing chip 30. During operation of the time-delayed integration scanning imaging system 10, reference light is emitted to the sample 20 to be tested on the sequencing chip 30. The sequencing chip 30 is placed on a movable platform 40. The movable platform 40 can control the relative movement of the sequencing chip 30 and the time-delayed integration scanning imaging system 10, thereby driving the relative movement of the sample 20 to be tested and the time-delayed integration scanning imaging system 10. That is, during the movement of the movable platform 40, the platform 40, the sequencing chip 30, and the sample 20 to be tested remain stationary. On the basis of maintaining the same emission direction of the reference light, by controlling the relative movement of the sequencing chip 30 and the time-delayed integration scanning imaging system 10, the reference light can be projected onto different areas on the sequencing chip 30 at different time periods. This process can also be referred to as "scanning." Since the field of view of the reference light on the sequencing chip 30 usually cannot completely cover the sequencing chip 30, by controlling the relative movement of the sequencing chip 30 and the time-delayed integration scanning imaging system 10, the time-delayed integration scanning imaging system 10 can scan the entire sequencing chip 30.

[0061] In this embodiment, different bases in the sample 20 on the sequencing chip 30 are labeled with different fluorescent substances. When reference light illuminates the sequencing chip 30, the different fluorescent substances are excited to produce fluorescence of different wavelengths. The time-delayed integration scanning imaging system 10 is used to obtain biological information of the sample 20 based on this fluorescence.

[0062] See also Figure 2 The time-delayed integral scanning imaging system 10 includes a light source module. The light source module is used to emit light source lasers of at least two wavelengths. For example, the light source module is used to emit red lasers and green lasers. In other embodiments, the light source module may include multiple lasers 111, each laser 111 is used to emit lasers of different wavelengths. The number of lasers 111 may depend on the type of fluorescent substance on the sample 20 to be tested. In this embodiment, the light source module includes a laser 111 and a multimode optical fiber 112 coupled to the laser.

[0063] The time delay integration scanning imaging system 10 further includes a light field control device 12 located on the propagation path of the light source laser. The light field control device 12 includes a switch array for modulating the light source laser to generate stripe structured light.

[0064] In this embodiment, the switch array in the light field control device 12 is as follows: Figure 3 The digital micro-mirror array shown includes a plurality of micro-mirrors 121 arranged in the same plane. The plurality of micro-mirrors 121 are arranged in multiple rows and columns. In this embodiment, each micro-mirror 121 is roughly rectangular. The light field control device 12 also includes necessary structures (not shown) such as electrical connection wiring arranged around the edges of the plurality of micro-mirrors 121. In other embodiments, the switch array in the light field control device 12 can also be a spatial light modulator (SLM).

[0065] See also Figure 4 Each micromirror 121 can deflect within a certain angle range. Only one micromirror 121 is shown in the figure as an example. In this embodiment, each micromirror 121 can deflect in two opposite directions around its axis, and the maximum deflection angles in the two opposite directions are the same. The maximum deflection angles in the two opposite directions are defined as α and -α, respectively. During operation of the time-delayed integration scanning imaging system 10, each micromirror 121 is controlled to be in a deflection angle α or a deflection angle -α. The micromirror 121 is defined as being in an "ON" state when its deflection angle is α, and as being in an "OFF" state when its deflection angle is -α. The "ON" state is a highly reflective state. When the micromirror 121 is in the "ON" state, it can project received light onto the sequencing chip 30 for illumination. The "OFF" state is a non-reflective state. When the micromirror 121 is in the "OFF" state, it cannot reflect received light onto the sequencing chip 30, and illumination cannot be achieved.

[0066] By adjusting the states of the micro-mirrors 121 in the light field control device 12 , the stripe shape (including stripe width, stripe spacing, stripe number, stripe extension direction, etc.) of the stripe structured light reflected by the light field control device 12 can be modulated.

[0067] Please refer to Figure 2 The sequencing chip 30 is located on the transmission path of the stripe structured light, and the stripe structured light reflected by the light field control device 12 is projected onto the sequencing chip 30. In the sample 20 to be tested on the sequencing chip 30, the fluorescent substance is excited to generate fluorescence.

