Line scanning-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 to Chinese Patent Application No. 202310207189.0, filed on March 6, 2023 In contrast and the entire disclosure of which is incorporated herein by reference. National phase of international application number PCT / CN2023 / 123332, filed on October 8, 2023. and Both of the above-mentioned applications is hereby incorporated by reference in its entirety.

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

Background Art

[0003] Conventional imaging technologies such as confocal imaging technology and wide-field imaging are limited by the Abbe diffraction limit, and thus their maximum imaging resolution is typically about 250 nanometers (nm), and structures with smaller sizes cannot be resolved. In view of this, super-resolution imaging technologies that can exceed the limitations of the diffraction limit have emerged, whereby the imaging resolution can reach 100 nm or less, providing important technical support for research on structures with micro-sizes.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

[0005] According to one aspect of the present disclosure, a line-scanning superresolution imaging method is provided, comprising: providing a plurality of types of structured line excitation light, each of which has a different irradiation mode; irradiating a target region by a line-by-line scan along a single first direction, while switching the plurality of types of structured line excitation light so that each line in the target region is irradiated by a corresponding type of structured line excitation light from the plurality of types of structured line excitation light; and detecting response light generated by each line in the target region in response to irradiation by the corresponding type of structured line excitation light.

[0006] According to another aspect of the present disclosure, a line-scanning super-resolution imaging device is provided, comprising: an illumination module configured to provide 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 scanning module configured to illuminate a target region 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 region is illuminated by a corresponding type of structured line excitation light from the plurality of types of structured line excitation light; and a detection module configured to detect response light generated by each row in the target region in response to illumination by the corresponding type of structured line excitation light.

[0007] Other features and advantages of this disclosure will become more apparent from the following detailed description of exemplary embodiments of this disclosure with reference to the accompanying drawings.

[0008] The aforementioned and other features and advantages of the present disclosure will become apparent from the following description of embodiments of the present disclosure shown with the drawings. The drawings are incorporated herein and constitute part of this specification and are used further to illustrate the principles of the present disclosure and to enable those skilled in the art to carry out and use the present disclosure. [Brief explanation of the drawing]

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

[0010] In the embodiments shown below, the same reference numeral may be used across different accompanying drawings to represent the same part or part having the same function, and it should be noted that repeated descriptions will be omitted. In some cases, similar items are indicated using the same reference number and letter, and therefore, once an item is defined in one drawing, it does not need to be discussed further in subsequent drawings.

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

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

[0013] In fact, the following description of at least one exemplary embodiment is merely illustrative and in no way limits the present disclosure and its application or use. In other words, the structures and methods in this specification 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 they are shown in an exemplary manner rather than in a comprehensive manner in which the present disclosure can be implemented. Further, the attached drawings are not necessarily drawn to scale, and some features may be enlarged to show details of specific components.

[0014] Furthermore, technologies, methods, and devices known to those skilled in the art may not be discussed in detail, but in appropriate situations, the technologies, methods, and devices will be considered part of this specification.

[0015] In all examples shown and discussed in this specification, any specific value should be interpreted as an example only and not as a limitation. Therefore, different values may exist in other examples of the exemplary embodiments.

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

[0017] Super-resolution imaging techniques based on wide-field imaging, such as structured light irradiation super-resolution imaging and single-molecule stereotactic imaging, have also been proposed. Super-resolution imaging techniques based on wide-field imaging require multiple imaging of the entire target region to capture multiple full-field 2D images, and finally, these multiple full-field 2D images are integrated into a super-resolution image through computation. However, this technique requires the target region to remain stationary throughout the entire imaging process; otherwise, it becomes impossible to reconstruct the super-resolution image.

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

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

[0020] The inventors of this disclosure have noted that line scanning is faster than point scanning. For example, referring to Figure 1B, during line scanning, the entire row (in the x-direction) within the target area is simultaneously illuminated by excitation light, and a detection device such as a camera is used to simultaneously detect the entire row. Then, by scanning row by row along the y-direction, the entire target area can be imaged in two dimensions. Therefore, comparing the point scanning process shown in Figure 1A with the line scanning process shown in Figure 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 from a single scan. To achieve resolution beyond the diffraction limit, existing super-resolution imaging techniques (e.g., the super-resolution imaging techniques based on wide-field imaging mentioned above) usually require acquiring multiple imaging results from multiple scans and integrating them into a super-resolution image. However, the imaging speed of such super-resolution imaging techniques is also low due to the long time interval between two adjacent scans.

