Single-detector single-scanning phase multiplexing multicolor imaging method and device

Abstract

The invention belongs to the technical field of multicolor imaging, and particularly discloses a single-detector single-scanning phase multiplexing multicolor imaging method and device. The method comprises the following steps: projecting a linear light spot to the surface of a to-be-imaged fluorescent sample by using a laser; the method comprises the following steps: detecting the surface of a to-be-imaged fluorescence sample by using a detector in a strip scanning manner to obtain a multi-layer fluorescence signal, determining a change period based on the single exposure time of the detector in the strip scanning process, and adjusting the intensity of a laser to change in a sinusoidal manner according to the change period to obtain the multi-layer fluorescence signal. Applying time delay of a preset wavelength to the intensity change waveform of the laser, and generating a phase with a wavelength identifier; and processing the multi-layer fluorescence signal to obtain a multi-color imaging result. According to the method, the time consumption of multicolor imaging can be greatly reduced.

Description

Technical Field

[0001] The present application belongs to the technical field of multi-color imaging, and more specifically, to a single-detector single-scan phase multiplexing multi-color imaging method and device. Background Art

[0002] Multicolor fluorescence microscopy analyzes the distribution of different substances by acquiring the spectral information of each sampling point in the sample. Existing technologies usually use multiple scanning methods to achieve multicolor imaging, including the following two categories: 1) Snapshot imaging: During a single exposure time, two-dimensional images at different wavelengths are simultaneously acquired through multiple array detectors; 2) Rotational imaging: Select the filter group according to the color channel, switch to another color channel after scanning one color, and perform imaging in sequence until all colors have been traversed.

[0003] Although both methods have their own advantages, they also have significant shortcomings: 1) Snapshot imaging: When the imaging range exceeds the size of a single field of view and mosaic scanning imaging is required, switching between different mosaic imaging, movement and stabilization of the sample requires a certain amount of time, and the overall imaging efficiency is reduced; 2) Alternate imaging: Different colors require switching different filter sets, and the imaging time increases exponentially with the increase in the number of color channels. Summary of the invention

[0004] In view of the defects of the prior art, the purpose of the present application is to provide a single-detector single-scan phase-multiplexed multi-color imaging method and device, aiming to solve the problems of the prior art in which the high-quality multi-color imaging range is limited by the imaging flux and the imaging efficiency is low due to multiple scanning.

[0005] To achieve the above objectives, in a first aspect, the present application provides a single-detector single-scan phase multiplexing multi-color imaging method, comprising: A line spot is projected onto the surface of the fluorescent sample to be imaged using a laser; Using a detector to detect the surface of the fluorescent sample to be imaged in a strip scanning manner to obtain a multi-layer fluorescence signal, and in the strip scanning process, determining a variation period based on a single exposure time of the detector, adjusting the intensity of the laser to change sinusoidally with the variation period, applying a time delay of a preset wavelength to the intensity variation waveform of the laser, and generating a phase with a wavelength mark; The multi-layer fluorescence signals are processed to obtain multi-color imaging results.

[0006] The present application uses a detector to perform a single scan on the surface of the fluorescent sample to be imaged in a strip scanning manner, and adjusts the intensity of the laser to change sinusoidally based on the single exposure time of the detector during scanning. A phase delay is applied to the laser based on the wavelength of the laser, and time and space are modulated simultaneously when the sample is scanned. Thus, the data required for multi-color imaging based on phase multiplexing can be obtained with a single scan, the imaging throughput of the system is improved, and the time consumption of the multi-color imaging method is reduced. In addition, the operation is performed on the original image of the online confocal, retaining the optical tomography capability, and can be combined with the line scanning method with the highest scanning flux, which is very suitable for large-scale sample imaging.

[0007] According to the single-detector single-scan phase multiplexing multi-color imaging method provided in the present application, the detector includes multiple sub-detectors. During the process of using the detector to detect the surface of the fluorescent sample to be imaged in a strip scanning manner, the multiple sub-detectors in the detector record the fluorescence signals of different sample points under the same light intensity modulation at the same time, and finally record the fluorescence signals of different sample points under different light intensity modulation.

