Line-scan-based super-resolution imaging method and apparatus

Abstract

Line-scan-based super-resolution imaging method 100 and apparatus. The line-scan-based super-resolution imaging method 100 includes: a step S102 of providing a plurality of types of structured line excitation light, each of the plurality of types of structured line excitation light having a different illumination mode; a step S104 of illuminating the target area by row-by-row scanning along a single first direction while switching among the plurality of types of structured line excitation light such that each row in the target area is illuminated by a corresponding type of structured line excitation light among the plurality of types of structured line excitation light; and a step S106 of detecting response light generated by each row in the target area in response to being illuminated by the corresponding type of structured line excitation light.

Description

[Technical Field]

[0001] This application claims priority based on Chinese Patent Application No. 202310207189.0, filed on March 6, 2023, the disclosure of which is incorporated herein in its entirety.

[0002] The present disclosure relates generally to the field of imaging technology, and more particularly to a line-scan based super-resolution imaging method and apparatus. [Background technology]

[0003] Conventional imaging techniques, such as confocal imaging and wide-field imaging, are limited by the Abbe diffraction limit, so their maximum imaging resolution is typically about 250 nanometers (nm), and they cannot resolve structures with smaller sizes. In light of this, super-resolution imaging techniques have emerged that can overcome the diffraction limit, enabling imaging resolutions to reach 100 nm or even smaller, providing important technical support for the study of microscopic structures. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Chinese Patent Application No. 202310207189.0 Summary of the Invention

[0005] According to one aspect of the present disclosure, there is provided a line-scanning-based super-resolution imaging method, which includes: providing a plurality of types of structured line excitation light, each of the plurality of types of structured line excitation light having a different illumination mode; illuminating the target area by row-by-row scanning along a single first direction while switching between the plurality of types of structured line excitation light so that each row in the target area is illuminated by a corresponding type of structured line excitation light among the plurality of types of structured line excitation light; and detecting response light generated by each row in the target area in response to being illuminated by the corresponding type of structured line excitation light.

[0006] According to another aspect of the present disclosure, there is provided a line-scanning-based super-resolution imaging device, comprising: an illumination module configured to provide multiple types of structured line excitation light, each of the multiple types of structured line excitation light having a different illumination mode; a scanning module configured to illuminate a target area by row-by-row scanning along a single first direction while switching between the multiple types of structured line excitation light so that each row in the target area is illuminated by a corresponding type of structured line excitation light from the multiple types of structured line excitation light; and a detection module configured to detect response light generated by each row in the target area in response to being illuminated by the corresponding type of structured line excitation light.

[0007] Other features and advantages of the present disclosure will become more apparent from the following detailed description of illustrative embodiments thereof, taken in conjunction with the accompanying drawings.

[0008] The foregoing and other features and advantages of the present disclosure will become apparent from the following description of embodiments of the present disclosure, illustrated in the accompanying drawings, which are incorporated in and constitute a part of this specification and further serve to explain the principles of the present disclosure and to enable those skilled in the art to make and use the same. [Brief explanation of the drawings]

[0009] [Figure 1A]1 is a schematic diagram showing the scanning process of an example of an imaging method based on point scanning; [Figure 1B] 1 is a schematic diagram showing the scanning process of an example of an imaging method based on line scanning; [Figure 2] 1 is a flowchart illustrating a line-scan based super-resolution imaging method, according to some embodiments of the present disclosure. [Figure 3A] 1 is a schematic diagram illustrating a scanning process of an example of a line-scan-based super-resolution imaging method, according to some embodiments of the present disclosure. [Figure 3B] FIG. 3B is a schematic diagram showing the illumination modes for the scanned rows in FIG. 3A combined for illustrative purposes. [Figure 4] 10 is a schematic diagram illustrating, for illustrative purposes, combined illumination modes for scanned rows in the scanning process of an example of a line-scan-based super-resolution imaging method according to some other embodiments of the present disclosure. [Figure 5] A schematic diagram showing, for illustrative purposes, combined illumination modes for scanned rows in the scanning process of an example of a line-scanning-based super-resolution imaging method according to some further other embodiments of the present disclosure. [Figure 6A] 10A and 10B are schematic diagrams illustrating the scanning process of an example of a line-scan-based super-resolution imaging method according to some other embodiments of the present disclosure. [Figure 6B] FIG. 6B is a schematic diagram showing the illumination modes for the scanned rows in FIG. 6A combined for illustrative purposes. [Figure 7] 10A and 10B are schematic diagrams illustrating the scanning process of an example of a line-scan-based super-resolution imaging method according to some other embodiments of the present disclosure. [Figure 8] 1 is a schematic block diagram illustrating a line-scan based super-resolution imaging device, according to some embodiments of the present disclosure. [Figure 9A] 9 is a schematic block diagram illustrating the illumination module of FIG. 8, according to some embodiments of the present disclosure. [Figure 9B] 9 is a schematic block diagram illustrating the illumination module of FIG. 8, according to some other embodiments of the present disclosure. [Figure 10]9 is an example optical path diagram illustrating the illumination module of FIG. 8, according to some embodiments of the present disclosure. [Figure 11] 9 is an example optical path diagram illustrating the sensing module and scanning module of FIG. 8, according to some embodiments of the present disclosure. [Figure 12] Part A of Figure 12 shows light intensity distribution maps at three two-dimensional cross sections xy, zy, and xz formed by the structured line excitation light 1 and the structured line excitation light 2 provided by the illumination module of Figure 10 on the sample at the focal plane of the microscope objective lens of Figure 11, and part B of Figure 12 shows light intensity distribution curves along selected cross-sectional lines y1, x2, y2, z1, and z2 in the light intensity distribution map shown in part A of Figure 12. [Figure 13] 11 is a schematic diagram showing, combined for illustrative purposes, illumination modes for a scanned row using structured line excitation light 1 and structured line excitation light 2 provided by the illumination module of FIG. 10. FIG. [Figure 14] Part A of Figure 14 shows the results of imaging fluorescent microbeads using the super-resolution imaging technique of the present disclosure, part B of Figure 14 shows the results of imaging fluorescent microbeads in the same region as shown in part A of Figure 14 using a conventional confocal microscope, and part C of Figure 14 shows the results of imaging neurons in the brain of an awake mouse using the super-resolution imaging technique of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0010] It should be noted that in the embodiments described below, the same reference symbols may be commonly used throughout different accompanying drawings to represent the same parts or parts having the same functions, and repeated descriptions thereof will be omitted. In some cases, similar items are indicated using similar reference numbers and letters, and therefore, once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0011] For ease of understanding, the positions, dimensions, ranges, etc. of structures shown in the accompanying drawings, etc. may not represent actual positions, dimensions, ranges, etc. Therefore, the present disclosure is not limited to the positions, dimensions, ranges, etc. disclosed in the accompanying drawings, etc.

