Multi-focus scanning and array-type multi-channel detection imaging method, device and medium

Abstract

This invention relates to a multifocal scanning and array-type multichannel detection imaging method, device, and medium, belonging to the field of laser scanning and fluorescence detection imaging technology. The method includes the following steps: multiple laser focal points each scan to form a laser scanning region; establishing a correspondence between the laser scanning region and the detection region formed by multiple detection channels; at each time point, based on the scanning position of each laser focal point, selecting only the detection channel corresponding to the current location of the image spot within the corresponding detection region; controlling the scanning phase of each laser focal point to maintain the scanning interval distance between adjacent laser focal points; when the scanning position of a laser focal point leaves the selected detection channel, closing that detection channel, and selecting only the detection channel within the detection region corresponding to the scanning region entered by the laser focal point. This invention effectively avoids fluorescence crosstalk while improving imaging throughput, and does not affect the two-photon imaging depth or the continuity of the imaging region.

Description

Technical Field

[0001] This invention relates to the field of laser scanning and fluorescence detection imaging technology, and in particular to a multifocal scanning and array-type multichannel detection imaging method, device and medium. Background Technology

[0002] Because two-photon microscopy can maintain excellent imaging resolution in thick biological tissues, it is increasingly being used in fields such as brain science research and tumor pathology.

[0003] Two-photon microscopy, similar to confocal microscopy, is a type of laser scanning fluorescence microscopy. It uses a laser beam to form a laser focus at the focal plane of the objective lens. It typically uses a point-by-point scanning method to scan the sample in two or three dimensions, exciting fluorescent molecules at each point. By using a detector on the image side to detect the intensity of the fluorescence signal, an image based on fluorescence intensity is formed, thereby reflecting the structural and functional information of the sample.

[0004] Point-by-point scanning limits the speed of two-photon imaging to some extent. Therefore, in recent years, people have increasingly adopted the method of scanning multiple laser focal points simultaneously to improve the throughput of two-photon imaging.

[0005] Multifocal scanning excitation creates multiple fluorescence sources, which need to be distinguished at the detector end (or image side). Otherwise, fluorescence crosstalk between the sources will affect the accuracy of the image information and cause image artifacts.

[0006] One detection method in multifocal scanning imaging is to use a camera (CCD or CMOS) instead of a conventional photomultiplier tube (PMT) as the detection device. The camera has a large and dense array of elements (typically millions to tens of millions of pixels), possessing spatial correspondence with the object-side field of view. That is, each pixel of the camera (image side) corresponds to a specific point in the object-side field of view, thus enabling the differentiation of the focal source of fluorescence. For example, the journal article "Kilohertz two-photon bra in imaging in awakening" (Tong Zhang et al., Nature Methods, 2019) used a microlens array to form a 20×20 laser focus and an sCMOS camera for detection, achieving an imaging frame rate of 1 kHz. However, as the imaging depth increases, the fluorescence excited at the focal point, after being scattered by upper tissue layers, reaches the image-side camera detection surface after being scattered into a diffuse image spot with a radius much larger than the ideal diffraction image spot. While camera detection has spatial correspondence, its temporal resolution is insufficient. Spatial structural information within the same exposure time will convolve with the diffuse image spot, ultimately reducing the spatial resolution of the imaging. Therefore, the imaging depth of multifocal two-photon imaging methods using camera detection schemes is limited.

[0007] Another detection method for multifocal scanning imaging is to use multi-channel or multi-element photodetectors, such as multi-anode photodetectors (PMTs) or detection modules assembled from multiple PMTs. Photodetectors, such as PMTs, have high temporal resolution and can deduce the scanning position by combining the laser focus scanning sequence. Therefore, the spatial resolution is not affected by the radius of the diffuse spot, and the optical resolution generally depends only on the focusing degree of the laser focus. Currently, in this multi-channel photodetector method, the scanning area of ​​each laser focus corresponds to the area of ​​one detection channel or detection element; that is, the number of laser focuses is equal to the number of detection channels or elements. When the laser focus is located at the boundary between two scanning areas, due to the large radius of the diffuse spot, fluorescent photons can easily cross over into the area of ​​adjacent detection elements, causing fluorescence crosstalk between fluorescence sources.

