Super-resolution microscopic imaging system, and imaging method

Abstract

The present invention relates to the technical field of gene sequencing, and aims to solve the problem of loss of light energy being large. Disclosed in the present invention are a super-resolution microscopic imaging system, and an imaging method. The imaging system comprises an objective bearing stage, a light source assembly, a drive assembly, a control assembly and an imaging assembly, wherein the objective bearing stage bears a sample to be tested; the light source assembly emits linear light to irradiate said sample, such that a linear region excites fluorescence; the drive assembly is connected to the objective bearing stage and drives the objective bearing stage to move; the control assembly controls the objective bearing stage to move and controls the light source assembly to emit the linear light at a preset on-off period; and the imaging assembly is configured to receive the fluorescence excited by the linear region and generate an area-array fluorescence image, and use the area-array fluorescence image to synthesize a super-resolution image. In the imaging system, a control assembly controls an objective bearing stage to move, such that linear light sequentially irradiates a plurality of linear regions of a sample to be tested, and a light source assembly can use fluorescence to perform imaging, so as to obtain a two-dimensional area-array fluorescence image of said sample, thereby realizing an imaging system of a simple structure, and also reducing the loss of light energy.

Description

Super-resolution microscopy imaging system and imaging method

[0001] The present invention claims priority to Chinese patent application number 202311009924.3, filed with the Patent Office of China on August 11, 2023, entitled “Super-resolution microscopy imaging system and imaging method”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of gene sequencing and biological sample technology, and in particular to a super-resolution microscopic imaging system and imaging method. Background Art

[0003] Currently, gene sequencers need to perform fluorescence imaging of the four ATCG bases separately during the sequencing process. Since each target point detected is in the submicron scale range, SIM (Structured Illumination Microscopy) can be used to achieve high-precision imaging when sequencing large amounts of DNA. In existing technologies, SIM solutions require diffraction and spectroscopy through devices such as gratings, spatial light modulators, and digital micromirrors, which wastes a lot of energy and also makes the lighting system relatively complex.

[0004] Summary of the Invention

[0005] The embodiments of the present invention provide a super-resolution microscopic imaging system and an imaging method to solve at least one of the above-mentioned technical problems.

[0006] A super-resolution microscopy system provided by an embodiment of the present invention includes:

[0007] a loading platform, the loading platform carrying a sample to be tested;

[0008] a light source assembly configured to emit linear light, the linear light irradiating the sample to be tested so as to excite fluorescence in a linear region of the sample to be tested, the linear region extending along a first direction;

[0009] a driving assembly connected to the stage and configured to drive the stage to move along a second direction, wherein the first direction is perpendicular to the second direction;

[0010] a control assembly electrically connected to the light source assembly and the drive assembly, the control assembly being configured to control the drive assembly to drive the stage to move along the second direction and to control the light source assembly to emit the linear light in a preset on / off cycle, so that the linear light illuminates the plurality of linear regions of the sample to be tested along the second direction, with at least one unilluminated linear region existing between the plurality of illuminated linear regions;

[0011] An imaging component is used to receive the fluorescence of a line area each time the linear light irradiates the line area and generate a planar array fluorescence image of a planar area, and synthesize a super-resolution image based on the planar array fluorescence images of multiple planar areas.

[0012] In the above-mentioned super-resolution microscopy imaging system, the control component can control the driving component to drive the stage to move along the second direction, so that the linear light can sequentially illuminate multiple line areas of the sample to be tested. The light source component can use the fluorescence excited by the multiple line areas for imaging, thereby obtaining a two-dimensional array fluorescence image of the sample to be tested. Ultimately, a super-resolution microscopy imaging system with a simple structure can be realized while reducing the loss of light energy.

[0013] In certain embodiments, the preset on-off cycle includes alternating on and off periods. The control component controls the light source component to emit the linear light according to the on-periods, causing the illuminated linear region to emit fluorescence. The control component controls the light source component to turn off the linear light according to the off-periods, resulting in an unilluminated linear region. The preset on-off cycles of the plurality of area array fluorescence images differ by a preset phase value. Thus, by reconstructing and calculating the area array fluorescence images with different phase values, a higher resolution image is obtained.

[0014] In some embodiments, the light source assembly includes a laser emitter and a beam shaper, wherein the laser emitter is used to emit laser light and the beam shaper is used to shape the laser light into the linear light. In this way, the structure of the light source assembly can be simplified.

[0015] In certain embodiments, the length of the image of the line region on the photosensitive element of the imaging assembly in the third direction is less than or equal to the length of the photosensitive element of the imaging assembly, thereby fully utilizing the imaging area of ​​the imaging assembly.

[0016] In some embodiments, the width of the image of the line region on the photosensitive element of the imaging assembly in the fourth direction is less than or equal to the width of a pixel of the photosensitive element of the imaging assembly. In this way, the resolution can be improved.

[0017] In certain embodiments, the super-resolution microscopy system includes a first dichroic mirror and an objective lens. The objective lens is configured to collect the fluorescence and transmit the fluorescence to the first dichroic mirror. The first dichroic mirror is configured to reflect the linear light toward the sample to be measured and transmit the fluorescence to the imaging component. This helps avoid interference from the linear light.