[0068] The time-delayed integration scanning imaging system 10 further includes an imaging module, which is located in the optical path of the fluorescence and is used to obtain biological information of the sample 20 to be tested based on the received fluorescence.

[0069] In this embodiment, the sample 20 to be tested generates fluorescence of four different wavelengths under the illumination of striped structured light. The imaging module includes four imaging channels, each of which is used to receive fluorescence of one of the wavelengths and perform subsequent data processing based on the received fluorescence. In other embodiments, the imaging module may include another number of imaging channels, the number of which is determined by the number of fluorescence wavelengths. Specifically, each imaging channel corresponds to a fluorescence of one wavelength; that is, each wavelength of fluorescence is incident on a unique imaging channel.

[0070] Each imaging channel includes a filter 131 and a time delay integration (TDI) camera 132, arranged in sequence along the fluorescence optical path. Filter 131 allows only one wavelength of fluorescence to pass through and enter TDI camera 132. The TDI camera generates an electrical signal based on the received fluorescence, which can be used to obtain biological information about the sample 20 being tested.

[0071] The time-delayed integration scanning imaging system 10 also includes a controller (not shown). The controller is electrically connected to the laser 111 in the light source module, the light field manipulation device 12, and the four time-delayed integration cameras 132 in the imaging module. The controller 15 is used to control the on / off state and light emission power of the laser 111, the deflection state of each micromirror 121 in the light field manipulation device 12, thereby controlling the morphology of the stripe structured light, and the operation of the four TDI cameras 132. In this embodiment, the controller also synchronizes the driving timing of the light field manipulation device 12 with the scanning timing of each time-delayed integration camera 132.

[0072] The time delay integration scanning imaging system 10 further includes a light guiding component, which may include a plurality of necessary optical elements of the same or different types to guide the light source laser, stripe structured light, and fluorescence to propagate in the time delay integration scanning imaging system 10 .

[0073] In this embodiment, the light guiding assembly includes a beam expanding and homogenizing lens 151, a total internal reflection mirror 152, four focusing lenses 153, four dichroic mirrors 154 and an objective lens 155. The beam expanding and homogenizing lens 151 is used to expand the received light beam and transmit it after homogenizing. The total internal reflection mirror 152 is used to reflect the received light source laser to the light field control device 12, and is also used to receive and transmit stripe structured light. Each focusing lens 153 is used to focus the received light and guide it to the subsequent light path. For example, the focusing lens 153 located between the total internal reflection mirror 152 and the sequencing chip 30 is used to focus the received stripe structured light onto the sequencing chip 30. Each dichroic mirror 154 is used to reflect or transmit the received light according to its wavelength. The objective lens 155 is used to focus the stripe structured light onto the sample 20 to be tested (or sequencing chip 30).

[0074] This embodiment further provides a time delay integration scanning imaging method, which adopts the above-mentioned time delay integration scanning imaging system 10.

[0075] See also Figure 5 The time delay integration scanning imaging method comprises the following steps:

[0076] Step S11, the light source module emits light source laser;

[0077] Step S12, the light source laser generates stripe structured light through a light field control device, and projects the stripe structured light onto the sample to be tested, so that the sample to be tested generates fluorescence according to the stripe structured light and forms a stripe pattern on the sample to be tested;

[0078] Step S13: an objective lens receives the fluorescence and transmits the fluorescence to at least one imaging channel; a time-delayed integration camera in the imaging channel generates a fluorescence image based on the fluorescence; the objective lens and the sample to be tested move relative to each other; a scanning rate of the time-delayed integration camera is synchronized with a movement rate of the sample to be tested; and a scanning direction of the time-delayed integration camera is perpendicular to an extension direction of the fringe pattern corresponding to the single fringe structured light;

[0079] Step S14, controlling the driving timing of the light field control device to be synchronized with the scanning timing of the time delay integration camera;

[0080] In step S15 , while the time-delayed integration camera performs synchronous scanning relative to the movement of the sample to be tested, the objective lens continuously receives the fluorescence, the time-delayed integration camera obtains a fluorescence image based on the fluorescence, and obtains biological information of the sample to be tested based on the fluorescence image.