[0008] To avoid crosstalk, one strategy is to create sufficient gaps between detector elements, but this causes discontinuities between imaging regions. Another strategy is to introduce a certain delay between laser pulses from different laser focal points. For example, the 80MHz repetition rate femtosecond laser commonly used in two-photon microscopy has a pulse period of 12.5ns, which can be divided into four time segments, meaning the pulse times of the four laser focal points differ by 3.125ns sequentially. The resulting fluorescence signals also have the same delay, thus further separating the fluorescence sources in time to avoid crosstalk. For instance, Chinese patent application CN202110897601.7 proposes a pulse four-time segmentation method combined with the "four-color boundary principle," further expanding the number of focal points allowed by the pulse delay method from the usual four focal points to a theoretically limitless number.

[0009] However, the pulse delay method requires advanced high-frequency digital-to-analog circuitry. For example, distinguishing a 3.125ns pulse delay necessitates a time resolution of 1ns, an analog circuit bandwidth of 1GHz, a gain-bandwidth product of at least 100GHz, and stringent requirements for cable impedance matching and even the consistency of each cable's length. Furthermore, the fluorescence lifetimes of different fluorescent proteins or probes vary considerably; not all fluorescence lifetimes exceed 3ns, and some phosphorescent probes even have millisecond-level lifetimes. This means that the pulse delay method limits the types of labeled substances that can be used. Summary of the Invention

[0010] In order to achieve the above-mentioned objectives and other advantages of the present invention, a first objective of the present invention is to provide a multifocal scanning and array-type multichannel detection imaging method, comprising the following steps:

[0011] Multiple laser focal points scan independently to form a laser scanning area, and the various laser scanning areas are arranged to form a continuous overall scanning field;

[0012] Establish the correspondence between the laser scanning area of ​​each laser focus and the detection area formed by multiple detection channels in the detection surface;

[0013] At each time point, based on the scanning position of each laser focus, the detection channel corresponding to the current location of the image spot is selected within the corresponding detection area, while the remaining detection channels within the detection area remain unselected.

[0014] The scanning phase of each laser focus is controlled to maintain the scanning interval between adjacent laser focuses;

[0015] When the scanning position of the laser focus leaves the selected detection channel, the detection channel is closed, and the detection channels in the detection area corresponding to the scanning area entered by the laser focus are selected, while the remaining detection channels in the detection area are kept in a deselected state.

[0016] Furthermore, the scanning of the multiple laser focal points to form a laser scanning area includes: the overall laser scanning field is divided into an N×M array-type laser scanning area formed by scanning multiple laser focal points, where N and M are arbitrary positive integers.

[0017] Furthermore, the laser scanning area of ​​each laser focus corresponds to a detection area.

[0018] Furthermore, the detection area is a detection area formed by 1×4 detection channels.

[0019] Furthermore, the detection area is a detection area formed by 2×2 detection channels.

[0020] Furthermore, controlling the scanning phase of each laser focus to maintain the scanning interval distance between adjacent laser focuses includes the following steps:

[0021] The scanning phases of adjacent laser focal points are controlled to be consistent in the horizontal and vertical directions, respectively, while maintaining the scanning interval between laser focal points; where the horizontal and vertical directions are the two scanning directions of the laser focal points.

[0022] Furthermore, controlling the scanning phase of each laser focus to maintain the scanning interval distance between adjacent laser focuses includes the following steps:

[0023] The scanning phases of adjacent laser focal points are controlled to differ by half a scanning cycle in the longitudinal direction, maintaining the scanning interval between laser focal points; wherein, the longitudinal direction is one of the scanning directions of the laser focal points.

[0024] Furthermore, the time resolution of switching the gating state of the detection channel is no less than 100 ns.

[0025] A second objective of the present invention is to provide an electronic device comprising: a memory storing program code thereon; a processor connected to the memory, wherein when the program code is executed by the processor, a multifocal scanning and array-type multichannel detection imaging method is implemented.

[0026] A third objective of the present invention is to provide a computer-readable storage medium having program instructions stored thereon, which, when executed, implement a multifocal scanning and array-type multichannel detection imaging method.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention provides a multi-focus scanning and array-type multi-channel detection imaging method. Each laser focus has a laser scanning area corresponding to multiple detection channels, such as 2×2 detection channels. Based on the current position of the laser focus, only one of the corresponding multiple detection channels is selected at the same time, while the other detection channels are not selected. At the same time point, the boundaries of the selected detection channels of each laser focus are not adjacent and are isolated by the unselected detection channels. Therefore, even if each laser focus is located near the boundary of the scanning area, the fluorescent photons excited by one laser focus cannot cross the isolation zone to enter the detection channels of other laser focuses that are in the selected state, thereby avoiding the generation of fluorescence crosstalk.

