CN-121994768-A - Multichannel fluorescence imaging system based on time sequence pulse excitation and asynchronous acquisition

Abstract

The invention discloses a multichannel fluorescence imaging system based on time sequence pulse excitation and asynchronous acquisition, in particular to the technical field of biomedical engineering and optical microscopy imaging, which comprises an inverted microscope main body, a multi-wavelength laser excitation module, a high-speed Scmos imaging device supporting hardware triggering, a lower computer time sequence control unit based on a microcontroller and an upper computer computing processing platform, wherein a common light path architecture and a static multi-band emission filter array are adopted, a lower computer generates a synchronous control signal with microsecond precision by utilizing a hardware timer, laser opening of different wavelengths is controlled by adopting a time division multiplexing strategy, and a camera is forced to perform global shutter synchronous exposure by matching with hardware handshake logic. The upper computer builds a memory asynchronous buffer architecture, decouples the high-bandwidth image acquisition and the low-speed hard disk storage process by using a producer-consumer model, and temporarily stores the original data by using a memory buffer pool.

Inventors

• ZHU XIAOLU
• XING JIALU

Assignees

• 河海大学

Dates

Publication Date

20260508

Application Date

20260410

Claims (6)

1. 1. The multichannel fluorescence imaging system based on time sequence pulse excitation and asynchronous acquisition is characterized by comprising the following components: the inverted microscope body is used for bearing a target sample and providing an optical imaging light path; The multi-wavelength laser excitation module is used for providing excitation lights with different wavelengths; the high-speed imaging device comprises a Scmos camera supporting hardware triggering and a global shutter mode, wherein the front end of the high-speed imaging device is provided with a static multiband emission filter array; The multi-wavelength laser excitation module outputs multiple paths of excitation lights with different wavelengths, and forms a coaxial light path after beam combination treatment, and the coaxial light path is incident to the inverted microscope main body to excite a target sample with multiple wavelengths; the high-speed imaging device adopts a Scmos camera to trigger synchronously with a laser excitation time sequence, and the global shutter performs synchronous exposure to complete high-speed and low-distortion imaging acquisition; The lower computer time sequence control unit is used for configuring a time division multiplexing strategy based on a micro controller MCU, controlling the multi-wavelength laser excitation module and the high-speed imaging device by concurrently outputting hardware pulse signals with microsecond precision, isolating excitation lights with different wavelengths on a physical time axis, realizing time-sharing starting of the excitation lights with different wavelengths on a millisecond time period, dividing an imaging period into time windows which are not overlapped with each other, and forcibly executing photosensitive reset by sending a global trigger level to an optocoupler isolation input end of the high-speed imaging device so as to realize alignment of a physical time sequence of laser excitation and a camera global exposure window; the upper computer computing processing platform is used for system configuration, data flow asynchronous processing and image storage.
2. 2. The multichannel fluorescence imaging system based on time series pulse excitation and asynchronous acquisition of claim 1, wherein the time division multiplexing strategy is completed through two sets of interrupt service logic and hardware handshake logic which are matched with each other.
3. 3. The multi-channel fluorescence imaging system based on time sequence pulse excitation and asynchronous acquisition according to claim 2, wherein hardware handshake links are built in the MCU for configuring the first timer control parameters, the time sequence constraint conditions, the second timer control parameters and the MCU input/output interface GPIO corresponding to each laser control channel and the photoelectric isolation circuit, and the hardware handshake links are connected to the photoelectric isolation input end of the high-speed imaging device.
4. 4. The multi-channel fluorescence imaging system based on sequential pulse excitation and asynchronous acquisition of claim 3, wherein a dual-timer cooperative control model based on a discrete time state machine is built in the MCU to realize interrupt service logic, specifically comprising the following steps: Setting the clock frequency of the MCU system as The timer prescaled coefficient is PSC, then the minimum time granularity of the hardware counter The method meets the following conditions: ; The MCU is internally provided with maintenance laser channel index variables k, k epsilon {0, 1, &..the MCU is provided with M-1}, and M is the total number of laser channels; the control model operation logic is as follows: (1) Excited State state_specification: Starting a first timer TIM_ON and setting its auto reload register value to At this time, the single channel is excited for a long time Defined by the following formula: ; MCU is based on the current index variable k from the preset light intensity mapping table Reads the corresponding