[0021] Therefore, this disclosure proposes a line-scanning-based super-resolution imaging technique that can acquire a super-resolution image in a single scan without repeating multiple scans and then integrating multiple imaging results. The line-scanning-based super-resolution imaging technique of this disclosure has a higher imaging speed, thereby having a greater tolerance for movement of the target area during the imaging process.

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

[0023] In this specification, "structured line excitation light" refers to excitation light having a linear structure whose projection onto the target area exhibits a linear distribution. In this specification, the "illumination mode" of "structured line excitation light" refers to the spatial distribution pattern of the light intensity of the structured line excitation light. More specifically, this may refer to the projection pattern of the structured line excitation light onto the target area (which can reflect the light intensity distribution of the structured line excitation light onto a two-dimensional cross-section in the plane on which the target area is located). The illumination modes of the two light beams are considered to be the same if the projection patterns of two light beams onto the target area perfectly coincide with each other, or if they perfectly coincide with each other after translation. Based on the principle of fluorescence imaging, after illumination by "excitation light," the target area emits "response light" accordingly, and the target area can be imaged according to that "response light." In this specification, "target area" refers to the area 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 irradiation mode that completely illuminates the entire row, and the second structured line excitation light may have an irradiation mode that does not completely illuminate the entire row. For example, the first structured line excitation light may have a spot that extends continuously in a second direction (e.g., the x-direction shown in Figure 3A) perpendicular to a first direction (e.g., the y-direction shown in Figure 3A) (e.g., structured line excitation light a having irradiation mode a shown in Figure 3A). The second structured line excitation light may have multiple spots arranged at substantially periodic intervals in the second direction (e.g., the x-direction shown in Figure 3A) (e.g., structured line excitation light b having irradiation mode b and structured line excitation light c having irradiation mode c shown in Figure 3A). In this specification, "substantially" periodic intervals mean that the interval variation is within ±20% of the design interval or target interval, for example, within ±15%, for example, within ±10%, or for example, within ±5%. In some embodiments, the multiple types of structured ray excitation light provided in step S102 may further include multiple types of second structured ray excitation light as additional or alternative, each type of second structured ray excitation light having a different combination of spot shape and interval period compared to other second structured ray excitation lights. For example, the spot shape and interval period of structured ray excitation light b having irradiation mode b shown in Figure 3A are different from the spot shape and interval period of structured ray excitation light c having irradiation mode c shown in Figure 3A. In this specification, a difference in interval periods can be understood as interval periods not being the same or not "substantially" the same. For example, the interval periods may differ by more than ±20%, for example, more than ±50%, or for example, more than ±70%. In some other embodiments, the second structured line excitation light may also have multiple spots arranged at a non-periodic intervals in a second direction (e.g., the x-direction shown in Figure 3A).

[0025] As a non-limiting example, Figures 3A and 3B show three types of structured line excitation light having different irradiation modes: structured line excitation light a with irradiation mode a, structured line excitation light b with irradiation mode b, and structured line excitation light c with irradiation mode c. It can be understood that any appropriate number of structured line excitation light types, including but not limited to two, three, four, or more, can be set as needed.

[0026] Figure 3 illustrates how steps S104 and S106 are carried out: (1) First, the scan begins at the first row of the target region, which is irradiated using structured line excitation light a with irradiation mode a, and the response light generated by the first row in response to the irradiation of the first row with structured line excitation light a is detected; (2) Next, the scan proceeds along the y-direction to the second row of the target region, which is irradiated by switching to structured line excitation light b with irradiation mode b, and the response light generated by the second row in response to the irradiation of the second row with structured line excitation light b is detected; (3) Next, the scan proceeds along the y-direction to the third row of the target region, which is irradiated by switching to structured line excitation light c with irradiation mode c, and the response light generated by the third row in response to the irradiation of the third row with structured line excitation light c is detected; (4) Next, the scan proceeds along the y-direction to the fourth row of the target region The scan then proceeds along the y-direction to the fifth row of the target region, where the scan is lit by switching to structured line excitation light a having irradiation mode a, and the response light generated by the fourth row in response to the illumination of the fourth row with structured line excitation light a is detected. (5) The scan then proceeds along the y-direction to the sixth row of the target region, where the scan is lit by switching to structured line excitation light b having irradiation mode b, and the response light generated by the fifth row in response to the illumination of the fifth row with structured line excitation light b is detected. (6) The scan then proceeds along the y-direction to the sixth row of the target region, where the scan is lit by switching to structured line excitation light c having irradiation mode c, and the response light generated by the sixth row in response to the illumination of the sixth row with structured line excitation light c is detected. ...and so on, with each subsequent row of the target region being continuously lit and detected by alternating use of structured line excitation light a, structured line excitation light b, and structured line excitation light c. Finally, after scanning the entire target region row by row, the super-resolution image can be directly reconstructed based on the detection results of the response light generated by each row of the target region.