[0012] Various exemplary embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. It should be noted that unless otherwise specifically indicated, the relative configurations of components and steps, formulas, and numerical values ​​described in these embodiments do not limit the scope of the present disclosure.

[0013] Indeed, the following description of at least one example embodiment is merely illustrative and in no way constitutes a limitation on the present disclosure and its application or uses. In other words, the structures and methods herein are shown in an exemplary manner to illustrate various embodiments of the structures and methods in the present disclosure. However, those skilled in the art will understand that these are merely illustrative ways in which the present disclosure may be practiced, and not comprehensive ways in which the present disclosure may be practiced. Further, the accompanying drawings are not necessarily drawn to scale, and some features may be enlarged to show details of particular components.

[0014] Additionally, techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but, where appropriate, the techniques, methods, and devices will be deemed to be part of this specification.

[0015] In all instances shown and discussed herein, any particular values ​​should be construed as examples only and not as limitations, and therefore, different values ​​may exist in other instances of the example embodiment.

[0016] Among existing super-resolution imaging technologies, point-scanning-based super-resolution imaging technologies have been proposed, such as stimulated emission depletion (STED) super-resolution imaging and point-scanning structured light super-resolution imaging. For example, referring to FIG. 1A , during point scanning, information is continuously collected by scanning a target area point by point, and then the information collected at each point in each row is reconstructed into an image of the target area. Therefore, the imaging speed of point-scanning-based super-resolution imaging technologies is usually low. If the target area is displaced when a row is scanned point by point compared with when the previous adjacent row is scanned point by point, the pixels of the two rows will be misplaced, resulting in inaccurate image information. Therefore, due to their low imaging speed, point-scanning-based super-resolution imaging technologies often can only tolerate very slow motion of the target area during imaging, or cannot tolerate any motion of the target area during imaging.

[0017] Super-resolution imaging techniques based on wide-field imaging, such as structured light illumination super-resolution imaging and single molecule localization imaging, have also been proposed. Super-resolution imaging techniques based on wide-field imaging require multiple imaging of the entire target area to capture multiple full-field two-dimensional images, which are finally integrated into a super-resolution image through computation. However, this technique requires the target area to remain stationary during the entire imaging process; otherwise, it is impossible to reconstruct the super-resolution image.

[0018] Current mainstream super-resolution imaging techniques typically require the sample, or more specifically, the target region in the sample, to remain stationary and are extremely sensitive to even minute movements of the target region during the imaging process. In light of this, in biological research where imaging techniques are widely used, existing super-resolution imaging techniques are mostly used to image cultured cells, but it is difficult to extend this to imaging of living animals. This is because physiological activities such as breathing and heartbeat of living animals (e.g., mice) inevitably cause minute movements of the target region. Some experimental requirements even require imaging living animals that are awake and active. During this process, the limb movements of living animals cause large movements of the target region, making existing super-resolution imaging techniques inapplicable.

[0019] Therefore, there is a need for improved super-resolution imaging techniques that can tolerate movement of the region of interest during the imaging process.

[0020] The inventors of the present disclosure have noted that line scanning has a faster speed than point scanning. For example, referring to FIG. 1B , during line scanning, an entire row (in the x direction) in a target area is simultaneously illuminated with excitation light, and a detection device such as a camera is used to simultaneously detect the entire row. Then, the entire target area can be imaged two-dimensionally by scanning row by row along the y direction. Therefore, comparing the point scanning process shown in FIG. 1A with the line scanning process shown in FIG. 1B , it can be seen that the imaging speed based on line scanning is much faster than the imaging speed based on point scanning. However, conventional confocal imaging techniques based on line scanning can only acquire images with diffraction-limited resolution through a single scan. To achieve resolution beyond the diffraction limit, existing super-resolution imaging techniques (e.g., the aforementioned super-resolution imaging techniques based on wide-field imaging) typically require multiple scans to acquire multiple imaging results and then integrating them into a super-resolution image. However, due to the long time interval between two adjacent scans, the imaging speed of such super-resolution imaging techniques is also low.

[0021] Therefore, the present disclosure proposes a line-scan-based super-resolution imaging technique that can acquire a super-resolution image by a single scan without repeating multiple scans and then integrating the multiple imaging results. The line-scan-based super-resolution imaging technique of the present disclosure has a higher imaging speed and therefore a greater tolerance to movement of the target region during the imaging process.

[0022] A line-scanning-based super-resolution imaging method 100 (hereinafter referred to as method 100) according to some embodiments of the present disclosure will be first described in detail below with reference to Fig. 2. As shown in Fig. 2, the method 100 includes: in step S102, providing a plurality of types of structured line excitation light, each of the plurality of types of structured line excitation light having a different illumination mode; in step S104, illuminating the target area by row-by-row scanning along a single first direction (e.g., the y direction shown in Fig. 3A ) while switching between the plurality of types of structured line excitation light such that each row in the target area is illuminated by a corresponding type of structured line excitation light among the plurality of types of structured line excitation light; and in step S106, detecting response light generated by each row in the target area in response to being illuminated by the corresponding type of structured line excitation light.

[0023] As used herein, "structured line excitation light" refers to excitation light having a linear structure whose projection onto a target region exhibits a linear distribution. As used herein, the "illumination mode" of "structured line excitation light" refers to the spatial distribution pattern of the light intensity of the structured line excitation light. Or, more specifically, it can refer to the pattern of projection of the structured line excitation light onto the target region (which can reflect the light intensity distribution of the structured line excitation light on a two-dimensional cross section in the plane where the target region is located). If the projection patterns of two light beams onto the target region perfectly match each other or perfectly match each other after translation, the illumination modes of the two light beams are considered to be the same. Based on the principle of fluorescence imaging, after being illuminated by "excitation light," the target region will emit "response light" accordingly, and the target region can be imaged according to the "response light." As used herein, the "target area" refers to the region to be imaged.