[0018] In certain embodiments, the imaging assembly includes multiple second dichroic mirrors that distribute the fluorescence to multiple imaging channels. Each imaging channel includes a converging lens and a line scan camera. The converging lens is configured to converge the fluorescence onto the line scan camera, which is configured to receive the fluorescence. This facilitates the detection of multiple fluorescence information.

[0019] In certain embodiments, the imaging component includes a filter, which is used to filter the fluorescence, thereby improving the imaging signal-to-noise ratio.

[0020] An imaging method for a sample to be tested provided by an embodiment of the present invention includes:

[0021] Emitting linear light in a preset on-off cycle and irradiating a linear region of the sample to be tested to excite the linear region of the sample to be tested to emit fluorescence, wherein the linear region extends along a first direction;

[0022] Moving the sample to be tested along a second direction so that the linear light illuminates a plurality of linear regions of the sample to be tested along the second direction, wherein at least one unilluminated linear region exists between the plurality of illuminated linear regions, and the first direction is perpendicular to the second direction;

[0023] receiving fluorescence excited by the linear light irradiating a line area each time and generating a planar array fluorescence image of a planar area according to the fluorescence;

[0024] A super-resolution image is reconstructed based on the area array fluorescence images of the plurality of area regions.

[0025] In the above-mentioned imaging method, the control component can control the driving component to drive the stage to move along the second direction, so that the linear light sequentially illuminates multiple line areas of the sample to be tested. The light source component can use the fluorescence excited by the multiple line areas for imaging, thereby obtaining a two-dimensional array fluorescence image of the sample to be tested. Ultimately, a simple super-resolution microscopic imaging system can be realized while reducing the loss of light energy.

[0026] In some embodiments, the preset on-off cycle includes alternating on periods and off periods, and the preset on-off periods of the plurality of area array fluorescence images differ by a preset phase value.

[0027] Emitting linear light with a preset on-off cycle to excite a linear area of ​​the sample to be tested, including:

[0028] When the preset on / off cycle is in the on period, emitting the linear light so that the illuminated linear area emits fluorescence;

[0029] When the preset on-off cycle is in the off period, the linear light is turned off to form the un-illuminated linear area, thereby improving the resolution.

[0030] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments with reference to the following drawings, in which:

[0032] FIG1 is a schematic diagram of a module of a super-resolution microscopy imaging system according to an embodiment of the present invention;

[0033] FIG2 is a schematic diagram of the composition of a super-resolution microscopy imaging system according to an embodiment of the present invention;

[0034] 3a to 3c are schematic diagrams of structured light illumination imaging according to an embodiment of the present invention;

[0035] 4a to 4f are schematic diagrams of linear light irradiation according to an embodiment of the present invention;

[0036] FIG5 is a schematic flow chart of an imaging method according to an embodiment of the present invention.

[0037] Explanation of the accompanying drawings: Super-resolution microscopy imaging system-100, sample to be measured-10, fluorescence-11, line area-12, illuminated line area-121, non-illuminated line area-122, light source assembly-20, linear light-21, laser emitter-22, beam shaper-23, multimode optical fiber-24, irradiation area-25, imaging assembly-30, second dichroic mirror-32, imaging channel-33, converging lens-331, linear array camera-332, filter-333, stage-40, first dichroic mirror-51, objective lens-52, drive assembly-60, control assembly-70. DETAILED DESCRIPTION

[0038] The embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention.

[0039] The disclosure below provides many different embodiments or examples for realizing different structures of the present invention. In order to simplify the disclosure of the present invention, the components and settings of specific examples are described below. Of course, they are merely examples and are not intended to limit the present invention. In addition, the present invention may repeat reference numbers and / or reference letters in different examples. Such repetition is for the purpose of simplicity and clarity and does not in itself indicate the relationship between the various embodiments and / or settings discussed. In addition, the present invention provides examples of various specific processes and materials, but those skilled in the art will recognize the application of other processes and / or the use of other materials.

[0040] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features being referred to. Thus, features defined as "first" or "second" may explicitly or implicitly include one or more features. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0041] Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that a specific feature, structure, material, or characteristic described in conjunction with an embodiment or example is included in at least one embodiment or example of the present invention. In this specification, illustrative descriptions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0042] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise" and the like to indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0043] In the present invention, unless otherwise expressly specified or limited, a first feature being "above" or "below" a second feature may include the first and second features being in direct contact, or may include the first and second features being in contact not directly but through another feature between them. Furthermore, a first feature being "above," "above," and "above" a second feature may include the first feature being directly above or obliquely above the second feature, or may simply mean that the first feature is higher in level than the second feature. A first feature being "below," "below," and "below" a second feature may include the first feature being directly below or obliquely below the second feature, or may simply mean that the first feature is lower in level than the second feature.

[0044] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood broadly. For example, they may refer to fixed connections, removable connections, or integral connections. They may refer to mechanical connections or electrical connections. They may refer to direct connections or indirect connections through an intermediary, and they may refer to internal communication between two components or interactions between two components. Those skilled in the art will understand the specific meanings of these terms in the present invention based on the specific circumstances.