pulse width modulation value The compare register CCR k is written such that the output duty cycle D k at the corresponding GPIO port satisfies: ; (2) Dead zone switch state_ DeadTime: When the first timer overflows to trigger the first interrupt, the MCU immediately closes the output of the first timer and starts the second timer TIM_OFF with reloading value of Dead zone interval duration The method meets the following conditions: ; (3) And the circulating polling logic is used for executing a channel index updating algorithm by the MCU when the second timer counts overflow to trigger second interrupt: ; Wherein mod is a modulo operation, The total number of the laser channels is the total number of the laser channels, the MCU updates CCR parameters of the next period according to k next , and the first timer is restarted so as to realize hardware closed-loop control without software intervention.
5. 5. The multi-channel fluorescence imaging system based on time sequence pulse excitation and asynchronous acquisition of claim 3, wherein the lower computer time sequence control unit is configured with hardware handshake logic constructed based on a general purpose input output interface GPIO and a photoelectric isolation circuit, and the GPIO pin of the MCU is connected to an external trigger input end of the imaging device to execute the following microsecond time sequence actions: (1) Triggering response, namely generating TTL synchronous trigger signals, namely pulse width, by the MCU at the initial moment of a single imaging period The effective edge of the signal directly drives an imaging device supporting a global shutter mode, and all pixel points of a sensor array are controlled to start photoelectric integration at the same time to form a common exposure window with an absolute time reference; (2) MCU outputs laser PWM modulation signal synchronously, namely effective luminous pulse width The system satisfies the timing envelope constraints to ensure the full energy of the laser excitation Global exposure window fully enveloped in camera [ , In the method, the lossless integration of photon energy is realized, and the time gating characteristic of the global shutter is utilized to effectively inhibit the ambient stray light noise outside the exposure window, wherein, It is the exposure start time that is set to be, Is the exposure end time; the timing envelope constraint inequality is as follows: ; Wherein, the For the inherent turn-on delay of the laser driver, Is a system timing safety margin.
6. 6. The system of claim 1, wherein the host computing platform is configured with an underlying memory asynchronous buffer architecture based on a producer-consumer concurrency model for decoupling image acquisition from hard disk writing, and the execution logic is as follows: (1) Physical memory pre-allocation and addressing topology: defining a single frame image pixel width as The height is Single pixel byte depth of Single frame image data amount ; The upper computer opens up a continuous memory space in the system physical memory in advance to be used as a ring data buffer area, and sets the total data block node quantity as The total memory size is pre-allocated Defining the memory block head address as ; System initialization and maintenance of independent and atomic write pointers And a read pointer The initial values are all set to 0; (2) Zero copy polar enqueue based on pointer offset: when the i-th frame of original image bare data triggers a data transmission interrupt, the producer thread performs the following state check and absolute address addressing: Firstly judging the idle node state, if the idle node state is satisfied Judging that the buffer area has a margin, and further calculating the memory physical absolute target address of the current node: ; Then, the translation instruction of the bottom memory block of the system is called, and the length is as follows From the sensor end source head address Original copy to To avoid heap object allocation in high-level language, after copying, the producer obtains high-precision system time stamp Team tuple containing pointer and time parameter ) Push into non-blocking queue Q and update write pointer: ; (3) Matrix mapping unmixing and pointer dequeuing asynchronous consumption: independently running consumer threads monitor the non-blocking queue Q and extract the queue tuple ) And synchronously update the read pointer To free the corresponding memory node, consumer based fetch Pointer direct access to original image matrix And sequentially performs: a) Color space conversion, performing matrix mapping using interpolation algorithm Reconstructing the Bayer array into an RGB matrix; b) Spectral unmixing, namely constructing a linear mixed mathematical model based on priori spectral observation Wherein Y is the current observation signal matrix A is a known end member spectrum matrix extracted by system calibration and comprises an autofluorescence background spectrum and a target probe spectrum, E is a system noise item, a consumer background solves a characteristic abundance matrix C through a non-negative matrix factorization algorithm, and a spontaneous background interference signal is stripped to reconstruct a pure multichannel target image ; C) Time sequence encapsulation and drop-out, time stamp of dequeue Encoding the target image into an image file header identifier, and unmixed target image And executing the asynchronous I/O writing of the hard disk according to a preset compression algorithm.