[0008] The present application uses multiple sub-detectors within the illumination range to simultaneously record the signal of the sample, so that a single camera can obtain all the data of the demodulated monochrome image in a single scan without additional pauses and time-consuming multiple scans.

[0009] According to the single-detector single-scan phase multiplexing multicolor imaging method provided in the present application, the multi-layer fluorescence signal is processed to obtain a multicolor imaging result, including: Processing the multi-layer fluorescence signals to obtain a mixed intensity map of all fluorescence channels and a phase map that can be used to distinguish the fluorescence channels; Based on the intensity map and the phase map, the fluorescence channel is split to obtain a multi-color imaging result.

[0010] According to the single-detector single-scan phase multiplexing multicolor imaging method provided by the present application, the multi-layer fluorescence signal is processed to obtain an intensity map of all fluorescence channels mixed and a phase map that can be used to distinguish the fluorescence channels, including: The multi-layer fluorescence signals are rearranged according to the time period law to generate four images taken in one cycle; Based on the four images, a four-step phase shift method is used to solve the intensity map of the mixture of all fluorescent channels and the phase map that can be used to distinguish the fluorescent channels.

[0011] According to the single-detector single-scan phase multiplexing multicolor imaging method provided in the present application, the fluorescence channel is split based on the intensity map and the phase map to obtain a multicolor imaging result, including: Binarizing the intensity map, multiplying the obtained mask by the phase map to obtain an aliased intensity map; splitting the multiplexed channels according to the phase delay applied to the laser and the phase diagram; The split phase map and the aliasing intensity map are intersected to obtain the multi-color imaging result.

[0012] According to the single-detector single-scan phase multiplexing multi-color imaging method provided in the present application, the detector includes four detectors working in a linear array mode, and the detector is used to detect the surface of the fluorescent sample to be imaged in a strip scanning manner, including: A detector is used to detect the fluorescent signal of the area illuminated by the line light spot on the surface of the fluorescent sample to be imaged in a direction parallel to the line light spot.

[0013] In a second aspect, the present application provides a single-detector single-scan phase multiplexing multi-color imaging device, comprising: A projection module, used for projecting a line spot onto the surface of a fluorescent sample to be imaged using a laser; A detection module, used to detect the surface of the fluorescent sample to be imaged in a strip scanning manner using a detector to obtain multi-layer fluorescence signals, and during the strip scanning process, determine the variation period based on the single exposure time of the detector, adjust the intensity of the laser to change sinusoidally with the variation period, apply a time delay of a preset wavelength to the intensity variation waveform of the laser, and generate a phase with a wavelength mark; The imaging module is used to process the multi-layer fluorescence signals to obtain multi-color imaging results.

[0014] In a third aspect, the present application provides an electronic device comprising: at least one memory for storing programs; and at least one processor for executing the programs stored in the memory. When the programs stored in the memory are executed, the processor is used to execute the single-detector single-scan phase multiplexing multi-color imaging method described in the first aspect or any possible implementation of the first aspect.

[0015] In a fourth aspect, the present application provides a computer-readable storage medium, which stores a computer program. When the computer program runs on a processor, the processor executes the single-detector single-scan phase multiplexing multi-color imaging method described in the first aspect or any possible implementation of the first aspect.

[0016] In a fifth aspect, the present application provides a computer program product, which, when executed on a processor, enables the processor to execute the single-detector single-scan phase multiplexing multi-color imaging method described in the first aspect or any possible implementation of the first aspect.

[0017] It can be understood that the beneficial effects of the second to fifth aspects mentioned above can be found in the relevant description of the first aspect mentioned above, and will not be repeated here.