[0045] Referring to Figures 1, 2, and 3a to 3c, a super-resolution microscopy imaging system 100 according to an embodiment of the present invention includes a stage 40, a light source assembly 20, a drive assembly 60, a control assembly 70, and an imaging assembly 30. The stage 40 supports a sample 10 to be tested. The light source assembly 20 is configured to emit linear light 21. The linear light 21 illuminates the sample 10, causing a linear region 12 of the sample 10 to excite fluorescence 11. The linear region 12 extends along a first direction X. The drive assembly 60 is connected to the stage 40 and is configured to drive the stage 40 to move along a second direction Y. The first direction X is perpendicular to the second direction Y. The control assembly 70 is electrically connected to the light source assembly 20 and the drive assembly 60. The control assembly 70 is configured to control the drive assembly 60 to drive the stage 40 to move along the second direction Y and to control the light source assembly 20 to emit linear light 21 in a predetermined on / off cycle, so that the linear light 21 illuminates multiple linear regions 12 of the sample 10 along the second direction Y. There is at least one un-irradiated linear region 122 between the multiple illuminated linear regions 121. The imaging component 30 is configured to receive the fluorescence 11 generated each time the linear light 21 illuminates a linear region 12 and generate an area array fluorescence image of a surface region, and synthesize a super-resolution image based on the area array fluorescence images of the multiple surface regions.

[0046] In the above-mentioned super-resolution microscopy imaging system 100, the control component 70 can control the drive component 60 to drive the stage 40 to move along the second direction Y, so that the linear light 21 sequentially illuminates multiple linear regions 12 of the sample to be tested 10. The imaging component 30 can use the fluorescence 11 excited by the multiple linear regions 12 for imaging, thereby obtaining a two-dimensional array fluorescence image of the sample to be tested 10. Ultimately, a super-resolution microscopy imaging system 100 with a simple structure can be realized while reducing the loss of light energy.

[0047] In some embodiments, the gene sequencer needs to perform fluorescence imaging of the four ATCG bases separately during the sequencing process. Since each target point detected is in the submicron scale range, the gene sequencer can use microscopic imaging to achieve the detection purpose. An important indicator of a gene sequencer is the point density on the sequencing chip, which determines the amount of data on a sequencing chip and also determines the output cost per unit data. Since the difference between high and low point density and the amount of reagent used per unit area is not significant, and the detection cost is positively correlated with the reagent volume, the higher the point density, the greater the amount of data on a single sequencing chip, the smaller the amount of reagent required per unit data volume, and thus the lower the detection cost. However, high-density chips require a higher-resolution microscope system. Traditional wide-field microscope systems are diffraction-limited, and the resolution generally reaches a maximum of around 200nm, which cannot meet the requirements of gene sequencers. Therefore, in order to obtain higher resolution and achieve higher chip point density, super-resolution optical methods can be used.

[0048] The optical means of super-resolution is to exceed the diffraction limit of the optical system, which can be achieved through means such as STED (Stimulated Emission Depletion), FPALM (Fluorescence Photoactivation Localization Microscopy) and SIM (Structured Illumination Microscopy).

[0049] Among them, STED requires the use of high-power lasers to suppress the fluorescence signals in non-target areas. Although this method can achieve super-resolution, it will inevitably damage the sample to be tested due to the high power. Therefore, it is not suitable for use in gene sequencers.

[0050] FPALM uses a time-scaling approach, allowing fluorescent proteins to light up randomly. This allows two fluorescent molecules, which are previously indistinguishable due to their inconsistent emission times, to be distinguished by separately capturing their signals. Ultimately, multiple captured images are reconstructed to create a single super-resolution image. However, this method requires thousands of images to fully detect all fluorescent molecules, which is time-consuming and unsuitable for use on gene sequencers.

[0051] Traditional SIM solutions require diffraction and splitting of light using devices such as gratings, spatial light modulators, and DMDs (digital micromirrors). The separated light beams then interfere on the focal plane of the objective lens to produce sinusoidal fringes, creating structured light illumination. This solution has two drawbacks: first, diffraction and splitting waste significant energy; second, the illumination system is relatively complex.

[0052] In summary, the super-resolution microscopy system 100, which uses linear light 21 for emission, can address the aforementioned issues. By moving the sample 10 and controlling the emission timing, the same structured light illumination effect can be achieved. Furthermore, the super-resolution microscopy system 100 has a simple structure and eliminates the loss of light energy due to grating diffraction. Furthermore, the stage 40 can continuously move the sample 10, resulting in fast scanning and imaging speeds, shortening the time required to generate sequencing reports.

[0053] In one embodiment, the stage 40 carries a gene sequencing chip, and the sequencing chip carries biological samples with fluorescent dyes, and the biological samples can be arranged in an array.

[0054] Referring to Figures 1 and 2, in one embodiment, a sample to be tested 10 is placed on a stage 40. The light source assembly 20 can emit linear light 21 to illuminate the sample to be tested 10 on the stage 40. The sample to be tested 10 can be excited by the linear light 21 and emit fluorescence 11. The imaging assembly 30 can receive the fluorescence 11 emitted by the sample to be tested 10. Specifically, the linear light 21 illuminates the linear region 12 of the sample to be tested 10, and the range of the light spot formed by the linear light 21 in the linear region 12 corresponds to the irradiation area 25. The size of the irradiation area 25 can be consistent with the size of the linear region 12. The length extension direction of the irradiation area 25 is consistent with the length extension direction of the linear region 12, and the length direction of the linear region 12 corresponds to the first direction X. In addition, the length of the irradiation area 25 can also be slightly larger than the linear region 12. The control assembly 70 can control the movement of the stage 40 through the drive assembly 60, thereby controlling the movement of the sample to be tested 10. While the light source assembly 20 remains stationary, the control assembly 70 controls the stage 40 to move along the second direction Y. That is, the sample 10 to be tested moves relative to the linear light 21 along the second direction Y, thereby causing the plurality of line regions 12 to sweep across the irradiation area 25 along the second direction Y. It will be appreciated that the linear light 21 may illuminate the sample 10 in a direction perpendicular to the surface of the sample 10 or in other directions, while maintaining the light spot of the linear light 21 on the sample 10 extending along the first direction X.