[0081] This embodiment further provides a super-resolution detection method using the above-mentioned time delay integration scanning imaging method, and employs the above-mentioned time delay integration scanning imaging system 10 .

[0082] See also Figure 6 , super-resolution detection methods include:

[0083] Step S21, a start step: the light source module emits a light source laser; the light source laser generates a stripe structured light through the switch array, and the stripe structured light is projected onto the sample to be tested, so that the sample to be tested generates fluorescence according to the stripe structured light and forms a stripe pattern on the sample to be tested;

[0084] Step S22, an image acquisition step: an objective lens receives the fluorescence and transmits the fluorescence to at least one imaging channel, a time-delayed integration camera in the imaging channel generates a fluorescence image based on the fluorescence, the objective lens and the sample to be tested move relative to each other, a scanning rate of the time-delayed integration camera is synchronized with the movement rate of the sample to be tested, and the scanning direction is perpendicular to the extension direction of the stripe pattern corresponding to the single stripe structured light; the driving timing of the switch array is controlled to be synchronized with the scanning timing of the time-delayed integration camera; while the time-delayed integration camera performs synchronous scanning relative to the movement of the sample to be tested, the objective lens continuously receives the fluorescence, and the time-delayed integration camera acquires the fluorescence image based on the fluorescence;

[0085] Step S23, adjusting the initial deflection state of the switch array, and repeating the starting step to generate the stripe structured light with different phases, and performing the image acquisition step under the stripe structured light with different phases to acquire a fluorescence image;

[0086] Step S24 , performing super-resolution reconstruction based on the fluorescence image, thereby obtaining a super-resolution image to obtain biological information of the sample to be tested.

[0087] In step S21, the controller controls the light source module to emit a laser beam. The laser beam is modulated by the light field control device 12 to produce striped structured light. The striped structured light is guided to the sequencing chip 30 through the total internal reflection mirror 152, the beam expansion and homogenization lens 151, the dichroic mirror 154, and the focusing lens 153, and then projected onto the surface of the sample 20 on the sequencing chip 30. When the striped structured light is projected onto the surface of the sample 20, it forms an illumination pattern consisting of alternating bright and dark stripes.

[0088] The following describes the formation process of stripe structured light with three phases.

[0089] In this embodiment, the specific form of the stripe structured light is modulated by controlling the deflection of each micro-mirror 121 in the light field control device 12. In order to achieve a three-step phase shift, at least four micro-mirrors 121 are used to represent one stripe period.

[0090] See also Figure 7 , Figure 7 In the micromirror array shown in Figure (a), each row has 16 micromirrors 121. The 16 micromirrors 121 in each row are divided into 8 modulation units, and each modulation unit includes two adjacent micromirrors 121. The deflection states of the micromirrors 121 in the two adjacent modulation units are opposite, which is used to represent a stripe cycle (a stripe cycle includes a dark stripe and a light stripe, and the dark stripe is represented as black / dark color in the figure, and the light stripe is represented as white / light color). Figure 7 A black micro-mirror 121 indicates that the micro-mirror 121 is in the "OFF" state, and a white micro-mirror 121 indicates that the micro-mirror 121 is in the "ON" state. The micro-mirrors 121 in the same column are in the same state at the same time.

[0091] First, by controlling the ON and OFF states of each micro-mirror 121, the light projected onto the micro-mirror 121 is reflected by the micro-mirror 121 to form a binary stripe. The light intensity distribution of the binary stripe is a square wave shape with a duty cycle of 50% (e.g. Figure 7 Due to the numerical aperture limitation of the objective lens 155, the objective lens MTF (Modulation Transfer Function) can only receive information within a certain spatial frequency range, so the objective lens 155 is equivalent to a low-pass filter (e.g. Figure 7 (c)). By utilizing the low-pass filtering effect of the objective lens MTF, when the binary stripes pass through the objective lens 155, the high-frequency information is filtered out, forming sinusoidal stripes (such as Figure 7 When the sinusoidal stripes are projected onto the sequencing chip 30, an illumination pattern of alternating bright stripes and dark stripes is formed (e.g. Figure 7 (middle (e)).