[0018] In general, the above technical solutions conceived by this application have the following beneficial effects compared with the prior art: The detector is used to perform a single scan of the surface of the fluorescent sample to be imaged in a strip scanning manner, and the intensity of the laser is adjusted to change sinusoidally with a period of four times the single exposure time of the detector during scanning. A phase delay is applied to the laser based on the wavelength of the laser, and time and space are modulated simultaneously when the sample is scanned. In this way, the data required for multi-color imaging based on phase multiplexing can be obtained with a single scan, which improves the imaging throughput of the system and reduces the time consumption of the multi-color imaging method. In addition, the operation is performed on the original image of the online confocal, retaining the optical tomography capability, and can be combined with the line scanning method with the highest scanning throughput, which is very suitable for imaging of large-scale samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present application or the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the prior art description. Obviously, the drawings described below are some embodiments of the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 It is a schematic flow chart of a single-detector single-scan phase multiplexing multi-color imaging method provided in an embodiment of the present application; Figure 2 This is a system optical path diagram of a single-scan phase multiplexing multi-color imaging method provided in an embodiment of the present application; Figure 3 It is a schematic diagram of the principle of laser modulation provided in an embodiment of the present application; Figure 4 This is a schematic diagram of acquiring multiple layers of fluorescence signals in a single scan provided in an embodiment of the present application; Figure 5 is a schematic diagram of rearranging multiple layers of fluorescent signals provided in an embodiment of the present application; Figure 6 It is a phase multiplexing multi-color imaging unmixing flow chart provided in an embodiment of the present application; Figure 7 It is a schematic diagram of the structure of a single-detector single-scan phase multiplexing multi-color imaging device provided in an embodiment of the present application; Figure 8 It is a schematic diagram of the structure of an electronic device provided in an embodiment of the present application.

[0021] Throughout the drawings, the same reference numerals are used to denote the same elements or structures, wherein: 1 is the laser of channel 1; 2 is the laser of channel 2; 3 is the dichroic mirror; 4 is the reflector; 5 is the beam expander 1; 6 is the beam expander 2; 7 is the cylindrical lens; 8 is the illumination tube lens; 9 is the dichroic mirror; 10 is the objective lens; 11 is the sample; 12 is the electric translation stage; 13 is the detection tube lens; 14 is the emission filter; 15 is the detector. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0023] The term "and / or" in this article is a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. The symbol " / " in this article indicates that the associated objects are in an or relationship, for example, A / B means A or B.

[0024] In the embodiments of the present application, words such as "exemplary" or "for example" are used to indicate examples, illustrations or descriptions. Any embodiment or design described as "exemplary" or "for example" in the embodiments of the present application should not be interpreted as being more preferred or more advantageous than other embodiments or designs. Specifically, the use of words such as "exemplary" or "for example" is intended to present related concepts in a specific way.

[0025] In the description of the embodiments of the present application, unless otherwise specified, "multiple" means two or more than two. For example, multiple processing units refer to two or more processing units, etc.; multiple elements refer to two or more elements, etc.

[0026] Next, combine Figure 1-Figure 6 The single-scan phase multiplexing multi-color imaging method provided in the embodiments of the present application is introduced.

[0027] Figure 1 is a flow chart of a single-scan phase multiplexing multi-color imaging method provided in an embodiment of the present application, such as Figure 1 As shown, the method comprises the following steps: Step 100, using a laser to project a line spot onto the surface of a fluorescent sample to be imaged; Optionally, a laser may be used to emit a light beam, and a cylindrical lens may be used to form a line light spot with a certain width, and the line light spot may be projected onto the surface of the fluorescent sample to be imaged through an illumination objective lens and a tube lens.

[0028] Figure 2 is a system optical path diagram of the single-scan phase multiplexing multi-color imaging method provided in an embodiment of the present application, such as Figure 2 As shown, in one embodiment of the present application, an optical imaging system is used to detect the surface of the fluorescent sample to be imaged. The outgoing light from lasers 1 and 2 is combined by a dichroic mirror 3, enters a beam expansion system composed of lenses 5 and 6, enters a cylindrical lens 7, and is then focused into a line spot, and then projected onto the sample surface by a focusing system composed of a double cemented lens 8 and an objective lens 10.