[0024] In some embodiments, the multiple types of structured line excitation light provided in step S102 may include a first structured line excitation light and a second structured line excitation light. The first structured line excitation light may have an illumination mode that completely illuminates the entire row, and the second structured line excitation light may have an illumination mode that does not completely illuminate the entire row. For example, the first structured line excitation light may have a spot that continuously extends in a second direction (e.g., the x direction shown in FIG. 3A) perpendicular to the first direction (e.g., the y direction shown in FIG. 3A) (e.g., structured line excitation light a having illumination mode a shown in FIG. 3A). The second structured line excitation light may have a plurality of spots arranged at substantially periodic intervals in the second direction (e.g., the x direction shown in FIG. 3A) (e.g., structured line excitation light b having illumination mode b and structured line excitation light c having illumination mode c shown in FIG. 3A). As used herein, "substantially" arranged at periodic intervals refers to intervals that vary within a range of ±20% of the design interval period or the target interval period, for example, within a range of ±15%, for example, within a range of ±10%, or for example, within a range of ±5%. In some embodiments, the multiple types of structured linear excitation light provided in step S102 may additionally or alternatively include multiple types of second structured linear excitation light, and each type of second structured linear excitation light may have a different combination of spot shape and interval period compared to other second structured linear excitation lights. For example, the spot shape and interval period of structured linear excitation light b having irradiation mode b shown in FIG. 3A are different from the spot shape and interval period of structured linear excitation light c having irradiation mode c shown in FIG. 3A. As used herein, a difference in interval period may be understood to mean that the interval periods are not the same or are not "substantially" the same. For example, the interval periods may differ by more than ±20%, for example, more than ±50%, or more than ±70%, etc. In some other embodiments, the second structured line excitation light may also have a plurality of spots arranged at non-periodic intervals in a second direction (eg, the x-direction shown in FIG. 3A).

[0025] 3A and 3B show three types of structured linear excitation light having different irradiation modes, i.e., structured linear excitation light a having irradiation mode a, structured linear excitation light b having irradiation mode b, and structured linear excitation light c having irradiation mode c. It can be understood that any suitable number of types of structured linear excitation light can be set as needed, including, but not limited to, two, three, four, or more.

[0026] FIG. 3 exemplarily shows how steps S104 and S106 are performed: (1) initially, scanning starts at a first row of the target area, where the first row is irradiated by using structured line excitation light a with irradiation mode a, and response light generated by the first row in response to irradiating the first row with structured line excitation light a is detected; (2) then scanning proceeds along the y direction to a second row of the target area, where the second row is irradiated by switching to using structured line excitation light b with irradiation mode b, and response light generated by the second row in response to irradiating the second row with structured line excitation light b is detected; (3) then scanning proceeds along the y direction to a third row of the target area, where the third row is irradiated by switching to using structured line excitation light c with irradiation mode c, and response light generated by the third row in response to irradiating the third row with structured line excitation light c is detected; (4) then scanning proceeds along the y direction to a fourth row of the target area, where the third row is irradiated by switching to using structured line excitation light c with irradiation mode c; (4) the scanning then advances along the y direction to a fourth row of the target area, which is illuminated by switching to use structured linear excitation light a with illumination mode a, and the response light generated by the fourth row in response to the illumination of the fourth row with structured linear excitation light a is detected; (5) the scanning then advances along the y direction to a fifth row of the target area, which is illuminated by switching to use structured linear excitation light b with illumination mode b, and the response light generated by the fifth row in response to the illumination of the fifth row with structured linear excitation light b is detected; (6) the scanning then advances along the y direction to a sixth row of the target area, which is illuminated by switching to use structured linear excitation light c with illumination mode c, and the response light generated by the sixth row in response to the illumination of the sixth row with structured linear excitation light c is detected; and so on, with each subsequent row of the target area being successively illuminated and detected by alternating between structured linear excitation light a, structured linear excitation light b, and structured linear excitation light c. Finally, after scanning the entire target area row by row, a super-resolution image can be directly reconstructed based on the detection of the response light generated by each row of the target area.

[0027] The multiple types of structured beam excitation light provided in step S102 may be switched according to a predetermined order to irradiate corresponding rows in the target area. For example, in the embodiment shown in FIG. 3A, the multiple types of structured beam excitation light provided in step S102 are traversed and used according to the same order in each cycle to irradiate corresponding rows in the target area. Alternatively, the multiple types of structured beam excitation light provided in step S102 may be traversed and used according to a different order in each cycle to irradiate corresponding rows in the target area. In some embodiments, this may only require that two adjacent rows in the target area be irradiated with different types of structured beam excitation light from the multiple types of structured beam excitation light. In some other embodiments, it is also possible to switch to different types of structured beam excitation light for irradiation every few rows. In short, the switching order may be specifically designed according to an actual application scenario. Various switching sequences such as "a,b,c,a,b,c......", "a,b,c,c,b,a,b,a,c......", "a,a,b,b,c,c,a,a,b,b,c,c......", "a,b,a,c,b,c......", etc. may all have their specific applicable application scenarios.

[0028] The time difference between the scanning of the two rows may be limited by the limitations of factors such as the detection speed of the detection device (e.g., a camera) for the response light, the switching speed of the structured line excitation light with different illumination modes (if it is necessary to switch between structured line excitation lights with different illumination modes), and the scanning speed. When possible, the time difference is set as short as possible, thereby making it possible to improve the imaging speed of method 100 as much as possible.

[0029] For illustrative purposes, FIG. 3B shows a hypothetical combined representation of the six views in FIG. 3A. In the example of FIG. 3B, during row-by-row scanning, the regions illuminated by two successive applications of structured beam excitation light in the target area do not overlap with each other. This is merely illustrative and not limiting. In some other examples, for example, as shown in FIG. 4, during row-by-row scanning, the regions illuminated by two successive applications of structured beam excitation light in the target area may partially overlap with each other. In some examples, the spacing between the regions illuminated by two successive applications of structured beam excitation light in the target area in a first direction (e.g., the y-direction) may be 5% to 50% of the design resolution (e.g., 100 nm). In some examples, the row-by-row scanning may be performed at equal intervals. In some other examples, the row-by-row scanning may be performed at unequal intervals.