[0027] The multiple types of structured line excitation light provided in step S102 can be switched according to a predetermined order to illuminate the corresponding rows in the target region. For example, in the embodiment shown in Figure 3A, the multiple types of structured line excitation light provided in step S102 are traversed and used in the same order in each cycle to illuminate the corresponding rows in the target region. Alternatively, the multiple types of structured line excitation light provided in step S102 may be traversed and used in a different order in each cycle to illuminate the corresponding rows in the target region. In some embodiments, this may only require that two adjacent rows in the target region be illuminated by different types of structured line excitation light from the multiple types of structured line excitation light. In some other embodiments, it is also possible to switch to different types of structured line excitation light every few rows for illumination. In short, the switching order may be specifically designed according to the 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......", and "a,b,a,c,b,c......" can all have their own specific applicable application scenarios.

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

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

[0030] Furthermore, in the embodiment shown in Figure 3B, the structured line excitation light that does not completely illuminate the entire row during row-by-row scanning (e.g., the aforementioned second structured line excitation light such as structured line excitation light b having irradiation mode b and structured line excitation light c having irradiation mode c) does not undergo any phase shift in the second direction (e.g., x direction) each time it is used for irradiation compared to the previous time it was used for irradiation. In some other embodiments, for example as shown in Figure 5, the structured line excitation light that does not completely illuminate the entire row during row-by-row scanning (e.g., the aforementioned second structured line excitation light such as structured line excitation light b having irradiation mode b and structured line excitation light c having irradiation mode c) undergoes a preset phase shift in the second direction (e.g., x direction) each time it is used for irradiation compared to the previous time it was used for irradiation. If this structured line excitation light is the aforementioned 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 this structured line excitation light. Such phase shifts can facilitate the collection of more information from the target area for imaging.

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

[0032] The imaging speed of Method 100 can also be further improved by the parallel processing method. Referring to Figures 6A and 6B, for example, in some embodiments, the target region may include a plurality of 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, for each target subregion, performing in parallel the following operations: illuminating the target subregion by row-by-row scanning along a single first direction (e.g., the y-direction), while switching between a plurality of 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 a plurality of types of structured line excitation light; and detecting the response light generated by each row in the target subregion in response to illumination by the corresponding type of structured line excitation light. In the embodiment shown in Figure 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 the first target sub-region may be opposite to the scanning direction in the second target sub-region.

[0033] In some embodiments, the first row in the first target subregion and the second row in the second target subregion may be irradiated simultaneously. In some examples, as shown, for example in Figure 6A, the first row and the second row may be irradiated with the same type of structured line excitation light from among several types of structured line excitation light. In some other examples, as illustrated, for example in Figure 7, the first row and the second row may be irradiated with different types of structured line excitation light from among several types of structured line excitation light. Although the examples in Figures 6A to 7 illustrate only cases where the target region is divided into two target subregions, it can be understood that the target region may be divided into any number of target subregions having any suitable shape as required for specific reasons.

[0034] Method 100 requires only single-line scanning imaging to accurately reconstruct the super-resolution image. This benefits from high-line scanning imaging speeds, which can effectively improve the tolerance of the super-resolution imaging method for movement of the target region during the imaging process. For example, with existing scientific-grade cameras, if only four rows of pixels are used to image the target region, the frame rate can exceed 40 kHz. If it is desired that the displacement of the target region occurring during information acquisition from 10 consecutive rows is less than 50 nm (half of the design resolution of 100 nm), the speed of target region movement 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 (for example, the speed of movement of the brain region in anesthetized mice is about 2 micrometers per second, and the speed of movement of the brain region in awake mice is about 50 micrometers per second). Even if the acquired image is significantly deformed due to rapid movement of the target region, this does not affect Method 100's ability to analyze fine structures at the micro-scale.