[0029] Step 110, using a detector to detect the surface of the fluorescent sample to be imaged in a strip scanning manner to obtain a multi-layer fluorescence signal, and in the strip scanning process, determining a variation period based on a single exposure time of the detector, adjusting the intensity of the laser to change sinusoidally with a variation period, applying a time delay of a preset wavelength to the intensity variation waveform of the laser, and generating a phase with a wavelength mark; Optionally, the detector may be a linear array detector or a dot array detector.

[0030] Optionally, if a linear array detector is used, the linear array detector includes 2048 sub-linear arrays, and 4 lines, 8 lines, or 16 lines can be used to detect the surface of the fluorescent sample to be imaged.

[0031] like Figure 2 As shown, the objective lens 10 and the tube lens 13 constitute an infinity correction system to image the fluorescence signal transmitted through the filter 14 onto the surface of the detector 15, so that the detector detects the fluorescence signal.

[0032] While the detector is scanning the strip, the illumination laser is modulated so that the laser intensity changes sinusoidally with the camera frequency. The laser intensity change period can be four, eight, or twelve times the camera exposure period, which plays a role in phase spectrum broadening. At the same time, a time delay of a preset wavelength is applied to the laser intensity change waveform to generate a phase with a wavelength mark to mark and distinguish the color channel. In this way, when the sample moves, the laser also changes regularly and periodically within 0-T.

[0033] In one embodiment of the present application, a period of four times the single exposure time of the detector is selected, that is, the intensity of the laser is adjusted to change sinusoidally with a period of four times the single exposure time of the detector.

[0034] In order to ensure the smooth execution of phase multiplexed multicolor imaging, precise synchronization between the components is key. This includes the synchronization between the detector photography and the laser intensity modulation, as well as the synchronization between the linear array detector photography and the movement of the translation stage during line scanning. In order to accurately control the timing of these devices, the translation stage is selected as the main control element.

[0035] Figure 3 Schematic diagram of the principle of laser modulation provided by the embodiment of the present application, such as Figure 3 As shown in the figure, when the translation stage reaches the predetermined position and starts to run stably, it will output a synchronization signal, which is used to trigger the detector to take pictures continuously. The detector will send a feedback signal at the beginning of each exposure, which is used as a synchronization signal between the camera and the NI acquisition card. The NI acquisition card outputs a voltage waveform with a specific frequency and a fixed delay based on this feedback signal, thereby realizing direct modulation of the laser intensity.

[0036] Optionally, the single-detector single-scan phase multiplexing multi-color imaging method provided in the present application can perform three-dimensional imaging, such as direct sample lifting imaging of thin slices and light-transparent samples, or cutting and lifting sample imaging of intact tissue organs. The multi-layer fluorescence signals collected by the detector can include three-dimensional data of fluorescence signals illuminated by Gaussian beam waist and three-dimensional data of fluorescence signals illuminated by a beam far from the beam waist.

[0037] Figure 4 is a schematic diagram of acquiring multiple layers of fluorescence signals by a single scan provided in an embodiment of the present application, such as Figure 4 As shown, in one embodiment of the present application, the fluorescent sample to be imaged is driven by a translation stage to obtain fluorescent signals within the entire sample range in a strip scanning manner. Specifically, first, a detector is used to detect the fluorescent signal of the area on the surface of the fluorescent sample to be imaged that is illuminated by the linear light spot, and a single-layer fluorescent signal is obtained, including spatially encoded data and color-time encoded data. Then, the fluorescent sample to be imaged is lifted by the translation stage, and the detector is used again to detect the fluorescent signal of the area on the surface of the fluorescent sample to be imaged that is illuminated by the linear light spot, and a single-layer fluorescent signal is obtained. The above steps are repeated until the fluorescent sample to be imaged is completely detected, and a multi-layer fluorescent signal can be obtained.

[0038] The above-mentioned space-time multiplexing process can obtain spatially encoded data and color time encoded data at the same time.

