A microscopic imaging system

Abstract

The present invention relates to the field of optical technology, and discloses a microscopic imaging system. The system includes: a light source module for generating a laser beam; a radio frequency drive generation module for generating radio frequency drive signals of multiple frequencies; a heterodyne interference optical path module for performing optical heterodyne interference processing on the laser beam according to the radio frequency drive signals of multiple frequencies, forming a light spot array and a reference light source respectively, and then combining the light spot array and the reference light source to generate a combined light beam incident on the sample to be tested; wherein different light spots in the light spot array correspond to different light frequencies; a spectral data receiving module for collecting light data generated after the sample to be tested reflects the combined light beam; wherein the sample to be tested is fluorescently labeled in advance; an image inversion module for receiving light data, and performing image inversion processing on the light data to generate imaging data of the sample to be tested. The above scheme improves the response speed of microscopic imaging, and has high efficiency and good accuracy.

Description

Technical Field

[0001] The present invention relates to the field of optical technology, and in particular to a microscopic imaging system. Background Art

[0002] Fluorescence Lifetime Imaging Microscopy (FLIM) is an imaging technique that uses the time that a fluorescent molecule remains in an excited state before emitting a photon (i.e., fluorescence lifetime) to generate images. Combining the spatial resolution of fluorescence lifetime microscopy with flow cytometry cell sorting has broad significance.

[0003] In the related art, when performing fluorescence lifetime microscopy imaging, cyclic scanning and addressing scanning are mostly adopted. An acousto-optic deflector or a mechanical galvanometer is used to apply a deflection angle to the laser beam each time, so that the laser images a point on the sample each time, and multiple scans are used to realize the acquisition of a frame of image. In addition to the acousto-optic deflector and the galvanometer, a clock synchronization counter is also required to count and synchronize with the light spot. Assuming that a frame of image consists of N pixels, it is necessary to scan at least N times to realize the scanning of the entire image. The frame rate increases with the increase in the number of image pixels, resulting in a limited frame rate and slow response speed, which is difficult to apply to high-throughput flow cytometry cell sorting.

[0004] Therefore, a microscopic imaging solution with faster response speed is urgently needed. Summary of the Invention

[0005] In view of this, an object of the present invention is to provide a microscopic imaging system to solve the problem of slow microscopic imaging response speed.

[0006] In a first aspect, the present invention provides a microscopic imaging system, comprising:

[0007] A light source module, used for generating a laser beam;

[0008] RF drive generation module, used to generate RF drive signals of multiple frequencies;

[0009] A heterodyne interference optical path module is used to perform optical heterodyne interference processing on the laser beams according to the radio frequency drive signals of the multiple frequencies, respectively forming a light spot array and a reference light source, and then combining the light spots to generate a combined light beam incident on the sample to be measured; wherein different light spots in the light spot array correspond to different light frequencies;

[0010] a spectral data receiving module, configured to collect light data generated by the sample to be tested after the sample to be tested reflects the combined light beam; wherein the sample to be tested is fluorescently labeled in advance;

[0011] The image inversion module is used to receive the light data and perform image inversion processing on the light data to generate imaging data of the sample to be tested.

[0012] In an optional embodiment, the heterodyne interference optical path module includes a beam splitter, a measuring arm and a reference arm; the beam splitter is used to split the laser beam into a first laser beam and a second laser beam; the measuring arm is used to optically modulate the first laser beam according to the RF drive signal of multiple frequencies to generate a light spot array; the reference arm is used to shape the second laser beam into a reference light source; wherein the reference light source covers the light spot array.

[0013] In an optional embodiment, the measuring arm includes a spatial light modulator, which is used to introduce a frequency offset into the first laser beam based on the frequency and amplitude of the corresponding radio frequency driving signal to generate a light spot array;

[0014] The reference arm includes a beam shaping device, which is used to shape the second laser beam into a reference light source.

[0015] In an optional embodiment, the system further includes a host computer; the host computer is used to send a signal adjustment instruction to the RF drive generation module, so that the RF drive generation module adjusts the intensity of the RF drive signal according to the signal adjustment instruction;

[0016] The host computer also receives the imaging data of the sample to be tested and displays it.