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

[0036] In some embodiments, for example with reference to Figure 9A, the irradiation module 220 may include a light source 221 configured to provide excitation light, an optical branching unit 222 configured to branch the excitation light from the light source 221 into a plurality of excitation light beams, a plurality of structured line excitation light generation units 2241, 2242, ..., 224N, each structured line excitation light generation unit configured to receive a corresponding excitation light beam from the optical branching unit 222 and generate a corresponding type of structured line excitation light based on the received corresponding excitation light beam, an optical coupling unit 225 configured to couple a plurality of types of structured line excitation light from the plurality of structured line excitation light generation units 2241, 2242, ..., 224N into a beam, and a plurality of optical switching units 2231, 2232, ..., 223N. Each optical switching unit may be placed, for example, between an optical branching unit 222 and a corresponding structured line excitation light generation unit 2241, 2242, ..., 224N, and configured to control whether the excitation light from the optical branching unit 222 is output to the corresponding structured line excitation light generation unit (for example, as shown in Figure 9A). Alternatively, each optical switching unit may be placed between a corresponding structured line excitation light generation unit 2241, 2242, ..., 224N and an optical coupling unit 225, and configured to control whether the structured line excitation light from the corresponding structured line excitation light generation unit is output to the optical coupling unit 225. The structured line excitation light generated by different structured line excitation light generation units 2241, 2242, ..., 224N has different irradiation modes.

[0037] In some other embodiments, referring to Figure 9B, for example, the irradiation 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 irradiation modes. The modulation unit 226 may include at least one of, for example, a spatial light modulator or a polarization modulator, and may output structured line excitation light having different irradiation modes depending on the configuration.

[0038] The excitation light provided by the light source 221 is not particularly limited and may include one or more wavelengths or ranges of wavelengths, as long as it can excite the target region and generate response light.

[0039] The irradiation module 220 shown in Figure 9B has a simpler structure and fewer components than the irradiation module 220 shown in Figure 9A. However, the irradiation module 220 shown in Figure 9A can switch between structured-ray excitation light having different irradiation modes faster than the irradiation module 220 shown in Figure 9B. Figures 9A and 9B are illustrative and not limiting, and it should be understood that any suitable irradiation module 220 may be employed or designed in accordance with the teachings of this disclosure to provide multiple types of structured-ray excitation light having different irradiation modes.

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

[0041] In some embodiments, the multiple types of structured line excitation light provided by the irradiation module 220 may include a third structured line excitation light and a fourth structured line excitation light. The irradiation mode of the third structured line excitation light is configured to improve imaging performance in a third direction. The irradiation mode of the fourth structured line excitation light is configured to improve imaging performance in a fourth direction different from the third direction. In some examples, the third direction is parallel to the first direction, and the fourth direction is perpendicular to the third direction. In some examples, the irradiation mode of the fourth structured line 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 line excitation light further include a fifth structured line excitation light. The irradiation mode of the fifth structured line 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 such that, during row-by-row scanning, the second structured line excitation light undergoes a preset phase shift in a second direction each time it is used for irradiation compared to the previous time it was used for irradiation, the phase shift distance being a non-integer multiple of the interval period of the spot of the second structured line excitation light. In some embodiments, the scanning module 240 may be further configured to irradiate two adjacent rows in the target region using different types of structured line excitation light from a plurality of types of structured line excitation light. In some embodiments, the scanning module 240 may be further configured to switch between a plurality of types of structured line excitation light in a preset order to irradiate each row in the target region. In some embodiments, the scanning module 240 may be further configured such that, during row-by-row scanning, the regions irradiated by two consecutive applications of structured line excitation light in the target region partially overlap each other.

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

[0044] Examples of apparatus 200 may be the same as those of the examples of method 100 described above and will not be repeated herein.