[0039] Step 120, processing the multi-layer fluorescence signals to obtain multi-color imaging results.

[0040] After acquiring multiple layers of fluorescence signal data in a single scan, the data is processed to obtain multi-color imaging results.

[0041] The single-scan phase-multiplexed multi-color imaging method provided by the present application uses a detector to perform a single scan on the surface of the fluorescent sample to be imaged in a strip scanning manner, and adjusts the intensity of the laser during scanning to change sinusoidally with a period of four times the single exposure time of the detector. A phase delay is applied to the laser based on the wavelength of the laser, and time and space are modulated simultaneously when the sample is scanned. Thus, the data required for multi-color imaging based on phase multiplexing can be obtained with a single scan, the imaging flux of the system is improved, and the time consumption of the multi-color imaging method is reduced. In addition, the operation is performed on the original image of the online confocal, which retains the optical tomography capability and can be combined with the line scanning method with the highest scanning flux, which is very suitable for large-scale sample imaging.

[0042] In some embodiments, the detector includes multiple sub-detectors. When using the detector to detect the surface of the fluorescent sample to be imaged in a strip scanning manner, the multiple sub-detectors in the detector record the fluorescence signals of different sample points under the same light intensity modulation at the same time, and finally record the fluorescence signals of different sample points under different light intensity modulation.

[0043] Multiple sub-detectors can be used to simultaneously record the signal of the sample within the illumination range. In this way, the signals of different sample points under the same light intensity modulation are recorded at the same time. When the sample passes through each sub-detector in turn, different sub-detectors record the signals of different sample points under different light intensity modulation in turn.

[0044] With this strategy, all the data of the demodulated monochrome image can be obtained with a single camera in a single scan, without any additional pauses or time-consuming multiple scans.

[0045] In some embodiments, step 120 specifically includes: Step 1201, processing the multi-layer fluorescence signals to obtain a mixed intensity map of all fluorescence channels and a phase map that can be used to distinguish the fluorescence channels; Step 1202: split the fluorescence channel based on the intensity map and the phase map to obtain a multi-color imaging result.

[0046] The multi-layer fluorescence signal is processed, which includes multi-color data encoded simultaneously in time and space, to obtain an intensity map of the color channel mixture and a phase map that can distinguish colors. After splitting the fluorescence channels, multi-color imaging results with improved signal-to-background ratio and imaging flux can be obtained.

[0047] Optionally, the multi-layer fluorescence signal can be processed by four-step phase shift solution, linear solution or deep learning to obtain an intensity map of the mixture of all fluorescence channels and a phase map that can be used to distinguish the fluorescence channels, which is not limited in this application.

[0048] In some embodiments, step 1201 specifically includes: Step 12011, rearrange the multi-layer fluorescence signals according to the time period rule to generate four images captured in one period; Step 12012, based on the four images, use the four-step phase shift method to solve the intensity map of all fluorescent channels mixed and the phase map that can be used to distinguish the fluorescent channels.

[0049] The optical imaging system imposes a specific phase delay φ on different lasers. 1 -φ 4 , and for a certain luminous point in space, the point may be excited by one or more of these lasers. The emitted fluorescence has φ 0 The phase of the light intensity changes in accordance with the following law:

[0050] in, , is the fluorescence intensity distribution, is the DC component, is the focal plane information, , is the sample surface coordinate, is the modulation frequency, For time.

[0051] In order to solve this phase related to the excitation light The phase of the multi-layer fluorescence signal is rearranged according to the time period law to generate four images taken in one cycle, which can be expressed as:

[0052] in, , , , are four images taken in one cycle, is the modulation period.

[0053] The four-step phase shift method is further used to solve an intensity map of all fluorescent channels mixed and a phase map that can be used to distinguish fluorescent channels, which can be expressed as:

[0054] Figure 5 is a schematic diagram of rearranging multi-layer fluorescence signals provided in an embodiment of the present application, such as Figure 5 As shown, the four-step phase shift method is used to solve the intensity aliasing images of all fluorescence channels and the phase images that can distinguish the fluorescence channels.