[0055] Furthermore, the surface of the sample 10 to be tested can be divided into multiple linear regions 12, with the width of the sample 10 corresponding to a first direction X and the length corresponding to a second direction Y. The control assembly 70 can control the continuous movement of the stage 40 along the second direction Y. Simultaneously, by controlling the linear light 21 of the light source assembly 20 to be turned on and off by the control assembly 70, a portion of the multiple linear regions 12 of the sample 10 to be illuminated by the linear light 21 can be illuminated by the linear light 21, while another portion is not. By controlling the light source assembly 20 according to a preset on / off cycle, the illuminated linear regions 121 can be distributed as stripes on the surface of the sample 10, with multiple unilluminated linear regions 122 interspersed between the illuminated linear regions 121. The distribution of the illuminated and unilluminated linear regions 121, 122 can be shown in Figure 3a. Thus, multiple illuminations of the linear light 21 can be combined to produce an effect equivalent to black and white striped structured light illumination through grating diffraction. The imaging assembly 30 receives the fluorescence 11 from each linear region 12 illuminated by the linear light 21, and combines the images from these multiple illuminations to produce a structured illumination image. The area array fluorescence image is composed of the fluorescence 11 from multiple illuminated linear regions 121 and unilluminated linear regions 122 within the sample 10. The imaging assembly 30 can reconstruct and calculate a super-resolution image of the sample 10 by combining multiple area array fluorescence images with different phase values.

[0056] In another embodiment, the drive assembly 60 can also be built into the stage 40, and the drive assembly 60 is connected to the tray on the stage 40 carrying the sample 10 to be tested. The stage 40 can control the drive assembly 60 to drive the tray according to the instructions of the control assembly 70, thereby controlling the movement of the sample 10 to be tested.

[0057] Referring to Figures 3a to 3c and Figures 4a to 4f, in some embodiments, the preset on-off cycle includes alternating on periods and off periods. The control component 70 controls the light source component 20 to emit linear light 21 according to the on period so that the illuminated line area 121 emits fluorescence 11. The control component 70 controls the light source component 20 to turn off the linear light 21 according to the off period to form an unilluminated line area 122. The preset on-off cycles of the multiple area array fluorescence images differ by a preset phase value.

[0058] In this way, the resolution can be improved.

[0059] Specifically, referring to Figures 3a to 3c and Figures 4a to 4f, in one embodiment, one on-period and one off-period constitute a minimum period of a preset on-off cycle. The duration of the on-period can be equal to the duration of the off-period. During an on-period, the light source assembly 20 emits linear light 21, sweeping across a plurality of linear regions 12 within the illumination area 25 corresponding to the light source assembly 20. The linear regions 12 swept sequentially during the on-period form an illuminated linear region 121. During an off-period, the light source assembly 20 turns off the linear light 21, sweeping across the same number of linear regions 12 within the illumination area 25 corresponding to the light source assembly 20. The linear regions 12 swept from the illumination area 25 during the off-period form an unilluminated linear region 122.

[0060] When the sample 10 to be tested moves along the second direction Y, the light source assembly 20 alternates between on and off periods according to a preset on / off cycle, scanning the entire linear region 12 of the sample 10. The imaging assembly 30 collects the fluorescence 11 from the illuminated linear region 121 of the sample 10 to generate a planar array fluorescence image. After completing the acquisition of one planar array fluorescence image, the control assembly 70 controls the drive assembly 60 to drive the stage 40 in the opposite direction of the second direction Y, so that the sample 10 to be tested once again scans the entire linear region 12 along the second direction Y and completes another planar array fluorescence image. Similarly, the imaging assembly 30 can acquire multiple planar array fluorescence images. The phases of these multiple planar array fluorescence images can be incremented by a preset phase value. The preset phase value can be determined based on a minimum period of the preset on / off cycle and the number of planar array fluorescence images. Thus, the illuminated linear region 121 within the multiple planar array fluorescence images can cover the entire surface of the sample 10 to be tested.

[0061] Referring to FIG. 2 , in some embodiments, the light source assembly 20 includes a laser emitter 22 and a beam shaper 23 . The laser emitter 22 is used to emit laser light, and the beam shaper 23 is used to shape the laser light into linear light 21 .

[0062] In this way, the structure of the light source assembly 20 can be simplified.