[0092] This embodiment uses a “three-step phase shift” method to scan the sequencing chip 30 (i.e., scan the sample 20 on the sequencing chip 30 ): the sequencing chip 30 is scanned in two different directions with three stripe structured lights of different phases in sequence.

[0093] See also Figures 7 to 9 There are three combinations of states of four adjacent micro-mirrors 121:

[0094] 1)OFF / OFF / ON / ON( Figure 7 ), the phase of the corresponding stripe structured light is 0;

[0095] 2)ON / OFF / OFF / ON( Figure 8 ), the phase of the corresponding stripe structured light is π / 2;

[0096] 3)ON / ON / OFF / OFF( Figure 9 ), the phase of the corresponding stripe structured light is π.

[0097] By modulating the stripe structured light separately Figures 7 to 9 The phase shown achieves a three-step phase shift. Figure 8 and Figure 9 The formation method of the stripe structured light shown in Figure 7 The main difference is the state of the micro-mirror 121, which will not be described in detail.

[0098] At different times, Figures 7 to 9 The three stripe structured lights shown scan the sequencing chip 30. When the sample 20 on the sequencing chip 30 is irradiated by the stripe structured light, fluorescence is generated. Figures 7 to 9 In the three stripe structured lights shown, the extension direction of the stripes is the same, and the definition is Figures 7 to 9 The stripes in the three stripe structured lights are along the first direction (the first direction is Figures 7 to 9 In this embodiment, the stripes are generated along the second direction (the second direction is the vertical direction of the orientation shown). Figures 7 to 9 The present invention provides a stripe structured light extending in the horizontal direction of the orientation shown in FIG1 , wherein the first direction and the second direction are perpendicular to each other. Similar to the stripe structured light extending in the first direction, this embodiment also generates three types of stripe structured light extending in the second direction with phases of 0, π / 2, and π, respectively. That is, by controlling the deflection state of each micro-reflector 121, this embodiment generates a total of six types of stripe structured light: stripe structured light with a phase of 0 and stripes extending in the first direction, stripe structured light with a phase of π / 2 and stripes extending in the first direction, stripe structured light with a phase of π and stripes extending in the first direction, stripe structured light with a phase of 0 and stripes extending in the second direction, stripe structured light with a phase of π / 2 and stripes extending in the second direction, and stripe structured light with a phase of π and stripes extending in the second direction. The above six types of stripe structured light are sequentially projected by the objective lens 155 onto the sequencing chip 30.

[0099] Typically, the sequencing chip 30 has a large area, while the illumination pattern formed by the stripe structured light on the sequencing chip 30 is relatively small, covering only a portion of the sequencing chip 30. Therefore, in step S2, it is necessary to control the sequencing chip 30 and the illumination pattern formed by the stripe structured light to produce relative displacement. By controlling the sequencing chip 30 and the illumination pattern formed by the stripe structured light to continuously produce relative displacement, the illumination pattern formed by the stripe structured light can completely scan the sequencing chip 30.

[0100] See also Figure 10 In this embodiment, the surface of the sequencing chip 30 illuminated by the stripe structured light is rectangular, with the lengths of two adjacent sides defined as W and H, respectively. The area of ​​the surface of the sequencing chip 30 illuminated by the stripe structured light is W×H. The sequencing chip 30 is divided into K+1 rectangular strip-shaped scanning areas, numbered sequentially as strip areas 1, 2, 3, ..., K+1. Of the K+1 scanning areas, the areas of K scanning areas are W×ΔH, and the area of ​​one scanning area is W×ΔH′. Where:

[0101] H=K·ΔH+ΔH′(0≤ΔH′<ΔH) (1),

[0102] Wherein ΔH is the scanning width of the illumination pattern formed by the stripe structured light on the sequencing chip 30 .