[0030] 3B , during row-by-row scanning, the structured line excitation light that does not completely illuminate the entire row (e.g., the second structured line excitation light, such as the structured line excitation light b having the illumination mode b and the structured line excitation light c having the illumination mode c) does not undergo any phase shift in the second direction (e.g., the x-direction) each time it is used for illumination, compared to the previous time it was used for illumination. In some other embodiments, for example, as shown in FIG. 5 , during row-by-row scanning, the structured line excitation light that does not completely illuminate the entire row (e.g., the second structured line excitation light, such as the structured line excitation light b having the illumination mode b and the structured line excitation light c having the illumination mode c) undergoes a preset phase shift in the second direction (e.g., the x-direction) each time it is used for illumination, compared to the previous time it was used for illumination. When the structured line excitation light is the second structured line excitation light, the phase shift distance for the preset phase shift may be a non-integer multiple of the interval period of the spot of the structured line excitation light. Such phase shifting can facilitate more complete collection of information from the region of interest for imaging.

[0031] In some examples, the multiple types of structured linear excitation light provided in step S102 may include a third structured linear excitation light and a fourth structured linear excitation light. The irradiation mode of the third structured linear excitation light may be configured to improve imaging performance in a third direction. The irradiation mode of the fourth structured linear excitation light may be configured to improve imaging performance in a fourth direction different from the third direction. In some examples, the third direction may be parallel to the first direction, and the fourth direction may be perpendicular to the third direction. In some examples, the irradiation mode of the fourth structured linear excitation light may be configured to improve imaging performance in both the fourth direction and a fifth direction perpendicular to both the third and fourth directions. In some examples, the multiple types of structured linear excitation light provided in step S102 may also include a fifth structured linear excitation light. The irradiation mode of the fifth structured linear excitation light may be configured to improve imaging performance in the fifth direction perpendicular to both the third and fourth directions. For example, as shown in Figure 12 described below, irradiation mode 1 of structured linear excitation light 1 is configured to improve imaging resolution in the y direction, and irradiation mode 2 of structured linear excitation light 2 is configured to improve imaging resolution in the x direction and z direction, so that the resulting image of the target area has high imaging resolution in each of three directions, i.e., the x direction, the y direction, and the z direction.

[0032] The imaging speed of method 100 can also be further improved by parallel processing. For example, referring to FIGS. 6A and 6B , in some embodiments, the target region can include multiple target subregions (e.g., a first target subregion and a second target subregion) arranged along a first direction (e.g., the y direction), and method 100 includes performing the following operations in parallel for each target subregion: illuminating the target subregion by row-by-row scanning along a single first direction (e.g., the y direction) while switching between the multiple types of structured line excitation light provided in step S102 so that each row in the target subregion is illuminated by a corresponding type of structured line excitation light from the multiple types of structured line excitation light; and detecting response light generated by each row in the target subregion in response to being illuminated by the corresponding type of structured line excitation light. In the embodiment shown in FIG. 6A , the scanning direction in the first target subregion is the same as the scanning direction in the second target subregion. In some other embodiments, the scanning direction in the first target subregion is different from the scanning direction in the second target subregion. For example, the scanning direction in a first target sub-region may be opposite to the scanning direction in a second target sub-region.

[0033] In some embodiments, a first row in a first target sub-region and a second row in a second target sub-region may be illuminated simultaneously. In some instances, for example, as shown in FIG. 6A, the first row and the second row may be illuminated by the same type of structured line excitation light among multiple types of structured line excitation light. In some other instances, for example, as illustrated in FIG. 7, the first row and the second row may be illuminated by different types of structured line excitation light among multiple types of structured line excitation light. Although the examples in FIGS. 6A to 7 only illustrate that the target region is divided into two target sub-regions, it can be understood that the target region may be divided into any suitable number of target sub-regions having any suitable shape according to specific needs.

[0034] Method 100 requires only a single line scan to accurately reconstruct a super-resolution image. This benefits from a high line scan imaging speed, which can effectively improve the tolerance of the super-resolution imaging method to movement of the target region during the imaging process. For example, in an existing scientific-grade camera, if only four rows of pixels are used to image the target region, the frame rate can exceed 40 kHz. If the desired displacement of the target region during information collection from 10 consecutive rows is less than 50 nm (half the design resolution of 100 nm), the speed of movement of the target region that can be tolerated by method 100 is as fast as 200 micrometers per second. This is sufficient to meet the needs of most application scenarios (e.g., the speed of movement of a brain region in an anesthetized mouse is approximately 2 micrometers per second, and the speed of movement of a brain region in an awake mouse is approximately 50 micrometers per second). Even if the acquired image is significantly deformed due to the intense movement of the target region, this does not affect the ability of method 100 to analyze fine structures at the microscale.

[0035] A line-scanning-based super-resolution imaging apparatus 200 (hereinafter referred to as apparatus 200) according to some embodiments of the present disclosure will be described in detail below with reference to FIG. 8 . As shown in FIG. 8 , the apparatus 200 includes an illumination module 220, a scanning module 240, and a detection module 260. The illumination module 220 is configured to provide multiple types of structured line excitation light, each of which has a different illumination mode. The scanning module 240 is configured to illuminate the target area by row-by-row scanning along a single first direction while switching between the multiple types of structured line excitation light such that each row in the target area is illuminated by a corresponding type of structured line excitation light among the multiple types of structured line excitation light. The detection module 260 is configured to detect response light generated by each row in the target area in response to being illuminated by the corresponding type of structured line excitation light.