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

[0046] The optical system shown in Figure 10 can be constructed to realize an illumination module that provides two types of structured line-excited light having different illumination modes. The input to the illumination module may be a parallel laser beam of a single wavelength or multiple wavelengths. The input light beam is split into two light beams having polarization perpendicular to each other by a polarizing beam splitter PBS1 (which can function as an optical splitting unit). These two light beams with polarization perpendicular to each other are controlled, respectively, 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 light within a specific wavelength range to pass through them, and allow for rapid control of the on / off of 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, which is illumination mode 1 shown in part A of Figure 12. In the optical path on the right in Figure 10, the parallel light beam reflected from the polarizing beam splitter PBS1 is first reflected by mirror M1, then passes through the acousto-optic variable 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, 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, such that the light is modulated to have illumination mode 2 shown in part A of Figure 12. In addition to using a transmission grating TG driven by a rotary motor, it is also possible to modulate the phase or intensity of the light using other solutions such as a digital micromirror device (DMD) or a liquid crystal spatial light modulator (SLM).While DMDs and SLMs can simply enable phase changes without being driven by a rotary motor, the transmission grating TG, which is driven by a rotary motor, can change phase faster than DMDs and SLMs. Therefore, cylindrical lens CL1 can function as a first structured line excitation light generation unit that provides structured line excitation light 1 having irradiation mode 1. Cylindrical lens CL2 and transmission grating TG can function as a second structured line excitation light generation unit that provides structured line excitation light 2 having irradiation mode 2. Structured line excitation light 1 having irradiation mode 1 and structured line excitation light 2 having irradiation mode 2 (reflected by mirror M2) are coupled into a beam by polarizing beam splitter PBS2 (which functions as an optical coupling unit). Furthermore, spherical lenses L1 and L3 constitute a relay imaging system and image the structured line excitation light 1 having irradiation mode 1 at the focal plane of spherical lens L3. Spherical lenses L2 and L3 also constitute a relay imaging system, and structured line excitation light 2 having irradiation mode 2 is imaged at the focal plane of spherical lens L3. Therefore, by controlling the acousto-optic variable filters AOTF1 and AOTF2, the irradiation module shown in Figure 10 can selectively output structured line excitation light 1 having irradiation mode 1 or structured line excitation light 2 having irradiation mode 2.

[0047] The optical system shown in Figure 11 can be constructed to realize a scanning module and a detection module, where solid arrows schematically show the optical path of the scanning module and dotted arrows schematically show the optical path of the detection module. Spherical lenses L4 and L5 can constitute a relay imaging system. Spherical lens L6 and a microscope objective lens can also constitute a relay imaging system. Structured line excitation light output by the illumination module is reflected by a dichroic mirror DM, then passes through spherical lens L4 to the scanning mirror SM. After being reflected by the scanning mirror SM, the structured line excitation light passes through spherical lens L5, spherical lens L6, and the microscope objective lens to reach the target area of ​​the sample. In the embodiment shown in Figure 11, scanning of the illumination position of the structured line excitation light in 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 illumination position of the structured line excitation light in the target area is achieved by displacing the sample. In this case, the scanning mirror SM can still be used or replaced with a fixed mirror. The response light emitted by the sample is collected by the microscope objective lens and then passes through the 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, it is also possible to detect the response light using other solutions such as photoelectric detectors and image sensors as alternatives. In the embodiment shown in Figure 11, the scanning module and the detection module share most of their optical elements and can be integrated into a single module called a line-scanning confocal imaging module.

[0048] Referring to Figure 12, the irradiation modes of structured line excitation light 1 and structured line excitation light 2, provided by the irradiation module in Figure 10 and projected onto the target area of ​​the sample by the scanning module in Figure 11, are specifically shown. As shown in Figure 12, irradiation 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 the imaging resolution in the y direction. Irradiation 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 the imaging resolution in the x and z directions.

[0049] During the line scanning process, alternating on / off switching of the acousto-optic variable filters AOTF1 and AOTF2 in the irradiation module of Figure 10 is achieved to irradiate the target region of the sample alternately with structured line excitation light 1 having irradiation mode 1 and structured line excitation light 2 having irradiation mode 2. During this period, the rotary motor can also be used to rotate the transmission grating TG in the irradiation module of Figure 10, thereby continuously moving the phase of irradiation mode 2 of structured line excitation light 2 along the x-direction, for example, as shown in Figure 13. Since the information thus collected with respect to the target region includes high-resolution information in the three directions x, y, and z, an image of the target region with high resolution in each of the three directions x, y, and z can be reconstructed in superresolution.