[0103] The stripe structured light is controlled to scan the K+1 scanning areas in sequence with the numbers from small to large.

[0104] Figure 11 The figure shows the process of scanning a certain scanning area on the sequencing chip 30 with stripe structured light. Figure 11 In the figure (a), in the first period, the light field control device 12 is controlled to modulate the light source laser to generate a stripe structured light with a phase of 0, and the stripe structured light with a phase of 0 is scanned. Figure 10 In the scanning area shown, the scanning direction is from left to right.

[0105] Please refer to Figure 11 In the second period, the light field control device 12 is controlled to modulate the light source laser to generate a stripe structured light with a phase of π / 2, and the stripe structured light with a phase of π / 2 is scanned. Figure 10 In the scanning area shown, the scanning direction is from right to left.

[0106] Please refer to Figure 11 In the figure (c), in the third period, the light field control device 12 is controlled to modulate the light source laser to generate a stripe structured light with a phase of π, and the stripe structured light with a phase of π is scanned. Figure 10 In the scanning area shown, the scanning direction is from left to right.

[0107] As the stripe structured light scans the sequencing chip 30, the emission direction of the stripe structured light remains unchanged. The sequencing chip 30 is driven to the left at a constant speed, causing the illumination pattern formed by the stripe structured light on the sequencing chip 30 to move relative to the sequencing chip 30. The direction of relative motion is the same as the extension direction of the side of the rectangular scanning area with a length of W.

[0108] In this embodiment, the above-mentioned "from left to right" or "from right to left" refers to the direction of the phase shift generated between the illumination pattern of the stripe structured light on the sequencing chip 30 and the sequencing chip. During the scanning process, the field of view of the stripe structured light remains unchanged, and only the sequencing chip 30 is driven to move to cause a relative displacement between the sequencing chip 30 and the illumination pattern.

[0109] Figure 11 The scanning process shown is only a scanning process for one scanning area. After the scanning is completed, the stripe structured light is controlled to continue scanning the next scanning area. The scanning process for each scanning area is roughly the same and will not be repeated here.

[0110] During operation of the time-delayed integration scanning imaging system 10, the field of view of the stripe structured light remains unchanged (i.e., the stripe structured light produces a "static" stripe image). The TDI camera also remains stationary. The sequencing chip 30 moves to produce a relative displacement with the illumination pattern, and the imaging module of the time-delayed integration scanning imaging system 10 receives the fluorescence generated by the sample 20 to be tested through the TDI camera and performs imaging. When the sequencing chip 30 is scanned with a "static" stripe pattern, if the sequencing chip 30 and the stripe structured light produce a relative displacement, the sequencing chip 30 is sequentially illuminated by bright stripes and dark stripes. The fluorescence generated by the sample 20 to be tested on the sequencing chip 30 based on the bright stripes and dark stripes is gradually accumulated by the TDI camera. Due to the existence of the accumulation effect, the intensity of the stripe structured light will be averaged. In the image generated by the TDI camera based on the fluorescence, the information of the stripe structured light will be lost, and super-resolution image reconstruction cannot be performed. In other words, the biological information of the sample 20 to be tested cannot be obtained.

[0111] Therefore, the super-resolution detection method and time-delayed integration scanning imaging system 10 provided in this embodiment use a "dynamic" stripe image to scan the sequencing chip 30, so that in the stripe image generated by the stripe structured light, the stripes and the sequencing chip 30 are synchronously displaced, thereby improving the technical problem of "information loss of the stripe structured light and inability to reconstruct a super-resolution image."

[0112] The following describes the “dynamic” fringe image and the synchronous displacement process of the fringe and sequencing chip 30 (ie, the image acquisition process in step S22 ) in this embodiment.

[0113] See also Figure 12 , the sequencing chip 30 and the TDI camera 132 are in the initial position, all the micro-mirrors 121 in the light field control device 12 are in the "OFF" state, no stripe structured light is formed, the sequencing chip 30 does not enter the illumination field of the stripe structured light, the sample to be tested 20 is not irradiated by the stripe structured light, and the TDI camera 132 does not receive any fluorescence.