[0036] In some embodiments, for example, referring to FIG. 9A , the illumination module 220 may include a light source 221 configured to provide excitation light, a light branching unit 222 configured to branch the excitation light from the light source 221 into multiple beams of excitation light, a plurality of structured line excitation light generating units 2241, 2242, ..., 224N, each structured line excitation light generating unit configured to receive a corresponding beam of excitation light from the light branching unit 222 and generate a corresponding type of structured line excitation light based on the received corresponding beam of excitation light, a light combining unit 225 configured to combine the multiple types of structured line excitation light from the plurality of structured line excitation light generating units 2241, 2242, ..., 224N into a beam, and a plurality of light switching units 2231, 2232, ..., 223N. Each light switching unit may be disposed, for example, between the light branching unit 222 and a corresponding one of the structured line pumping light generating units 2241, 2242, ..., 224N, and configured to control whether the pumping light from the light branching unit 222 is output to the corresponding one of the structured line pumping light generating units (e.g., as shown in FIG. 9A ). Alternatively, each light switching unit may be disposed between a corresponding one of the structured line pumping light generating units 2241, 2242, ..., 224N, and the light combining unit 225, and configured to control whether the structured line pumping light from the corresponding one of the structured line pumping light generating units is output to the light combining unit 225. The structured line pumping light generated by different structured line pumping light generating units among the plurality of structured line pumping light generating units 2241, 2242, ..., 224N has different irradiation modes.

[0037] 9B , the illumination module 220 may also include a light source 221 configured to provide excitation light, and a single modulation unit 226 configured to modulate the excitation light from the light source 221 into structured line excitation light having different illumination modes. The modulation unit 226 may include, for example, at least one of a spatial light modulator or a polarization modulator, and may output structured line excitation light having different illumination modes depending on settings.

[0038] It should be noted that the excitation light provided by light source 221 is not particularly limited and may include one or more wavelengths or ranges of wavelengths as long as it is capable of exciting the target area to produce response light.

[0039] The illumination module 220 shown in Figure 9B has a simpler structure and fewer components than the illumination module 220 shown in Figure 9A. However, the illumination module 220 shown in Figure 9A can switch between structured line excitation light having different illumination modes faster than the illumination module 220 shown in Figure 9B. Figures 9A and 9B are merely exemplary and not limiting, and it can be understood that any suitable illumination module 220 can be adopted or designed in accordance with the teachings of the present disclosure to provide multiple types of structured line excitation light having different illumination modes.

[0040] In some embodiments, the multiple types of structured linear excitation light provided by the irradiation module 220 may include a first structured linear excitation light and a second structured linear excitation light. The first structured linear excitation light has a spot that extends continuously in a second direction perpendicular to the first direction. The second structured linear excitation light has a plurality of spots arranged at substantially periodic intervals in the second direction. In some embodiments, the multiple types of structured linear excitation light may additionally or alternatively include multiple types of second structured linear excitation light having a plurality of spots arranged at substantially periodic intervals in the second direction perpendicular to the first direction. Each of the multiple types of second structured linear excitation light has a different combination of spot shape and interval duration compared to the others of the multiple types of second structured linear excitation light.

[0041] In some embodiments, the multiple types of structured linear excitation light provided by the illumination module 220 may include a third structured linear excitation light and a fourth structured linear excitation light. The illumination mode of the third structured linear excitation light is configured to improve imaging performance in a third direction. The illumination mode of the fourth structured linear excitation light is configured to improve imaging performance in a fourth direction different from the third direction. In some instances, the third direction is parallel to the first direction, and the fourth direction is perpendicular to the third direction. In some instances, the illumination mode of the fourth structured linear excitation light is configured to improve imaging performance in both the fourth direction and a fifth direction perpendicular to both the third and fourth directions. In some embodiments, the multiple types of structured linear excitation light further include a fifth structured linear excitation light. The illumination mode of the fifth structured linear excitation light is configured to improve imaging performance in a fifth direction perpendicular to both the third and fourth directions.

[0042] In some embodiments, the scanning module 240 may be further configured to, during the row-by-row scanning, subject the second structured line excitation light to a predetermined phase shift in a second direction each time it is used for illumination compared to the previous time it was used for illumination, where the phase shift distance is a non-integer multiple of the interval duration of the spots of the second structured line excitation light. In some embodiments, the scanning module 240 may be further configured to illuminate two adjacent rows in the target region with different types of structured line excitation light from the multiple types of structured line excitation light. In some embodiments, the scanning module 240 may be further configured to switch between the multiple types of structured line excitation light according to a predetermined sequence to illuminate each row in the target region. In some embodiments, the scanning module 240 may be further configured to, during the row-by-row scanning, subject regions irradiated by two successive applications of the structured line excitation light in the target region to partially overlap each other.

[0043] To further improve the imaging speed of the device 200, the target region can be divided into multiple target sub-regions, and parallel processing can then be performed. In some embodiments, the target region can include multiple target sub-regions arranged along a first direction. The scanning module 240 can be configured to perform the following operation in parallel for each target sub-region: irradiating the target sub-region by row-by-row scanning along a single first direction while switching between multiple types of structured line excitation light such that each row in the target sub-region is irradiated with a corresponding type of structured line excitation light from the multiple types of structured line excitation light. The detection module 260 can be configured to perform the following operation in parallel for each target sub-region: detecting response light generated by each row in the target sub-region in response to being irradiated with the corresponding type of structured line excitation light. In some embodiments, the multiple target sub-regions can include a first target sub-region and a second target sub-region. A first row in the first target sub-region and a second row in the second target sub-region can be simultaneously illuminated. The scanning module 240 may be configured to illuminate the first row and the second row with the same or different types of structured line excitation light from the multiple types of structured line excitation light provided by the illumination module 220.

[0044] The implementation of the apparatus 200 may be similar to the implementation of the method 100 described above and will not be repeated here.

[0045] For illustrative purposes, non-limiting specific examples in which the super-resolution imaging techniques of the present disclosure may be applied are described in detail below with reference to Figures 10-14. It should be understood that other optical elements may be present in an actual optical system, and that these other optical elements will not be discussed herein or shown in the accompanying drawings in order to avoid obscuring the points herein.