Description

Multichannel fluorescence imaging system based on time sequence pulse excitation and asynchronous acquisition Technical Field The invention relates to the technical field of biomedical engineering and optical microscopy imaging, in particular to a multichannel fluorescence imaging system based on time sequence pulse excitation and asynchronous acquisition. Background Fluorescence microscopy is a central tool in life science research to observe cell microstructure (e.g. mitochondria, lysosomes) and dynamic physiological processes (e.g. neurite transfer, cytoautophagy). To resolve complex biological processes, researchers often need to label different biomolecules simultaneously using multiple different colored fluorescent probes, i.e., polychromatic/multichannel imaging. The existing multichannel fluorescence imaging technology mainly faces the following technical bottlenecks: Spectral crosstalk (Spectral Crosstalk) because of the broadband nature of the emission spectrum of fluorophores, the emitted light from short wavelength channels tends to "leak" into long wavelength channels (e.g., GFP signal crosstalk into YFP channels), resulting in reduced imaging signal-to-noise ratios, severely affecting the accuracy of co-localization analysis. The time resolution is insufficient, the traditional wide-field microscope relies on a mechanical filter wheel driven by a stepping motor to switch channels, the switching time is usually between tens of milliseconds and hundreds of milliseconds, sometimes even seconds, and with physical vibration, rapid biological dynamics in millisecond level are difficult to capture. Data throughput bottleneck-with the increasing number of pixels of scientific grade CMOS (cameras), the data stream at full resolution is extremely large (e.g., 1200 kilopixels @100fps produces about 1.2GB/s of data). The traditional upper computer software adopts a synchronous acquisition and direct disk drop architecture, is limited by the writing speed of a SATA or USB interface, and is extremely easy to cause data congestion and frame dropping. While acousto-optic tunable filters (AOTF) solve part of the problem, their expensive cost limits popularity in the general laboratory. Thus, there is a need for a low cost, high performance solution based on general purpose hardware. Disclosure of Invention Therefore, the invention provides a multichannel fluorescence imaging system based on sequential pulse excitation and asynchronous acquisition, which utilizes an embedded microcontroller to realize microsecond photoelectric synchronization and combines an upper computer memory buffer technology to realize high-speed, low-crosstalk and multichannel fluorescence imaging so as to solve the problems in the background technology. The technical scheme is that the multichannel fluorescence imaging system based on time sequence pulse excitation and asynchronous acquisition comprises: the inverted microscope body is used for bearing a target sample and providing an optical imaging light path; The multi-wavelength laser excitation module is used for providing excitation lights with different wavelengths; the high-speed imaging device comprises a Scmos camera supporting hardware triggering and a global shutter mode, wherein the front end of the high-speed imaging device is provided with a static multiband emission filter array; The multi-wavelength laser excitation module outputs multiple paths of excitation lights with different wavelengths, and forms a coaxial light path after beam combination treatment, and the coaxial light path is incident to the inverted microscope main body to excite a target sample with multiple wavelengths; the high-speed imaging device adopts a Scmos camera to trigger synchronously with a laser excitation time sequence, and the global shutter performs synchronous exposure to complete high-speed and low-distortion imaging acquisition; The lower computer time sequence control unit is used for configuring a time division multiplexing strategy based on a micro controller MCU, controlling the multi-wavelength laser excitation module and the high-speed imaging device by concurrently outputting hardware pulse signals with microsecond precision, isolating excitation lights with different wavelengths on a physical time axis, realizing time-sharing starting of the excitation lights with different wavelengths on a millisecond time period, dividing an imaging period into time windows which are not overlapped with each other, and forcibly executing photosensitive reset by sending a global trigger level to an optocoupler isolation input end of the high-speed imaging device so as to realize alignment of a physical time sequence of laser excitation and a camera global exposure window; the upper computer computing processing platform is used for system configuration, data flow asynchronous processing and image storage. Preferably, the time division multiplexing strategy is completed through two sets of interr