[0114] See also Figure 13 The leftmost row of micro-mirrors 121 of the light field control device 12 are all in the "ON" state, and the light field control device 12 begins to generate stripe structured light. At this time, the platform drives the sequencing chip 30 to move, and a small area on the rightmost side of the sequencing chip 30 enters the illumination field of the stripe structured light. The DNA nanospheres on the sample 20 to be tested that are illuminated by the stripe structured light produce fluorescence, and the leftmost primary sensor on the target surface of the TDI camera 132 receives the fluorescence and generates a sensing electrical signal.

[0115] See also Figure 14-17The platform continuously drives the sequencing chip 30 to move, causing the sequencing chip 30 and the stripe structured light to continuously move relative to each other. The area of ​​the rightmost portion of the sequencing chip 30 that enters the stripe structured light's field of view gradually increases. The stripe structured light generated by the light field control device 12 continuously shifts rightward, and the platform drives the sequencing chip 30 to continuously shift rightward. The TDI camera 132 gradually shifts and accumulates the electrical signals sensed by the fluorescence. The displacement of the stripe structured light, the displacement of the sequencing chip 30, and the reading of the electrical signals by the TDI camera 132 are synchronized.

[0116] exist Figure 17 At this moment, all the micro-mirrors 121 on the light field control device 12 are in the ON / OFF working state. The stripe structured light generated by the light field control device 12 shifts to the right and covers the entire illumination field of the stripe structured light. All stages of the TDI camera 132 receive fluorescence and generate sensing electrical signals.

[0117] See also Figure 18 , continue scanning the sequencing chip 30 until the leftmost side of the sequencing chip 30 is flush with the left side of the illumination field of the stripe structured light.

[0118] See also Figure 19 , the sequencing chip 30 is continuously scanned. There are no DNA nanoballs on the left side of the illumination field of the stripe structured light. All micro-mirrors 121 corresponding to the left area are in the "OFF" state, and no stripe pattern is generated.

[0119] See also Figure 20 The sequencing chip 30 continues to be scanned until the leftmost portion of the sequencing chip 30 has moved out of the field of view of the stripe illumination light. At this point, all micromirrors 121 of the light field control device 12 are in the "OFF" state, and no illumination stripes are visible on the entire sequencing chip 30. At this point, the scanning of a stripe-shaped scanning area on the sequencing chip 30 is complete (this refers to the process of scanning a scanning area with stripe structured light of the same phase. The phase of the stripe structured light needs to be changed in the next period to continue scanning the scanning area).

[0120] Figure 12-Figure 20 The scanning process shown is only the scanning process under the illumination of one of the six stripe structured lights mentioned above. In this embodiment, the scanning process is completed. Figure 12-Figure 20 After the scanning process is shown, in step S23 , the deflection states of the plurality of micro-mirrors 121 are adjusted to generate other types of stripe structured light to scan the sequencing chip 30 .

[0121] During the stripe structured light scanning of sequencing chip 30, the generated fluorescence is received by TDI camera 132, generating a corresponding fluorescence image. The fluorescence image generated by illumination with stripe structured light of three phases extending along a first direction is defined as a first fluorescence image, while the fluorescence image generated by illumination with stripe structured light of three phases extending along a second direction is defined as a second fluorescence image.

[0122] In step S24 , the controller performs super-resolution reconstruction based on the first fluorescence image and the second fluorescence image generated by the four TDI cameras 132 , thereby acquiring a super-resolution image to obtain biological information of the sample 20 to be tested.

[0123] The scanning process for the remaining scanning areas is basically the same as above and will not be described in detail. Figures 12 to 20 It can be seen that by changing the deflection state of each micro-mirror 121, the distribution positions of the bright stripes and the dark stripes in the stripe pattern generated by the stripe structured light are different, so that when observed with the naked eye, a visual effect of the stripes "flowing" in a preset direction is achieved, that is, a "dynamic" stripe image is generated.