[0046] The optical system shown in Figure 10 can be constructed to realize an illumination module that provides two types of structured line excitation light with different illumination modes. The input of the illumination module can be a parallel laser beam of a single wavelength or multiple wavelengths. The input light beam is split into two light beams with perpendicular polarizations by a polarizing beam splitter PBS1 (which can function as an optical splitting unit). These two light beams with perpendicular polarizations are controlled by two acousto-optic tunable filters AOTF1 and AOTF2 (which can function as optical switching units). The acousto-optic tunable filters AOTF1 and AOTF2 selectively allow light of a specific wavelength or within a specific wavelength range to pass through them, allowing for quick on / off control of the light. In the optical path on the left side of Figure 10, the parallel light beam emitted from the polarizing beam splitter PBS1 passes through the acousto-optic tunable filter AOTF1 and is then focused into a line along the x-direction by a cylindrical lens CL1, i.e., illumination mode 1 shown in part A of Figure 12. In the optical path on the right side of Figure 10, the collimated light beam reflected from the polarizing beam splitter PBS1 is first reflected by mirror M1, then passes through the acousto-optic tunable filter AOTF2, and is then focused into a line along the x-direction by the cylindrical lens CL2. Unlike the optical path on the left side, a transmission grating TG driven by a rotary motor is placed at the focal plane of the cylindrical lens CL2. The transmission grating TG modulates the phase or intensity of the linearly focused light emitted from the cylindrical lens CL2 along the x-direction, so that the light is modulated to have illumination mode 2, as shown in part A of Figure 12. In addition to using the transmission grating TG driven by a rotary motor, other solutions such as a digital micromirror device (DMD) or a liquid crystal spatial light modulator (SLM) can also be used to modulate the phase or intensity of light.While DMDs and SLMs can simply change the phase without being driven by a rotary motor, transmission gratings TG, driven by a rotary motor, can change the phase faster than DMDs and SLMs. Therefore, cylindrical lens CL1 can function as a first structured line excitation light generating unit that provides structured line excitation light 1 with illumination mode 1. Cylindrical lens CL2 and transmission grating TG can function as a second structured line excitation light generating unit that provides structured line excitation light 2 with illumination mode 2. Structured line excitation light 1 with illumination mode 1 and structured line excitation light 2 with illumination mode 2 (reflected by mirror M2) are combined into a beam by polarizing beam splitter PBS2 (which functions as a light combining unit). Furthermore, spherical lenses L1 and L3 form a relay imaging system, imaging structured line excitation light 1 with illumination mode 1 at the focal plane of spherical lens L3. The spherical lens L2 and the spherical lens L3 also constitute a relay imaging system, and image the structured line excitation light 2 having the illumination mode 2 at the focal plane of the spherical lens L3. Therefore, by controlling the acousto-optic tunable filters AOTF1 and AOTF2, the illumination module shown in FIG. 10 can selectively output the structured line excitation light 1 having the illumination mode 1 or the structured line excitation light 2 having the illumination mode 2.

[0047] The optical system shown in FIG. 11 can be constructed to realize a scanning module and a detection module, where the solid arrows schematically indicate the optical path of the scanning module and the dotted arrows schematically indicate the optical path of the detection module. The spherical lenses L4 and L5 can form a relay imaging system. The spherical lens L6 and the microscope objective lens can also form a relay imaging system. The structured line excitation light output by the illumination module is reflected by the dichroic mirror DM and then passes through the spherical lens L4 to reach the scanning mirror SM. After being reflected by the scanning mirror SM, the structured line excitation light passes through the spherical lenses L5, L6, and the microscope objective lens to reach the target area of ​​the sample. In the embodiment shown in FIG. 11, scanning of the irradiation position of the structured line excitation light on the target area is achieved by rotating the scanning mirror SM. In this case, the sample can be fixed. Additionally or alternatively, the sample can be placed on a displacement stage for sample scanning, and scanning of the irradiation position of the structured line excitation light on the target area is achieved by displacing the sample. In this case, the scanning mirror SM can still be used or can be replaced with a fixed mirror. The response light emitted by the sample is collected by the microscope objective, then passes through spherical lens L6, spherical lens L5, scanning mirror SM, spherical lens L4, and dichroic mirror DM, and is finally captured by the camera. In addition to using a camera, other solutions, such as photoelectric detectors and image sensors, can alternatively be used to detect the response light. In the example shown in Figure 11, the scanning module and detection module share most of the optical elements and can be integrated into a single module called a line-scanning confocal imaging module.

[0048] 12, illumination modes of structured line excitation light 1 and structured line excitation light 2 provided by the illumination module of FIG. 10 and projected onto a target area of ​​a sample by the scanning module of FIG. 11 are specifically shown. As shown in FIG. 12, illumination mode 1 is a line that is uniform in the x direction and maximally focused in the y direction, which creates the fastest change in intensity in the y direction, thereby improving imaging resolution in the y direction. Illumination mode 2 is a row of spots arranged at substantially periodic intervals along the x direction, which creates the fastest change in intensity in the x and z directions, thereby improving imaging resolution in the x and z directions.

[0049] During the line scanning process, by alternately controlling the on / off of the acousto-optic tunable filters AOTF1 and AOTF2 in the illumination module of Fig. 10, alternate illumination of the target area of ​​the sample with the structured line excitation light 1 having illumination mode 1 and the structured line excitation light 2 having illumination mode 2 is achieved. During this period, the rotary motor can also be used to rotate the transmission grating TG in the illumination module of Fig. 10, so that the phase of the illumination mode 2 of the structured line excitation light 2 moves continuously along the x direction, for example, see Fig. 13. Since the information collected in this way about the target area includes high-resolution information in the three directions of x, y, and z, an image of the target area with high resolution in each of the three directions of x, y, and z can be reconstructed with super-resolution.

[0050] 14, part A of Fig. 14 shows the results of imaging fluorescent microbeads using the super-resolution imaging technique of the present disclosure, part B of Fig. 14 shows the results of imaging fluorescent microbeads in the same region as shown in part A using a conventional confocal microscope, and part C of Fig. 14 shows the results of imaging neurons in the brain of an awake mouse using the super-resolution imaging technique of the present disclosure. The imaging performance of the super-resolution imaging technique of the present disclosure was found to be significantly better than that of conventional imaging techniques, and is highly suitable for imaging living animals because it can resist movement of the target region during the imaging process.

[0051] The terms "left," "right," "front," "rear," "top," "bottom," "above," "under," "upper," "lower," and the like in this specification and claims, when present, are used for descriptive purposes and not necessarily to describe fixed relative positions. Terms so used are interchangeable in appropriate circumstances, so that it is understood that the embodiments of the disclosure described herein are operable, for example, in other orientations different from those shown or otherwise described herein. For example, if a device in the accompanying drawings is turned upside down, a feature originally described as being "above" another feature may, in this case, be described as being "under" that other feature. The device may also be otherwise oriented (rotated 90 degrees or otherwise), in which case the relative spatial relationships would be interpreted accordingly.