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

[0051] The terms “left,” “right,” “front,” “rear,” “top,” “bottom,” “above,” “under,” “upper,” “lower,” and similar terms used herein and in the claims are used for descriptive purposes, where applicable, and are not necessarily used to describe immutable relative positions. Since such terms are interchangeable in appropriate contexts, it should be understood that the embodiments of this disclosure described herein may operate in orientations different from those shown herein or otherwise described. For example, if the apparatus in the accompanying drawings is inverted, a feature originally described as being “above” another feature may now be described as being “under” another feature. The apparatus may also be oriented in other ways (rotated 90 degrees or in other orientations), in which case the relative spatial relationships will be interpreted accordingly.

[0052] Where in this specification and claims an element is referred to as being “above” another element, “attached” to another element, “connected” to another element, “coupled” to another element, “linked” to another element, or “in contact” to another element, that element may be directly above, directly attached to, directly connected to, directly coupled to, directly linked to, or directly in contact with, or there may be one or more intermediate elements. 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 is no intermediate element. In this specification and claims, when a feature is said to be "adjacent" to another feature, it may mean that the feature has an overlapping portion 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,” not as a “model” to be exactly imitated. Any embodiment described herein by example should not necessarily be construed as being preferable or advantageous to other embodiments. Furthermore, this disclosure is not limited by any express or implied theory provided in the art, background art, summary of the invention, or in the form for carrying out the invention.

[0054] As used herein, the term "substantially" includes any minor variations caused by design or manufacturing defects, tolerances of a device or component, environmental impacts, and / or other factors. The term "substantially" also allows for deviations from the perfect or ideal state caused by parasitic effects, noise, and other practical considerations that may exist in actual implementation.

[0055] Furthermore, terms such as “first” and “second” may also be used herein for reference purposes only and are therefore not intended to be limiting. For example, terms relating to structures or elements such as “first,” “second,” and other such numerical terms do not imply order or sequence unless the context explicitly indicates otherwise.

[0056] It should be further understood that, as used herein, the term "comprise / include" indicates the presence of the described features, integers, steps, actions, units, and / or components, but does not preclude the presence or addition of one or more other features, integers, steps, actions, units, and / or components, and / or combinations thereof.

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

[0058] As used herein, the terms "and / or" include any and all combinations of one or more of the relevant descriptions. The terms used herein are solely for the purpose of describing specific embodiments and are not intended to limit the disclosure. As used herein, the singular forms "a," "an," and "the" are intended to include the plural form unless otherwise explicitly stated in the context.

[0059] Those skilled in the art will recognize that the boundaries between the operations described above are merely illustrative. Multiple 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. Furthermore, alternative embodiments may include multiple examples of a particular operation, and a certain order of operations may be modified in various other embodiments. However, other modifications, changes, and substitutions are also possible. All embodiments and elements of the embodiments disclosed above may be combined in any way to provide multiple additional embodiments and / or combined with embodiments or elements of other embodiments. Accordingly, this specification and the accompanying drawings should be considered illustrative and not limiting.

[0060] While some specific embodiments of this disclosure are described in detail by example, those skilled in the art should understand that such examples are used solely for illustrative purposes and not to limit the scope of this disclosure. The embodiments disclosed herein can be combined in any way without departing from the spirit and scope of this disclosure. Those skilled in the art should also understand that various modifications can be made to the embodiments without departing from the spirit and scope of this disclosure. The scope of this disclosure is defined by the appended claims.

Claims

1. To provide multiple types of structured line excitation light, each of which has a different spatial distribution pattern of light intensity, The target region is irradiated by a row-by-row scan along a single first direction, while switching between the multiple types of structured line excitation light so that each row in the target region is irradiated by the corresponding type of structured line excitation light from among the multiple types of structured line excitation light, A super-resolution imaging method based on line scanning, comprising detecting response light generated by each row in the target region in response to irradiation by the corresponding type of structured line excitation light.

2. The super-resolution imaging method according to claim 1, wherein the plurality of 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 stripe extending 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. The super-resolution imaging method according to claim 1, wherein the plurality of types of structured line excitation light includes a plurality of types of second structured line 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 line excitation light has a different combination of spot shape and interval period compared to the others of the plurality of types of second structured line excitation light.