[0055] Find the phase After the phase, we can Unscramble the fluorescence channels.

[0056] In particular, the present application is robust to excitation light crosstalk and multi-color samples with spatial overlap, and can distinguish excitation light crosstalk by phase peak shift and use linear unmixing in the phase image to unmix spatial co-localization information.

[0057] In some embodiments, step 1202 specifically includes: Step 12021, binarize the intensity map, multiply the obtained mask with the phase map, and obtain an aliased intensity map; Step 12022, splitting the multiplexed channel according to the phase delay and phase diagram applied to the laser; Step 12023, taking the intersection of the split phase map and the aliasing intensity map to obtain a multi-color imaging result.

[0058] Figure 6 is a phase multiplexing multi-color imaging unmixing flow chart provided in an embodiment of the present application, such as Figure 6 As shown in the figure, when splitting different channels, the aliasing intensity map is first binarized, and the obtained mask is multiplied with the phase image to reduce the influence of the background phase on the channel splitting. Then, the multiplexed channel is split according to the phase delay and phase map applied to the laser. Then, the split phase map and the aliasing intensity map are intersected to obtain the final single channel intensity map, realizing phase multiplexing multi-color imaging.

[0059] The single-detector single-scan phase multiplexing multicolor imaging method provided in this application can greatly reduce the time consumed by multiple scans of the phase multiplexing multicolor imaging method, and the time consumed by the multicolor imaging method of changing filters for scanning is reduced exponentially. In addition, this method operates on the original image of the online confocal, retains the optical tomography capability, and can be combined with the line scanning method with the highest scanning flux, which is very suitable for large-scale sample imaging.

[0060] The single-detector single-scan phase multiplexing multicolor imaging method provided in the present application can effectively distinguish fluorescent dyes with excitation light crosstalk. At the same time, when there are multiple substances emitting light in a space, the phase linear mixing ratio law can be used to separate the intensities of fluorescence of different colors.

[0061] In some embodiments, the detector includes four detectors operating in a linear array mode, and step 120 specifically includes: A detector is used to detect the fluorescent signal of the area illuminated by the line light spot on the surface of the fluorescent sample to be imaged in a direction parallel to the line light spot.

[0062] The present application uses a linear array detector comprising four working in a linear array mode, and the single-layer fluorescence signal detected by the linear array detector comprises the original data of multi-line linear array detection: the fluorescence sample signal detected by the linear array detector 1, the fluorescence sample signal detected by the linear array detector 2, the fluorescence sample signal detected by the linear array detector 3 and the fluorescence sample signal detected by the linear array detector 4.

[0063] The detector works in the linear array mode, detecting the fluorescence signal of the area illuminated by the linear light spot parallel to the direction of the linear light spot.

[0064] Figure 7 is a schematic diagram of the structure of a single-detector single-scan phase multiplexing multi-color imaging device provided in an embodiment of the present application, such as Figure 7 As shown, the device includes a projection module 710, a detection module 720 and an imaging module 730, wherein: A projection module 710 is used to project a line spot onto the surface of the fluorescent sample to be imaged using a laser; The detection module 720 is used to detect the surface of the fluorescent sample to be imaged in a strip scanning manner using a detector to obtain multi-layer fluorescence signals, and in the strip scanning process, determine the variation period based on the single exposure time of the detector, adjust the intensity of the laser to change sinusoidally with the variation period, apply a time delay of a preset wavelength to the intensity variation waveform of the laser, and generate a phase with a wavelength mark; The imaging module 730 is used to process the multi-layer fluorescence signals to obtain multi-color imaging results.

[0065] It should be understood that the above-mentioned device is used to execute the method in the above-mentioned embodiment. The implementation principle and technical effect of the corresponding program module in the device are similar to those described in the above-mentioned method. The working process of the device can refer to the corresponding process in the above-mentioned method, which will not be repeated here.