[0029] Compared to the pulse delay method, the method provided by this invention also effectively avoids the generation of fluorescence crosstalk. The time resolution requirement of the gating logic circuit is within 100 ns, which is much lower than the requirement of within 1 ns for the pulse delay method. The overall technical difficulty is greatly reduced, and there are no restrictions on the types of fluorescently labeled proteins and fluorescence lifetime.

[0030] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0031] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0032] Figure 1 This is a schematic diagram showing the correspondence between the scanning array region and the detector array region in a conventional multifocal scanning detection imaging method.

[0033] Figure 2 This is a flowchart of a multifocal scanning and array-type multichannel detection imaging method according to Example 1;

[0034] Figure 3 This is a schematic diagram showing the correspondence between the laser scanning area and the detection area in Example 1, and when the detection channels in the selected state are A1, B1, C1, and D1.

[0035] Figure 4 This is a schematic diagram showing the correspondence between the laser scanning area and the detection area in Example 1, and when the detection channels in the selected state are A2, B2, C2, and D2.

[0036] Figure 5 This is a schematic diagram showing the correspondence between the laser scanning area and the detection area in Example 1, and when the detection channels in the selected state are A3, B3, C3, and D3.

[0037] Figure 6 This is a schematic diagram showing the correspondence between the laser scanning area and the detection area in Example 1, and when the detection channels in the selected state are A4, B4, C4, and D4.

[0038] Figure 7 This is a schematic diagram showing the correspondence between the laser scanning area and the detection area in Example 1, with each laser focus located at the edge of the scanning area.

[0039] Figure 8 This is a schematic diagram of the 3×2 array laser scanning area in Example 1, with each laser scanning area corresponding to a 1×4 detection channel;

[0040] Figure 9 This is a schematic diagram of the electronic device in Example 2;

[0041] Figure 10 This is a schematic diagram of a computer-readable storage medium according to Example 3. Detailed Implementation

[0042] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0043] like Figure 1 As shown, in conventional multifocal scanning detection imaging methods, the laser scanning area of ​​one laser focus corresponds to one detection channel. The figure shows laser scanning areas with four laser focuses (A, B, C, and D), corresponding to four detection channels (array elements) A1, B1, C1, and D1, respectively. When each laser focus is located at the boundary of the laser scanning area, the radius of the diffuse image spot is large, and it will simultaneously fall into two adjacent detection channels. For example, the fluorescence excited by the laser focus in area A is detected simultaneously by A1 and B1, and the fluorescence excited by the laser focus in area C is detected simultaneously by C1 and D1, thus resulting in fluorescence crosstalk.

[0044] Example 1

[0045] A multifocal scanning and array-type multichannel detection imaging method, such as Figure 2 As shown, it includes the following steps:

[0046] Multiple laser focal points scan independently to form a laser scanning area, and the various laser scanning areas are arranged to form a continuous overall scanning field (or object-side field of view);

[0047] In this embodiment, the overall laser scanning field is divided into an N×M array-type laser scanning region formed by multiple laser focal scans, where N and M are arbitrary positive integers. For example, a 2×2 array-type laser scanning region would be as follows: Figures 3-7 The laser scanning areas A, B, C, and D can be square or other shapes, and can be designed according to actual needs.

[0048] Establish a correspondence between the laser scanning area of ​​each laser focus and the detection area formed by multiple detection channels in the detection plane (or image-side field of view). In this embodiment, the laser scanning area of ​​each laser focus corresponds to one detection area. For example, each laser scanning area is mapped to the detection area formed by 2×2 detection channels. Figures 3-7 As shown, scan area A corresponds to four detection channels A1, A2, A3, and A4; scan area B corresponds to four detection channels B1, B2, B3, and B4; scan area C corresponds to four detection channels C1, C2, C3, and C4; and scan area D corresponds to four detection channels D1, D2, D3, and D4.

[0049] At each time point, based on the scanning position of each laser focus, the detection channel corresponding to the current location of the image spot is selected within the corresponding detection area, while the remaining detection channels within the detection area remain unselected; that is, one detection channel corresponding to the current location of the image spot is selected within the detection area formed by the 2×2 detection channels, while the other three detection channels remain unselected. Figure 3 In the selection process, the detection channels in the gated state are A1, B1, C1, and D1; Figure 4 In the list, A2, B2, C2, and D2 are in the gating state. Figure 5 In the list, A3, B3, C3, and D3 are in the gating state; Figure 6 In the diagram, A4, B4, C4, and D4 are in the selected state. The specific selection method can be achieved by providing an enable signal to the corresponding detection channel.