[0063] Specifically, referring to FIG. 2 , in one embodiment, the laser emitter 22 can emit laser light of a specific wavelength corresponding to a wavelength at which the sample 10 to be tested can be excited. The light source assembly 20 can also include a multimode optical fiber 24, through which the laser emitter 22 is connected to a beam shaper 23. The multimode optical fiber 24 is used to transmit the laser light emitted by the laser emitter 22 to the beam shaper 23. The beam shaper 23 is used to shape the laser light into linear light 21 and irradiate the linear light toward the sample 10 to form a linear irradiation area 25 on the sample 10 to be tested.

[0064] 2 and 3 a , in some embodiments, the length of the image of the line region 12 on the photosensitive element (not shown) of the imaging assembly 30 in the third direction is less than or equal to the length of the photosensitive element of the imaging assembly 30 .

[0065] In this way, the imaging area of ​​the imaging component 30 can be fully utilized.

[0066] Specifically, referring to Figures 2 and 3a, in one embodiment, linear light 21 is irradiated onto the sample 10 to excite fluorescence 11 within a linear region 12. The fluorescence 11 within the linear region 12 can be fully received by the photosensitive element of the imaging assembly 30. The image of the fluorescence 11 within the linear region 12 on the photosensitive element is also linear, extending in a third direction. The length of the linear region 12 in the first direction X is positively correlated with the length of the image of the fluorescence 11 within the linear region 12 on the photosensitive element in the third direction. The image of the linear region 12 can be equal to the length of the photosensitive element. Under this condition, the fluorescence 11 within the linear region 12 can be fully received by the photosensitive element of the imaging assembly 30, and the image of the fluorescence 11 on the photosensitive element is maximized. Of course, the image of the linear region 12 can also be less than the length of the photosensitive element. Furthermore, the illumination length of the linear light 21 emitted by the light source assembly 20 can be greater than or equal to the length of the linear region 12.

[0067] 2 and 3 a , in some embodiments, the width of the line region 12 in the fourth direction of the imaging of the photosensitive element of the imaging component 30 is less than or equal to the width of a pixel of the photosensitive element of the imaging component 30 .

[0068] In this way, the resolution can be improved.

[0069] Specifically, referring to Figures 2 and 3a, in one embodiment, linear light 21 is irradiated onto the sample to be tested 10 so that a linear region 12 excites fluorescence 11. The fluorescence 11 within the linear region 12 can all be received by the photosensitive element of the imaging component 30. The illumination range of the fluorescence 11 within the linear region 12 on the photosensitive element is also linear, and its vertical direction corresponds to the fourth direction. The illumination ranges of the linear light 21, the linear region 12, and the fluorescence 11 all have a certain width. The wider the linear region 12, the wider the width of the illumination range of the fluorescence 11 in the fourth direction. When the width of the illumination range of the fluorescence 11 in the fourth direction is greater than the width of the pixel in the fourth direction, the fluorescence 11 of one linear region 12 will also interfere with the imaging effect of the adjacent linear region 12, thereby reducing the frequency of the stripes of the area array fluorescence image and reducing the resolution of the synthesized super-resolution image. Therefore, when the width of the image of the linear region 12 in the second direction Y is equal to the width of the pixels of the photosensitive element of the imaging assembly 30 in the fourth direction, the width of the illumination range of the fluorescent light 11 in the fourth direction is equal to the width of the smallest pixel in the fourth direction, and the imaging effect is optimal in this case. Of course, the width of the image of the linear region 12 in the second direction Y can also be smaller than the width of the pixels of the photosensitive element of the imaging assembly 30 in the fourth direction.

[0070] Please refer to Figure 2. In some embodiments, the super-resolution microscopy imaging system 100 includes a first dichroic mirror 51 and an objective lens 52. The objective lens 52 is used to collect fluorescence 11 and transmit the fluorescence 11 to the first dichroic mirror 51. The first dichroic mirror 51 is used to reflect the linear light 21 to the sample to be measured 10 and to transmit the fluorescence 11 to the imaging component 30.

[0071] This helps to avoid interference from the linear light 21 .

[0072] Specifically, referring to FIG. 2 , in one embodiment, the sample 10, objective lens 52, first dichroic mirror 51, and imaging assembly 30 are located on the same linear path, which corresponds to the propagation path of the fluorescence 11 from the sample 10 to the imaging assembly 30. The first dichroic mirror 51 can transmit light of a certain wavelength while reflecting light of another wavelength, thereby enabling the light source assembly 20 to emit linear light 21 from other directions toward the first dichroic mirror 51, which can then be reflected onto the linear path by the first dichroic mirror 51. Furthermore, the first dichroic mirror 51 can also reflect the linear light 21 reflected by the sample 10, thereby preventing the linear light 21 from being directly transmitted to the imaging assembly 30 and affecting the imaging effect. Furthermore, the objective lens 52 can also converge the linear light 21 onto the sample 10 and converge the fluorescence 11 onto the first dichroic mirror 51. By changing the magnification of the objective lens 52, the magnification of the super-resolution microscopy imaging system 100 can also be changed.

[0073] Please refer to Figure 2. In some embodiments, the imaging component 30 includes multiple second dichroic mirrors 32, which distribute the fluorescence 11 to multiple imaging channels 33. Each imaging channel 33 includes a converging lens 331 and a line array camera 332. The converging lens 331 is used to converge the fluorescence 11 to the line array camera 332, and the line array camera 332 is used to receive the fluorescence 11.

[0074] This is beneficial for detecting multiple fluorescence information.