[0066] Based on the method in the above embodiment, Figure 8 An example of a physical structure diagram of an electronic device is shown in FIG. Figure 8 As shown, an embodiment of the present application provides an electronic device, which may include: a processor (processor) 810, a communication interface (Communications Interface) 820, a memory (memory) 830 and a communication bus 840. The processor 810, the communication interface 820, and the memory 830 communicate with each other through the communication bus 840. The processor 810 can call the logic instructions in the memory 830 to execute the single-detector single-scan phase multiplexing multi-color imaging method in the above embodiment.

[0067] In addition, the logic instructions in the above-mentioned memory 830 can be implemented in the form of a software functional unit and can be stored in a computer-readable storage medium when it is sold or used as an independent product. Based on this understanding, the technical solution of the present application is essentially or the part that contributes to the prior art or the part of the technical solution can be embodied in the form of a software product. The computer software product is stored in a storage medium, including a number of instructions for enabling a computer device (which can be a personal computer, a server, or a network device, etc.) to perform all or part of the steps of the single-detector single-scan phase multiplexing multi-color imaging method described in each embodiment of the present application.

[0068] Based on the method in the above embodiment, an embodiment of the present application provides a computer-readable storage medium, which stores a computer program. When the computer program runs on a processor, the processor executes the single-detector single-scan phase multiplexing multi-color imaging method in the above embodiment.

[0069] Based on the method in the above embodiment, an embodiment of the present application provides a computer program product. When the computer program product runs on a processor, the processor executes the single-scan phase multiplexing multi-color imaging method in the above embodiment.

[0070] It is understandable that the processor in the embodiment of the present application may be a central processing unit (CPU), or other general-purpose processors, digital signal processors (DSP), application specific integrated circuits (ASIC), field programmable gate arrays (FPGA) or other programmable logic devices, transistor logic devices, hardware components or any combination thereof. The general-purpose processor may be a microprocessor or any conventional processor.

[0071] The method steps in the embodiments of the present application can be implemented by hardware or by a processor executing software instructions. The software instructions can be composed of corresponding software modules, and the software modules can be stored in random access memory (RAM), flash memory, read-only memory (ROM), programmable read-only memory (PROM), erasable programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), registers, hard disks, mobile hard disks, CD-ROMs, or any other form of storage medium known in the art. An exemplary storage medium is coupled to the processor so that the processor can read information from the storage medium and write information to the storage medium. Of course, the storage medium can also be a component of the processor. The processor and the storage medium can be located in an ASIC.

[0072] In the above embodiments, it can be implemented in whole or in part by software, hardware, firmware or any combination thereof. When implemented by software, it can be implemented in whole or in part in the form of a computer program product. The computer program product includes one or more computer instructions. When the computer program instructions are loaded and executed on a computer, the process or function described in the embodiment of the present application is generated in whole or in part. The computer may be a general-purpose computer, a special-purpose computer, a computer network, or other programmable device. The computer instructions may be stored in a computer-readable storage medium or transmitted through the computer-readable storage medium. The computer instructions may be transmitted from a website site, computer, server or data center to another website site, computer, server or data center by wired (e.g., coaxial cable, optical fiber, digital subscriber line (DSL)) or wireless (e.g., infrared, wireless, microwave, etc.). The computer-readable storage medium may be any available medium that a computer can access or a data storage device such as a server or data center that includes one or more available media integrated. The available medium may be a magnetic medium (e.g., a floppy disk, a hard disk, a tape), an optical medium (e.g., a DVD), or a semiconductor medium (e.g., a solid state disk (SSD)), etc.

[0073] It should be understood that the various numerical numbers involved in the embodiments of the present application are only used for the convenience of description and are not used to limit the scope of the embodiments of the present application.