[0050] Controlling the scanning phase of each laser focus maintains the scanning interval between adjacent laser focuses; such as Figures 3 to 6As shown, the boundaries of the detection channels in the selected state corresponding to each laser focus are not adjacent, and are separated by the isolation zone formed by the detection channels in the non-selected state.

[0051] When the detection area is formed by 2×2 detection channels, the scanning phases of adjacent laser focal points are controlled to be consistent in both the horizontal and vertical directions, while maintaining the scanning interval between laser focal points approximately equal to the side length of the laser scanning area. The horizontal and vertical directions are the two scanning directions of the laser focal points. Figures 3-7 As shown, the horizontal direction (column direction) is the scanning direction of the laser focus from left to right, and the vertical direction (row direction) is the scanning direction of the laser focus from top to bottom. This ensures that the boundaries of the detection channels in the selected state corresponding to each laser focus are not adjacent, but separated by isolation zones formed by the non-selected detection channels.

[0052] like Figure 7 As shown, when each laser focus is located at the boundary of the scanning area, due to the existence of the isolation zone, the scattered fluorescence will enter the non-gated channel, but will not cause crosstalk. Figure 7 The fluorescence excited by the laser focus in region A falls into the detection channels A2 and B1. A2 is the detection channel corresponding to region A and is in a normal gating state, while B1 does not correspond to region A and is in a non-gating state, so it will not cause image fluorescence crosstalk. The fluorescence excited by the laser focus in region C falls into the detection channels C2 and D1. C2 is the detection channel corresponding to region C and is in a normal gating state, while D1 does not correspond to region C and is in a non-gating state, so it will not cause image fluorescence crosstalk.

[0053] It should be understood that this invention does not limit the number or arrangement of laser scanning areas, including but not limited to the aforementioned 2×2 array laser scanning areas. Similarly, the specific arrangement of detection channels within the detection area includes, but is not limited to, the aforementioned 2×2 detection channel-formed detection area, and can be designed according to actual needs. For example... Figure 8 As shown, the overall scanning field is divided into 3×2 array laser scanning regions, namely A, B, C, D, E, and F. Each laser scanning region corresponds to a detection region formed by 1×4 detection channels. For the detection region formed by 1×4 detection channels, it is necessary to control the scanning phase difference between the even-numbered and odd-numbered laser focal points in the longitudinal direction (row direction) to be half a scanning cycle, thereby maintaining a distance of twice the narrow side length of the detection channel at the laser focal point in that direction. Similarly, only the detection channel corresponding to the current position of the laser focal point is selected, such as... Figure 8 In the non-gated state, the detection channels A1, B3, C1, D1, E3, and F1 can still form an isolation band to avoid fluorescence crosstalk. When a 1×4 detection channel forms the detection area, there are no special requirements for the scanning phase of the laser focus in the horizontal (column) direction.

[0054] It should be noted that, according to the "four-color boundary principle," any region distributed in a plane can always be divided into four types. Therefore, by not selecting approximately 3 / 4 of the total number of detection channels, an isolation zone avoiding fluorescence crosstalk can always be formed. In summary, the laser scanning area can be designed with any array size and arrangement. When the scanning position of the laser focus leaves the selected detection channel, that detection channel is closed, and the detection channels within the detection area corresponding to the scanning area entered by the laser focus are selected, keeping the remaining detection channels within that detection area in a non-selected state. That is, only one detection channel within the detection area is always selected, while the other three detection channels remain non-selected. Considering the performance of current common scanning devices, the time resolution for switching the selection state is typically required to be no less than 100 ns.

[0055] like Figures 3-7 As shown, the overall scanning field formed by the arrangement of laser scanning areas is continuous without obvious gaps, and the image-side field of view formed by the arrangement of each detection area is also continuous without obvious gaps, thus maintaining the continuity of the imaging area. In actual implementation, the image brightness at the boundary of each detection area may be slightly darker than that at the center of the area, but there is no loss of image structure or functional information at that location, and the uniformity of image brightness can be further corrected by algorithms.

[0056] In practice, if there are large gaps between the detector array elements, a lens array can be added in front of the detector array for pupil matching. The lens array is located near the image plane, and the lens gaps can be made very small.