4. The super-resolution imaging method according to claim 2, wherein during each row scan, the second structured line excitation light undergoes a preset phase shift in the second direction each time it is used for irradiation compared to the previous time the second structured line excitation light was used for 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.

5. The plurality of types of structured line excitation light include a third structured line excitation light and a fourth structured line excitation light, wherein the spatial distribution pattern of the light intensity of the third structured line excitation light is configured to improve imaging performance in a third direction, and the spatial distribution pattern of the light intensity of the fourth structured line excitation light is configured to improve imaging performance in a fourth direction different from the third direction. The super-resolution imaging method according to claim 1, wherein the third direction is parallel to the first direction and the fourth direction is perpendicular to the third direction.

6. The super-resolution imaging method according to claim 5, wherein the spatial distribution pattern of the light intensity of the fourth structured line 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.

7. The super-resolution imaging method according to claim 5, wherein the plurality of types of structured line excitation light further includes a fifth structured line excitation light, and the spatial distribution pattern of the light intensity of the fifth structured line excitation light is configured to improve imaging performance in a fifth direction perpendicular to both the third and fourth directions.

8. The super-resolution imaging method according to claim 1, wherein the plurality of types of structured line excitation light are switched according to a predetermined order to irradiate each row in the target region.

9. The super-resolution imaging method according to claim 1, wherein during the scanning of each row, portions of the target region irradiated by two consecutive applications of structured line excitation light partially overlap with each other.

10. The target region includes a plurality of target subregions arranged along the first direction, and the method performs the following operations on each of the plurality of target subregions, namely, The target subregion is irradiated by scanning each row along the single first direction, while switching between the multiple types of structured line excitation light so that each row in the target subregion is irradiated by the corresponding type of structured line excitation light from the multiple types of structured line excitation light. The super-resolution imaging method according to claim 1, comprising, in parallel, detecting response light generated by each row in the target subregion in response to irradiation with the corresponding type of structured line excitation light.

11. An irradiation module configured to provide multiple types of structured line excitation light, wherein each of the multiple types of structured line excitation light has a different spatial distribution pattern of light intensity, A scanning module configured to illuminate the target region 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 region is illuminated by the corresponding type of structured line excitation light from the plurality of types of structured line excitation light, A line-scanning super-resolution imaging apparatus comprising: a detection module configured to detect response light generated by each row in the target region in response to irradiation by the corresponding type of structured line excitation light;

12. The plurality of types of structured line excitation light include a first structured line excitation light and a second structured line excitation light, wherein the first structured line excitation light has a stripe extending in a second direction perpendicular to the first direction, and the second structured line excitation light has a plurality of spots arranged at substantially periodic intervals in the second direction. The super-resolution imaging apparatus according to claim 11, wherein the scanning module is further configured such that, during each row scan, the second structured line excitation light undergoes a preset phase shift in the second direction each time it is used for irradiation compared to when it was used for the previous irradiation, the phase shift distance being a non-integer multiple of the interval period of the spot of the second structured line excitation light.

13. The irradiation module is A light source configured to provide excitation light, A light splitter unit configured to split the excitation light from the light source into multiple beams of excitation light, A plurality of structured line excitation light generation units, each of which is configured to receive a corresponding excitation light beam from the optical branching unit and generate a corresponding type of structured line excitation light based on the received corresponding excitation light beam, A photocoupler configured to couple multiple types of structured line excitation light from the multiple structured line excitation light generation units into a beam, A plurality of optical switching units, each of which is placed between an optical branching unit and a corresponding one of the structured line excitation light generation units and configured to control whether the excitation light from the optical branching unit is output to the corresponding one of the structured line excitation light generation units, or each of which is placed between a corresponding one of the structured line excitation light generation units and an optical coupling unit and configured to control whether the structured line excitation light from the corresponding one of the structured line excitation light generation units is output to the optical coupling unit, comprising: The super-resolution imaging apparatus according to claim 11, wherein the structured line excitation light generated by different structured line excitation light generation units among the plurality of structured line excitation light generation units has different spatial distribution patterns of light intensity.

14. The irradiation module is A light source configured to provide excitation light, The super-resolution imaging apparatus according to claim 11, comprising a single modulation unit configured to modulate the excitation light from the light source into structured line excitation light having different spatial distribution patterns of light intensity.

Images