[0052] In this specification and claims, when an element is referred to as being "above," "attached," "connected," "coupled," "linked," "in contact," or the like, the element may be directly above, directly attached to, directly connected to, directly coupled to, directly linked to, or in direct contact with the other element, or one or more intermediate elements may be present. In contrast, when an element is referred to as being "directly above," "directly attached," "directly connected," "directly coupled," "directly linked," or "in direct contact" with another element, there are no intervening elements present. In this specification and claims, a feature located "adjacent" another feature may mean that the feature has an overlap with the adjacent feature or that the feature is located above or below the adjacent feature.

[0053] As used herein, the term "exemplary" means "used as an example, instance, or illustration" rather than as a "model" to be exactly imitated. Any embodiment described herein as exemplary is not necessarily to be construed as preferred or advantageous over other embodiments. Furthermore, this disclosure is not limited by any expressed or implied theory presented in the technical field, background, brief summary, or detailed description.

[0054] As used herein, the term "substantially" is meant to include any minor variations caused by design or manufacturing imperfections, device or element tolerances, environmental influences, and / or other factors. The term "substantially" also allows for variations from perfect or ideal situations caused by parasitic effects, noise, and other practical considerations that may be present in actual implementation.

[0055] Additionally, terms such as "first" and "second" may be used herein for reference purposes only and are therefore not intended to be limiting. For example, the terms "first," "second," and other such numerical terms in reference to structures or elements do not imply a sequence or order unless the context clearly dictates otherwise.

[0056] It is to be further understood that the term "comprise / include", when used herein, indicates the presence of stated features, integers, steps, operations, units, and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, units, and / or components and / or combinations thereof.

[0057] In this disclosure, the term "provide" is used broadly to encompass all manners of obtaining an object. Thus, "providing an object" includes, but is not limited to, "purchasing," "preparing / manufacturing," "arranging / setting," "installing / assembling," and / or "ordering" the object.

[0058] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. The terms used herein are for the purpose of describing particular examples only and are not intended to be limiting of the disclosure. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0059] Those skilled in the art will recognize that the boundaries between operations described above are merely exemplary. Operations may be combined into a single operation, a single operation may be distributed among additional operations, and operations may be performed with at least partial overlap in time. Moreover, alternative embodiments may include multiple instances of a particular operation, and the order of certain operations may be changed in various other embodiments. However, other modifications, variations, and substitutions are possible. Aspects and elements of all embodiments disclosed above may be combined in any manner and / or combined with aspects or elements of other embodiments to provide multiple additional embodiments. Accordingly, the present specification and accompanying drawings should be considered illustrative and not restrictive.

[0060] Although some specific embodiments of the present disclosure have been described in detail by way of example, those skilled in the art should understand that the above examples are merely used for illustration purposes and are not intended to limit the scope of the present disclosure. The embodiments disclosed herein may be combined in any manner without departing from the spirit and scope of the present disclosure. Those skilled in the art should also understand that various modifications may be made to the embodiments without departing from the spirit and scope of the present disclosure. The scope of the present disclosure is defined by the appended claims.

Claims

1. providing a plurality of types of structured line excitation light, each of the plurality of types of structured line excitation light having a different irradiation mode; irradiating the target area by scanning row by row along a single first direction while switching the plurality of types of structured beam excitation light so that each row in the target area is irradiated with a corresponding type of structured beam excitation light among the plurality of types of structured beam excitation light; and detecting response light generated by each row in the target area in response to being illuminated by the corresponding type of structured line excitation light.

2. 2. The super-resolution imaging method of claim 1, wherein the multiple types of structured line excitation light include a first structured line excitation light and a second structured line excitation light, the first structured line excitation light having a spot extending continuously in a second direction perpendicular to the first direction, and the second structured line excitation light having a plurality of spots arranged at substantially periodic intervals in the second direction.

3. 3. The super-resolution imaging method of claim 1, wherein the plurality of types of structured linear excitation light include a plurality of types of second structured linear excitation light having a plurality of spots arranged at substantially periodic intervals in a second direction perpendicular to the first direction, and each of the plurality of types of second structured linear excitation light has a different combination of spot shape and interval period compared to the other of the plurality of types of second structured linear excitation light.

4. 3. The super-resolution imaging method of claim 2, wherein during the row-by-row scanning, the second structured line excitation light undergoes a preset phase shift in the second direction each time it is used for illumination compared to the previous time the second structured line excitation light was used for illumination, and the phase shift distance is a non-integer multiple of the interval period of the spots of the second structured line excitation light.

5. 2. The super-resolution imaging method of claim 1, wherein the plurality of types of structured linear excitation light include a third structured linear excitation light and a fourth structured linear excitation light, the irradiation mode of the third structured linear excitation light is configured to improve imaging performance in a third direction, and the irradiation mode of the fourth structured linear excitation light is configured to improve imaging performance in a fourth direction different from the third direction.

6. The super-resolution imaging method according to claim 5 , wherein the third direction is parallel to the first direction, and the fourth direction is perpendicular to the third direction.

7. The super-resolution imaging method of claim 6, wherein the irradiation mode of the fourth structured linear excitation light is configured to improve imaging performance in both the fourth direction and a fifth direction perpendicular to both the third direction and the fourth direction.

8. 7. The super-resolution imaging method of claim 6, wherein the plurality of types of structured linear excitation light further includes a fifth structured linear excitation light, and the irradiation mode of the fifth structured linear excitation light is configured to improve imaging performance in a fifth direction perpendicular to both the third direction and the fourth direction.

9. The super-resolution imaging method according to claim 1 , wherein two adjacent rows in the target region are illuminated with different types of structured beam excitation light from among the plurality of types of structured beam excitation light.

10. The super-resolution imaging method according to claim 1 , wherein the plurality of types of structured line excitation light are switched in a predetermined order to illuminate respective rows in the target region.

11. The method of claim 1 , wherein during the row-by-row scanning, regions illuminated by two successive applications of structured line excitation light in the target area partially overlap each other.