[0057] This invention employs multiple laser focal points for multi-focal synchronous scanning to improve imaging throughput. The scanning area corresponding to each laser focal point corresponds to a 2×2 or 1×4 detector channel array on the detector plane (image-side field of view). Based on the current scanning position of each laser focal point, only the detector channel corresponding to the position in the 4 detector channel array is selected, while the other 3 detector channels are in a non-selected state. Due to the existence of the isolation zone formed by the non-selected detector channels, the fluorescence generated by each focal point can only be detected by the selected detector channel corresponding to that laser focal point, thereby avoiding the generation of fluorescence crosstalk between focal points.

[0058] This invention improves imaging throughput while effectively avoiding fluorescence crosstalk, without affecting two-photon imaging depth or imaging region continuity. Moreover, the method is easy to implement and does not limit the types of fluorescently labeled proteins or the length of fluorescence lifetime.

[0059] Example 2

[0060] An electronic device 200, such as Figure 9As shown, the method includes, but is not limited to: a memory 201 storing program code; and a processor 202 connected to the memory, which, when the program code is executed by the processor, implements a multifocal scanning and array-type multichannel detection imaging method. For a detailed description of the method, please refer to the corresponding description in the above method embodiments, which will not be repeated here.

[0061] Example 3

[0062] A computer-readable storage medium, such as Figure 10 As shown, it stores program instructions, which, when executed, implement a multifocal scanning and array-type multichannel detection imaging method. For a detailed description of the method, please refer to the corresponding description in the above method embodiments; it will not be repeated here.

[0063] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0064] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0065] The above are merely embodiments of this specification and are not intended to limit the scope of the one or more embodiments herein. For those skilled in the art, various modifications and variations can be made to the one or more embodiments of this specification. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the one or more embodiments of this specification should be included within the scope of the claims of the one or more embodiments of this specification.

Claims

1. A multifocal scanning and array-type multichannel detection imaging method, characterized in that, Includes the following steps: Multiple laser focal points scan independently to form a laser scanning area, and the various laser scanning areas are arranged to form a continuous overall scanning field; Establish the correspondence between the laser scanning area of ​​each laser focus and the detection area formed by multiple detection channels in the detection surface; At each time point, based on the scanning position of each laser focus, the detection channel corresponding to the current location of the image spot is selected within the corresponding detection area, while the remaining detection channels within the detection area remain unselected. The scanning phase of each laser focus is controlled to maintain the scanning interval between adjacent laser focuses; When the scanning position of the laser focus leaves the selected detection channel, the detection channel is closed, and the detection channels in the detection area corresponding to the scanning area entered by the laser focus are selected, while the remaining detection channels in the detection area are kept in a non-selected state. The laser scanning area formed by the scanning of the multiple laser focal points includes: the overall laser scanning field is divided into an N×M array-type laser scanning area formed by the scanning of multiple laser focal points, where N and M are any positive integers; Each laser focus corresponds to a detection area.

2. The multifocal scanning and array-type multichannel detection imaging method according to claim 1, characterized in that: The detection area is a detection area formed by 1×4 detection channels.

3. The multifocal scanning and array-type multichannel detection imaging method according to claim 1, characterized in that: The detection area is a detection area formed by 2×2 detection channels.

4. The multifocal scanning and array-type multichannel detection imaging method according to claim 3, characterized in that, The process of controlling the scanning phase of each laser focus to maintain the scanning interval between adjacent laser focuses includes the following steps: The scanning phases of adjacent laser focal points are controlled to be consistent in the horizontal and vertical directions, respectively, while maintaining the scanning interval between laser focal points; where the horizontal and vertical directions are the two scanning directions of the laser focal points.

5. The multifocal scanning and array-type multichannel detection imaging method according to claim 2, characterized in that: The process of controlling the scanning phase of each laser focus to maintain the scanning interval between adjacent laser focuses includes the following steps: The scanning phases of adjacent laser focal points are controlled to differ by half a scanning cycle in the longitudinal direction, maintaining the scanning interval between laser focal points; wherein, the longitudinal direction is one of the scanning directions of the laser focal points.

6. The multifocal scanning and array-type multichannel detection imaging method according to claim 1, characterized in that: The time resolution for switching the gating state of the detection channel is no less than 100 ns.

7. An electronic device, characterized in that, include: A memory that stores program code; A processor, which is connected to the memory, and which, when the program code is executed by the processor, implements the method as described in any one of claims 1 to 6.

8. A computer-readable storage medium, characterized in that, It stores program instructions that, when executed, implement the method as described in any one of claims 1 to 6.

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