[0124] The time-delayed integration scanning imaging system 10 and the super-resolution detection method applied thereto provided in this embodiment employ "dynamic" fringe image scanning of the sequencing chip 30. This ensures that, in the fringe image generated by the fringe structured light, the fringes and the sequencing chip 30 shift synchronously (with the same displacement direction and speed). This displacement speed is also the same as the speed at which the TDI camera 132 reads data (i.e., the scanning speed of the TDI camera). In other words, the fringe displacement, the sequencing chip 30 displacement, and the data reading speed of the TDI camera 132 are all synchronized. The movement directions of the sequencing chip 30 and the TDI camera 132 are perpendicular to the extension direction of each fringe in the current fringe structured light. This ensures that the fringe structured light information is fully retained in the image output by the TDI camera 132, rather than being accumulated and averaged by the TDI camera 132. This helps resolve the technical issue of "loss of fringe structured light information and inability to perform super-resolution image reconstruction."

[0125] Those skilled in the art should recognize that the above embodiments are only used to illustrate the present invention and are not intended to limit the present invention. As long as they are within the scope of the essential spirit of the present invention, appropriate changes and modifications to the above embodiments are within the scope of protection claimed by the present invention.

Claims

1. A time-delayed integration scanning imaging system, characterized in that: include: A light source module, used for emitting light source laser; A light field control device is located in the optical path of the light source laser and is used to receive and modulate the light source laser to emit stripe structured light. The light field control device includes a switch array. The stripe structured light is projected onto the sample to be tested so that the sample to be tested produces fluorescence under the illumination of the stripe structured light. The stripe structured light forms a stripe pattern on the sample to be tested, and the sample to be tested is a nucleic acid sample. An imaging module, comprising an objective lens disposed relative to the sample to be tested and at least one imaging channel, each imaging channel having a time-delayed integration camera, the objective lens being configured to receive the fluorescence and transmit the fluorescence to the time-delayed integration camera, the time-delayed integration camera being configured to generate a fluorescence image based on the fluorescence, the fluorescence image being configured to obtain biological information of the sample to be tested, the objective lens and the sample to be tested being in relative motion, the scanning rate of the time-delayed integration camera being synchronized with the movement rate of the sample to be tested, the scanning direction of the time-delayed integration camera being perpendicular to the extension direction of the stripe pattern corresponding to a single stripe of structured light, and the biological information being base sequence information of the sample to be tested; as well as A controller is connected to the switch array and the time delay integration camera, and is used to control the synchronization of the drive timing of the switch array and the scanning timing of the time delay integration camera, and the synchronization of the stripe displacement in the stripe pattern, the displacement of the sample to be measured, and the speed at which the time delay integration camera reads data.

2. The time-delayed integration scanning imaging system according to claim 1, wherein: The switch array is a digital micro-mirror array.

3. The time-delayed integration scanning imaging system according to claim 2, wherein: The striped structured light forms an illumination pattern on the surface of the sample to be tested in which bright stripes and dark stripes are arranged alternately and parallel to each other, and the bright stripes and the dark stripes are arranged alternately along the movement direction of the sample to be tested; the light field control device includes a plurality of micro-mirrors, and the plurality of micro-mirrors are divided into a plurality of modulation units, each modulation unit includes two micro-mirrors, and the deflection states of the micro-mirrors of adjacent modulation units are opposite, and the controller adjusts the deflection state of each modulation unit according to the driving timing to control the striped morphology of the illumination pattern.

4. The time-delayed integration scanning imaging system according to claim 3, wherein: The controller is further configured to control the initial deflection states of the plurality of micro-mirrors to control the phase of the stripe structured light, so that stripe structured light of different phases is projected onto the sample to be measured.

5. The time-delayed integration scanning imaging system according to claim 4, wherein: The time delay integration camera includes a linear array CCD image sensor, and the pixel units of the CCD image sensor correspond one-to-one to the modulation units.

6. The time-delayed integration scanning imaging system according to claim 4, wherein: The time-delayed integral scanning imaging system includes multiple imaging channels, and the time-delayed integral scanning imaging system also includes a light guiding component located between the objective lens and the multiple imaging channels. The sample to be tested generates fluorescence of multiple wavelengths under the irradiation of the striped structured light, and the light guiding component is used to guide the fluorescence of the multiple wavelengths to different imaging channels respectively. The multiple imaging channels correspond one-to-one to the multiple wavelengths of the fluorescence.