[0074] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A single-detector single-scan phase multiplexing multi-color imaging method, characterized in that: include: A line spot is projected onto the surface of the fluorescent sample to be imaged using a laser; Using a detector to detect the surface of the fluorescent sample to be imaged in a strip scanning manner to obtain a multi-layer fluorescence signal, and in the strip scanning process, determining a variation period based on a single exposure time of the detector, adjusting the intensity of the laser to change sinusoidally with the variation period, applying a time delay of a preset wavelength to the intensity variation waveform of the laser, and generating a phase with a wavelength mark; The multi-layer fluorescence signals are processed to obtain multi-color imaging results.

2. The single-detector single-scan phase multiplexing multi-color imaging method according to claim 1, characterized in that: The detector includes multiple sub-detectors. When using the detector to detect the surface of the fluorescent sample to be imaged in a strip scanning manner, the multiple sub-detectors in the detector record the fluorescence signals of different sample points under the same light intensity modulation at the same time, and finally record the fluorescence signals of different sample points under different light intensity modulation.

3. The single-detector single-scan phase multiplexing multi-color imaging method according to claim 1, characterized in that: The processing of the multi-layer fluorescence signals to obtain a multi-color imaging result includes: Processing the multi-layer fluorescence signals to obtain a mixed intensity map of all fluorescence channels and a phase map that can be used to distinguish the fluorescence channels; Based on the intensity map and the phase map, the fluorescence channel is split to obtain a multi-color imaging result.

4. The single-detector single-scan phase multiplexing multi-color imaging method according to claim 3, characterized in that: The processing of the multi-layer fluorescence signals to obtain a mixed intensity map of all fluorescence channels and a phase map that can be used to distinguish the fluorescence channels includes: The multi-layer fluorescence signals are rearranged according to the time period law to generate four images taken in one cycle; Based on the four images, a four-step phase shift method is used to solve the intensity map of the mixture of all fluorescent channels and the phase map that can be used to distinguish fluorescent channels.

5. The single-detector single-scan phase multiplexing multi-color imaging method according to claim 4, characterized in that: The step of splitting the fluorescence channel based on the intensity map and the phase map to obtain a multicolor imaging result includes: Binarizing the intensity map, multiplying the obtained mask by the phase map to obtain an aliased intensity map; splitting the multiplexed channels according to the phase delay applied to the laser and the phase diagram; The split phase map and the aliasing intensity map are intersected to obtain the multi-color imaging result.

6. The single-detector single-scan phase multiplexing multi-color imaging method according to claim 1, characterized in that: The detector includes four detectors working in a linear array mode, and the detector is used to detect the surface of the fluorescent sample to be imaged in a strip scanning manner, including: A detector is used to detect the fluorescent signal of the area illuminated by the line light spot on the surface of the fluorescent sample to be imaged in a direction parallel to the line light spot.

7. A single-detector single-scan phase multiplexing multi-color imaging device, characterized in that: include: A projection module, used for projecting a line spot onto the surface of a fluorescent sample to be imaged using a laser; A detection module, used to detect the surface of the fluorescent sample to be imaged in a strip scanning manner using a detector to obtain multi-layer fluorescence signals, and during the strip scanning process, determine the variation period based on the single exposure time of the detector, adjust the intensity of the laser to change sinusoidally with the variation period, apply a time delay of a preset wavelength to the intensity variation waveform of the laser, and generate a phase with a wavelength mark; The imaging module is used to process the multi-layer fluorescence signals to obtain multi-color imaging results.

8. An electronic device, characterized in that: include: at least one memory for storing a computer program; At least one processor is used to execute the program stored in the memory. When the program stored in the memory is executed, the processor is used to execute the single-detector single-scan phase multiplexing multi-color imaging method as described in any one of claims 1-6.

9. A computer-readable storage medium storing a computer program, characterized in that: When the computer program runs on a processor, the processor is enabled to execute the single-detector single-scan phase multiplexing multi-color imaging method according to any one of claims 1 to 6.

10. A computer program product, characterized in that When the computer program product runs on a processor, the processor is enabled to execute the single-detector single-scan phase multiplexing multi-color imaging method according to any one of claims 1 to 6.

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