[0075] Specifically, referring to FIG2 , in one embodiment, the imaging channel 33 can obtain different fluorescence 11 information. A second dichroic mirror 32 can reflect and transmit the incident light to the two imaging channels 33 at different wavelengths respectively. By setting a plurality of second dichroic mirrors 32, a plurality of imaging channels 33 can be divided to obtain different fluorescence information respectively. For example, in gene sequencing, the sample 10 to be tested can be DNA, and the fluorescence information can correspond to the base type of DNA. By setting three second dichroic mirrors 32, four fluorescence channels corresponding to different bases can be divided. The light source assembly 20 can emit linear light 21 of a specific wavelength to respectively excite the four bases of the sample 10 to be tested. The wavelengths of the fluorescence 11 excited by the four bases are different, so that the fluorescence information of the four bases can be obtained respectively according to the wavelength of the fluorescence 11, and then the image of the DNA can be synthesized. Simply put, the imaging channel 33 can be composed of a converging lens 331 and a line scan camera 332. The converging lens 331 can converge the fluorescent light 11 to the line scan camera 332, and the line scan camera 332 can sequentially capture the fluorescent light 11 of each illuminated linear region 121. In another embodiment, the imaging assembly 30 can further include a processor connected to the line scan camera 332 to synthesize a super-resolution image based on the fluorescent light 11 of the linear region 12 captured by the line scan camera 332.

[0076] Referring to FIG. 2 , in some embodiments, the imaging assembly 30 includes a filter 333 , which is used to filter the fluorescence 11 .

[0077] In this way, the imaging signal-to-noise ratio can be improved.

[0078] Specifically, referring to FIG2 , in one embodiment, some stray light may enter the imaging assembly 30. This stray light passes through the second dichroic mirror 32 and enters the imaging channel 33. By placing a filter 333 within the imaging channel 33, stray light can be filtered out, allowing only the fluorescent light 11 to pass through, thereby preventing the stray light from affecting the imaging. The filter 333 can be positioned between the converging lens 331 and the line array camera 332.

[0079] In another embodiment, the super-resolution microscopy system 100 can use the first dichroic mirror 51 to reflect the linear light 21 to prevent it from entering the imaging assembly 30. Because the power of the linear light 21 is much greater than that of the fluorescent light 11, some of the linear light 21 may pass through the first dichroic mirror 51 and enter the imaging assembly 30. The filter 333 can also filter the linear light 21 that enters the imaging assembly 30, thereby improving the imaging signal-to-noise ratio.

[0080] Referring to FIG. 5 , an imaging method for a sample 10 according to an embodiment of the present invention includes:

[0081] Step 01: emit linear light 21 with a preset on / off cycle and illuminate a linear region 12 of a sample 10 to stimulate the linear region 12 of the sample 10 to emit fluorescence 11, where the linear region 12 extends along a first direction X;

[0082] Step 02: Move the sample 10 to be tested along the second direction Y so that the linear light 21 illuminates multiple linear regions 12 of the sample 10 along the second direction Y. There is at least one unilluminated linear region 122 between the multiple illuminated linear regions 121. The first direction X is perpendicular to the second direction Y.

[0083] Step 03: receiving the fluorescence 11 excited by the linear light 21 irradiating a line area 12 each time and generating a planar array fluorescence image of a planar area according to the fluorescence 11;

[0084] Step 04: Reconstruct a super-resolution image based on the array fluorescence images of multiple surface areas.

[0085] The angle control method of the embodiment of the present invention can be implemented by the super-resolution microscopy imaging system 100 of the embodiment of the present invention. Specifically, referring to FIG1 , the super-resolution microscopy imaging system 100 includes a control component 70 and an imaging component 30. The control component 70 is configured to emit linear light 21 with a preset on / off cycle and illuminate a linear region 12 of the sample 10 to stimulate the linear region 12 of the sample 10 to emit fluorescence 11, wherein the linear region 12 extends along a first direction X. The sample 10 is moved along a second direction Y so that the linear light 21 illuminates multiple linear regions 12 of the sample 10 along the second direction Y, with at least one unilluminated linear region 122 existing between the multiple illuminated linear regions 121. The first direction X is perpendicular to the second direction Y. The imaging component 30 is configured to receive the fluorescence 11 excited by each illumination of the linear light 21 on a linear region 12 and generate a planar array fluorescence image of a planar region based on the fluorescence 11. A super-resolution image is reconstructed based on the planar array fluorescence images of the multiple planar regions.

[0086] In the above-mentioned imaging method and super-resolution microscopy imaging system 100, the control component 70 can control the drive component 60 to drive the stage 40 to move along the second direction Y, so that the linear light 21 sequentially illuminates multiple linear regions 12 of the sample to be tested 10. The imaging component 30 can use the fluorescence 11 excited by the multiple linear regions 12 for imaging, thereby obtaining a two-dimensional array fluorescence image of the sample to be tested 10. Ultimately, a super-resolution microscopy imaging system 100 with a simple structure can be realized while reducing the loss of light energy.