12. The target region includes a plurality of target subregions arranged along the first direction, and the method comprises performing the following operations for each of the plurality of target subregions: irradiating the target partial region by scanning each row along the single first direction while switching the plurality of types of structured beam excitation light so that each row in the target partial region is irradiated with a corresponding type of structured beam excitation light among the plurality of types of structured beam excitation light; 2. The super-resolution imaging method of claim 1, further comprising: detecting response light generated by each row in the target partial region in response to being illuminated by the corresponding type of structured line excitation light, in parallel.

13. a first row in a first target sub-area of ​​the plurality of target sub-areas and a second row in a second target sub-area of ​​the plurality of target sub-areas are simultaneously illuminated; the first row and the second row are irradiated with the same type of structured line excitation light among the plurality of types of structured line excitation light; or The super-resolution imaging method according to claim 12 , wherein the first row and the second row are illuminated by different types of structured line excitation light among the plurality of types of structured line excitation light.

14. an irradiation module configured to provide a plurality of types of structured linear excitation light, each of the plurality of types of structured linear excitation light having a different irradiation mode; a scanning module configured to illuminate the target area by scanning row by row along a single first direction while switching among the plurality of types of structured beam excitation light such that each row in the target area is illuminated by a corresponding type of structured beam excitation light among the plurality of types of structured beam excitation light; a detection module configured to detect response light generated by each row in the target area in response to being illuminated by the corresponding type of structured line excitation light.

15. 15. The super-resolution imaging device of claim 14, wherein the multiple types of structured line excitation light include a first structured line excitation light and a second structured line excitation light, the first structured line excitation light having a spot extending continuously in a second direction perpendicular to the first direction, and the second structured line excitation light having a plurality of spots arranged at substantially periodic intervals in the second direction.

16. The super-resolution imaging device of claim 14 or 15, wherein the multiple types of structured linear excitation light include multiple types of second structured linear excitation light having multiple spots arranged at substantially periodic intervals in a second direction perpendicular to the first direction, and each of the multiple types of second structured linear excitation light has a different combination of spot shape and interval period compared to the other multiple types of second structured linear excitation light.

17. 16. The super-resolution imaging device of claim 15, wherein the scanning module is further configured to, during the row-by-row scanning, subject the second structured line excitation light to a predetermined phase shift in the second direction each time it is used for irradiation compared to when it was used for the previous irradiation, and the phase shift distance is a non-integer multiple of the interval period of the spot of the second structured line excitation light.

18. 15. The super-resolution imaging device of claim 14, wherein the plurality of types of structured linear excitation light include a third structured linear excitation light and a fourth structured linear excitation light, the irradiation mode of the third structured linear excitation light is configured to improve imaging performance in a third direction, and the irradiation mode of the fourth structured linear excitation light is configured to improve imaging performance in a fourth direction different from the third direction.

19. The super-resolution imaging device of claim 18 , wherein the third direction is parallel to the first direction, and the fourth direction is perpendicular to the third direction.

20. 20. The super-resolution imaging device of claim 19, wherein the irradiation mode of the fourth structured linear excitation light is configured to improve imaging performance in both the fourth direction and a fifth direction perpendicular to both the third direction and the fourth direction.

21. 20. The super-resolution imaging device of claim 19, wherein the plurality of types of structured linear excitation light further includes a fifth structured linear excitation light, and the irradiation mode of the fifth structured linear excitation light is configured to improve imaging performance in a fifth direction perpendicular to both the third direction and the fourth direction.

22. The super-resolution imaging device of claim 14 , wherein the scanning module is further configured to illuminate two adjacent rows in the target area with different types of structured line excitation light from the plurality of types of structured line excitation light.

23. The super-resolution imaging device of claim 14 , wherein the scanning module is further configured to switch among the plurality of types of structured line excitation light according to a preset sequence to illuminate each row in the target region.

24. 15. The super-resolution imaging device of claim 14, wherein the scanning module is further configured such that during the row-by-row scanning, regions illuminated by two successive applications of structured line excitation light in the target area partially overlap each other.

25. the target region includes a plurality of target subregions arranged along the first direction; The scanning module performs the following operations for each of the plurality of target sub-regions: the scanning of the target partial region by each row along the single first direction is performed in parallel while switching the plurality of types of structured beam excitation light so that each row in the target partial region is illuminated by a corresponding type of structured beam excitation light among the plurality of types of structured beam excitation light; The detection module performs the following operations on each of the plurality of target sub-regions: The super-resolution imaging device of claim 14, configured to perform in parallel the detection of response light generated by each row in the target partial area in response to being illuminated by the corresponding type of structured line excitation light.

26. a first row in a first target sub-area of ​​the plurality of target sub-areas and a second row in a second target sub-area of ​​the plurality of target sub-areas are simultaneously illuminated; the scanning module is configured to illuminate the first row and the second row with the same type of structured line excitation light among the plurality of types of structured line excitation light; or 26. The super-resolution imaging device of claim 25, wherein the scanning module is configured to illuminate the first row and the second row with different types of structured line excitation light from among the plurality of types of structured line excitation light.

27. The illumination module comprises: a light source configured to provide excitation light; a light branching unit configured to branch the excitation light from the light source into a plurality of excitation light beams; a plurality of structured line excitation light generating units, each of which is configured to receive a corresponding beam of excitation light from the light branching unit and generate a corresponding type of structured line excitation light based on the received corresponding beam of excitation light; a light combining unit configured to combine the plurality of types of structured linear excitation light from the plurality of structured linear excitation light generating units into a beam; a plurality of optical switching units, each of which is disposed between the optical branching unit and a corresponding one of the structured linear pumping light generating units and configured to control whether the pumping light from the optical branching unit is output to the corresponding one of the structured linear pumping light generating units, or each of which is disposed between the corresponding one of the structured linear pumping light generating units and the optical combining unit and configured to control whether the structured linear pumping light from the corresponding one of the structured linear pumping light generating units is output to the optical combining unit; The super-resolution imaging device according to claim 14 , wherein the structured line excitation light generated by different structured line excitation light generating units among the plurality of structured line excitation light generating units has different irradiation modes.

28. The illumination module comprises: a light source configured to provide excitation light; and a single modulation unit configured to modulate the excitation light from the light source into structured line excitation light having different illumination modes.

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