7. A scanning imaging method, characterized in that: Using the time-delayed integration scanning imaging system according to any one of claims 1 to 6, the scanning imaging method includes: The light source module emits light source laser; The light source laser generates stripe structured light through a light field control device, and projects the stripe structured light onto a sample to be tested, so that the sample to be tested generates fluorescence according to the stripe structured light and forms a stripe pattern on the sample to be tested; An objective lens receives the fluorescence and transmits the fluorescence to at least one imaging channel. A time-delayed integration camera in the imaging channel generates a fluorescence image based on the fluorescence. The objective lens and the sample to be tested move relative to each other. The scanning rate of the time-delayed integration camera is synchronized with the movement rate of the sample to be tested. The scanning direction of the time-delayed integration camera is perpendicular to the extension direction of the stripe pattern corresponding to the single stripe structured light. Controlling the driving timing of the light field control device to be synchronized with the scanning timing of the time delay integration camera, and synchronizing the displacement of the stripes in the stripe pattern, the displacement of the sample to be measured, and the speed of reading data by the time delay integration camera; During the period of synchronous scanning of the time-delayed integration camera relative to the movement of the sample to be tested, the objective lens continuously receives the fluorescence, the time-delayed integration camera obtains a fluorescence image based on the fluorescence, and obtains biological information of the sample to be tested based on the fluorescence image; the sample to be tested is a nucleic acid sample, and the biological information is the base sequence information of the sample to be tested.

8. The scanning imaging method according to claim 7, wherein: Also includes: forming an illumination pattern on the surface of the sample to be tested, wherein light stripes and dark stripes are arranged alternately and parallel to each other, wherein the light stripes and the dark stripes are arranged alternately along the moving direction of the sample to be tested; The deflection states of the micro-mirrors in adjacent modulation units in the light field control device are controlled to be opposite, and the deflection state of each modulation unit is adjusted according to the driving timing to control the stripe shape of the illumination pattern.

9. The scanning imaging method according to claim 8, wherein: The time delay integration camera includes a linear array CCD image sensor, and the pixel units of the CCD image sensor correspond one-to-one to the modulation units.

10. A super-resolution detection method, characterized in that: Using the time-delayed integration scanning imaging system according to any one of claims 1 to 6, the super-resolution detection method comprises: Starting step: the light source module emits a light source laser; the light source laser generates a stripe structured light through the switch array, and the stripe structured light is projected onto the sample to be tested, so that the sample to be tested generates fluorescence according to the stripe structured light and forms a stripe pattern on the sample to be tested; Adjusting the initial deflection state of the switch array, and repeating the starting step to generate the stripe structured light with different phases, and performing the image acquisition step under the stripe structured light with different phases to acquire a fluorescence image; and Performing super-resolution reconstruction based on the fluorescence image to obtain a super-resolution image to obtain biological information of the sample to be tested, wherein the sample to be tested is a nucleic acid sample, and the biological information is the base sequence information of the sample to be tested; The image acquisition step includes: an objective lens receives the fluorescence and transmits the fluorescence to at least one imaging channel, a time-delayed integration camera in the imaging channel generates a fluorescence image based on the fluorescence, the objective lens and the sample to be tested move relative to each other, the scanning rate of the time-delayed integration camera is synchronized with the movement rate of the sample to be tested, and the scanning direction is perpendicular to the extension direction of the stripe pattern corresponding to the single stripe structured light; controlling the driving timing of the switch array to be synchronized with the scanning timing of the time-delayed integration camera, and synchronizing the stripe displacement in the stripe pattern, the displacement of the sample to be tested, and the speed of reading data by the time-delayed integration camera; during the period when the time-delayed integration camera performs synchronous scanning relative to the movement of the sample to be tested, the objective lens continuously receives the fluorescence, and the time-delayed integration camera obtains the fluorescence image based on the fluorescence.

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