[0087] Specifically, referring to Figures 3a to 3c and Figures 4a to 4f, in one embodiment, the sample 10 to be tested may include multiple linear regions 12 extending from region A to region L. The super-resolution microscopy system 100 may emit linear light 21 to illuminate one of regions A to L at a time, or may turn off the linear light 21 and not illuminate any of the linear regions 12. Figures 4a to 4c are schematic diagrams of the super-resolution microscopy system 100 emitting linear light 21 to illuminate a single linear region 12, while Figures 4d to 4f are schematic diagrams of the super-resolution microscopy system 100 emitting linear light 21 to illuminate a linear region 12, while Figures 4d to 4f are schematic diagrams of the super-resolution microscopy system 100 emitting linear light 21 to illuminate a linear region 12 when the linear light 21 is turned off. When the linear light 21 is emitted, the spot formed by the linear light 21 on the sample 10 is the illumination region 25. The sample 10 to be tested may be moved along a first direction X so that regions A to L sequentially pass through the illumination region 25. By adjusting the preset on / off cycle of the linear light 21, some of the linear regions 12 in regions A to L can be caused to emit fluorescent light 11. The area array fluorescence image shown in FIG3a can be synthesized by acquiring images of the fluorescence 11 from area A to area L. By moving the sample 10 multiple times, multiple area array fluorescence images can be acquired and synthesized into a super-resolution image of the sample 10.

[0088] 3a to 3c and 4a to 4f, in some embodiments, the preset on-off cycle includes alternating on periods and off periods. The preset on-off cycles of the plurality of area array fluorescence images differ by a preset phase value.

[0089] Step 02 (emitting linear light 21 with a preset on / off cycle and irradiating a linear region 12 of the sample 10 to stimulate the linear region 12 of the sample 10 to emit fluorescence 11) includes:

[0090] When the preset on / off cycle is in the on period, the linear light 21 is emitted so that the illuminated linear area 121 emits fluorescence 11;

[0091] When the preset on-off cycle is in the off period, the linear light 21 is turned off to form an un-illuminated linear region 122 .

[0092] The angle control method of the embodiment of the present invention can be implemented using the super-resolution microscopy imaging system 100 of the embodiment of the present invention. Specifically, referring to FIG1 , the super-resolution microscopy imaging system 100 includes a control component 70. The control component 70 is configured to emit linear light 21 during the on period of a preset on / off cycle, causing an illuminated linear region 121 to emit fluorescence 11; and to turn off the linear light 21 during the off period of the preset on / off cycle, resulting in an unilluminated linear region 122.

[0093] In this way, the resolution can be improved.

[0094] Specifically, referring to Figures 3a to 3c and Figures 4a to 4f, in one embodiment, one on period and one off period constitute a minimum period of the predetermined on-off cycle. The duration T of the on period and the off period is the same and can correspond to the time required for the illumination area 25 to sweep across the three linear areas 12.

[0095] For example, the duration T of an on period or an off period may satisfy: T = 3*S / M / V,

[0096] Wherein, S is the pixel size of the imaging component 30 of the super-resolution microscopic imaging system 100 , M is the magnification of the super-resolution microscopic imaging system 100 , and V is the moving speed of the sample 10 to be measured.

[0097] Figures 4a to 4f illustrate the illumination of the linear region 12 as regions A to F sequentially pass through the illumination region 25 within a minimum period. Regions A to C illustrate the range of the illuminated linear region 121 during the on-period, and regions D to F illustrate the range of the unilluminated linear region 122 during the off-period. Continuing to move the sample 10 can scan all linear regions 12 of the sample 10 and generate the area array fluorescence image shown in Figure 3a. After completing the area array fluorescence image shown in Figure 3a, the sample 10 can be moved again so that the illumination region 25 scans from region A to region C, while simultaneously controlling the emission of the linear light 21 with a preset on / off cycle that increases by a preset phase value, thereby generating the area array fluorescence image shown in Figure 3b. Similarly, multiple area array fluorescence images can be generated as shown in Figure 3c, thereby generating a super-resolution image based on the multiple area array fluorescence images, thereby improving the image resolution in the first direction X.

[0098] In certain embodiments of the present invention, the module involved may be a single-chip microcomputer chip that integrates a processor, memory, communication module, etc. The processor may be a central processing unit (CPU), or other general-purpose processors, digital signal processors (DSP), application-specific integrated circuits (ASIC), field-programmable gate arrays (FPGA), or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, etc.

[0099] Any process or method description in a flowchart or otherwise described herein may be understood to represent a module, segment or portion of code comprising one or more executable instructions for implementing the steps of a specific logical function or process, and the scope of the preferred embodiments of the present invention includes alternative implementations in which functions may be performed out of the order shown or discussed, including performing functions in a substantially simultaneous manner or in the reverse order depending on the functions involved, which should be understood by those skilled in the art to which the embodiments of the present invention pertain.

[0100] The logic and / or steps represented in the flowchart or otherwise described herein, for example, can be considered as an ordered list of executable instructions for implementing logical functions, and can be embodied in any computer-readable medium for use by an instruction execution system, apparatus or device (such as a computer-based system, a system including a processing module, or other system that can fetch instructions from an instruction execution system, apparatus or device and execute instructions), or used in conjunction with such instruction execution systems, apparatuses or devices.

[0101] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to the embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the claims and their equivalents.

Claims

1. A super-resolution microscopic imaging system, characterized in that: include: A stage, the stage carrying a sample to be tested; A light source assembly, the light source assembly is used to emit linear light, the linear light illuminates the sample to be tested, so that a linear area of ​​the sample to be tested excites fluorescence, the linear area extends along a first direction; A driving assembly, the driving assembly is connected to the stage and is used to drive the stage to move along a second direction, the first direction being perpendicular to the second direction; a control component, the control component being electrically connected to the light source component and the driving component, the control component being used to control the driving component to drive the stage to move along the second direction and to control the light source component to emit the linear light with a preset on-off cycle, so that the linear light respectively illuminates a plurality of the linear regions of the sample to be tested along the second direction, and there is at least one un-illuminated linear region between the plurality of illuminated linear regions; An imaging component is used to receive the fluorescence of a line area each time the linear light irradiates the line area and generate a planar array fluorescence image of a planar area, and synthesize a super-resolution image based on the planar array fluorescence images of multiple planar areas.

2. The super-resolution microscopic imaging system according to claim 1, characterized in that: The preset on-off cycle includes alternating on periods and off periods. The control component controls the light source component to emit the linear light according to the on period so that the illuminated line area emits fluorescence. The control component controls the light source component to turn off the linear light according to the off period to form the unilluminated line area. The preset on-off cycles of the multiple area array fluorescence images differ by a preset phase value.

3. The super-resolution microscopic imaging system according to claim 1, characterized in that: The duration T of the on period or the off period satisfies: T=3×S / M / V, wherein S is the pixel size of the imaging component of the super-resolution microscopy imaging system, M is the magnification of the super-resolution microscopy imaging system, and V is the moving speed of the sample to be tested.

4. The super-resolution microscopic imaging system according to claim 1, characterized in that: The light source assembly includes a laser emitter and a beam shaper, wherein the laser emitter is used to emit laser light, and the beam shaper is used to shape the laser light into the linear light.

5. The super-resolution microscopic imaging system according to claim 1, characterized in that: The length of the image of the line area on the photosensitive element of the imaging component in the third direction is less than or equal to the length of the photosensitive element of the imaging component.

6. The super-resolution microscopic imaging system according to claim 1, characterized in that: The width of the image of the line area on the photosensitive element of the imaging component in the fourth direction is less than or equal to the width of a pixel of the photosensitive element of the imaging component.

7. The super-resolution microscopic imaging system according to claim 1, characterized in that: The super-resolution microscopic imaging system includes a first dichroic mirror and an objective lens, wherein the objective lens is used to collect the fluorescence and transmit the fluorescence to the first dichroic mirror, and the first dichroic mirror is used to reflect the linear light to the sample to be measured and to transmit the fluorescence to the imaging component.

8. The super-resolution microscopic imaging system according to claim 1, characterized in that: The imaging component includes a plurality of second dichroic mirrors, which distribute the fluorescence to a plurality of imaging channels. Each of the imaging channels includes a converging lens and a line array camera. The converging lens is used to converge the fluorescence to the line array camera, and the line array camera is used to receive the fluorescence.

9. The super-resolution microscopic imaging system according to claim 8, characterized in that: The imaging component includes a filter, and the filter is used to filter the fluorescence.

10. The super-resolution microscopic imaging system according to claim 8, characterized in that: The wavelengths of the fluorescence respectively correspond to the fluorescence information of the four ATCG bases, and the multiple second dichroic mirrors respectively distribute the fluorescence to the imaging channels corresponding to the four ATCG bases.

11. A method for imaging a sample to be tested, characterized in that: include: Emitting linear light in a preset on-off cycle and irradiating a linear region of the sample to be tested to excite the linear region of the sample to be tested to emit fluorescence, wherein the linear region extends along a first direction; Move the sample to be tested along a second direction so that the linear light illuminates a plurality of line regions of the sample to be tested along the second direction, respectively, and there is at least one un-illuminated line region between the plurality of illuminated line regions, and the first direction is perpendicular to the second direction; receiving fluorescence of a line area each time the linear light irradiates the line area and generating a planar array fluorescence image of a planar area; A super-resolution image is synthesized based on the plurality of planar array fluorescence images of the planar regions.

12. The imaging method according to claim 11, characterized in that: The preset on-off cycle includes alternating on periods and off periods, and the preset on-off periods of the plurality of area array fluorescence images differ by a preset phase value. Emitting linear light with a preset on-off cycle to excite a linear region of the sample to be tested, including: When the preset on / off cycle is in the on period, emitting the linear light so that the illuminated linear area emits fluorescence; When the preset on-off cycle is in the off period, the linear light is turned off to form the line area which is not illuminated.

13. The imaging method according to claim 12, characterized in that: The duration T of the on period or the off period satisfies: T=3×S / M / V, wherein S is the pixel size of the imaging component of the super-resolution microscopy imaging system, M is the magnification of the super-resolution microscopy imaging system, and V is the moving speed of the sample to be tested.

14. The imaging method according to claim 11, characterized in that: In the preset on-off cycle, the wavelengths of fluorescence emitted by the linear area of ​​the sample to be tested by the linear light laser correspond to the fluorescence information of the four bases of ATCG.

15. The imaging method according to claim 14, characterized in that: Each time the linear light irradiates the fluorescence of one of the linear regions, the fluorescence is respectively distributed to the imaging channels corresponding to the four bases of ATCG through a plurality of second dichroic mirrors in the imaging component.

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