[0017] In an optional embodiment, the system further includes a light path beam reduction module, which is used to scale the combined light beam to a target size and then inject the combined light beam onto the sample to be measured.

[0018] In an optional embodiment, the spectral data receiving module includes a multi-channel unit point detector; the multi-channel unit point detector is used to collect optical data of multiple channels generated after the sample to be tested reflects the combined light beam; wherein different channels correspond to optical data of different bands.

[0019] In an optional embodiment, the image inversion module includes a data acquisition card and a high-speed logic device; the data acquisition card is used to receive the optical data, convert the optical data into a digital signal, and then scale it; the high-speed logic device is used to convert the scaled digital signal from the time domain to the frequency domain to perform image inversion processing and generate a pixel signal as imaging data of the sample to be tested.

[0020] In an optional embodiment, the light source module includes multiple lasers; wherein different lasers correspond to different laser bands, laser powers and working times.

[0021] In an optional embodiment, the light source module further includes a bright field illumination light source; the bright field illumination light source is used to generate a bright field illumination beam incident on the sample to be tested;

[0022] The system further comprises an imaging camera, which is used to acquire a bright field image of the sample to be tested.

[0023] In an optional embodiment, the sample to be tested is a single cell suspension; the system further includes a fluid system pipeline path; the cells in the single cell suspension flow through the fluid system pipeline path in sequence for imaging.

[0024] The technical solution provided by the present invention can have the following beneficial effects:

[0025] The microscopic imaging system provided by the present invention includes a light source module for generating a laser beam; a radio frequency drive generation module for generating radio frequency drive signals of multiple frequencies; a heterodyne interference optical path module for performing optical heterodyne interference processing on the laser beam according to the radio frequency drive signals of multiple frequencies, forming a light spot array and a reference light source respectively, and then combining the beams to generate a combined beam that is incident on a sample to be measured; wherein different light spots in the light spot array correspond to different light frequencies; a spectral data receiving module for collecting light data generated after the sample to be measured reflects the combined beam; wherein the sample to be measured is pre-fluorescently labeled; and an image inversion module for receiving the light data and performing image inversion processing on the light data to generate imaging data of the sample to be measured. By setting up a radio frequency drive generation module to generate radio frequency drive signals of multiple frequencies, and using a heterodyne interference optical path module to perform optical heterodyne interference processing on the laser beam according to the radio frequency drive signals of multiple frequencies, radio frequency multiplexing technology is introduced to achieve marking of the spatial position of the sample to be measured with different radio frequency frequencies. Different spatial positions correspond to different pixels of imaging data, which eliminates the speed limit of fluorescence microscopy imaging in related technologies and enables the transition from point scanning to multi-dimensional scanning during microscopy imaging, thereby improving the response speed of microscopy imaging, high efficiency and good accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 is a schematic structural diagram of a microscopic imaging system according to an embodiment of the present invention;

[0028] Figure 2 is a schematic structural diagram of another microscopic imaging system according to an embodiment of the present invention;

[0029] Figure 3 is a schematic diagram of an optical beat signal according to an embodiment of the present invention;

[0030] Figure 4 2 is a schematic diagram of image inversion according to an embodiment of the present invention.

[0031] Description of reference numerals:

[0032] 1- Light source module; 2- First glass slide; 3- Second glass slide; 4- Third glass slide; 5- Fourth glass slide; 6- Fifth glass slide; 7- Spatial light modulator in the reference arm; 8- Spatial light modulator in the measurement arm; 9- First reflector; 10- Second reflector; 11- Beam splitter; 12- Beam combiner; 13- Beam expander; 14- Homogenizer; 15- Optical path beam reduction module; 16- Liquid flow system pipeline; 17- Dichroic mirror; 18- Lens; 19- Unit point detector; 20- RF drive generation module; 21- Image inversion module; 22- Host computer; 23- Filter. DETAILED DESCRIPTION

[0033] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts shall fall within the scope of protection of the present invention.

[0034] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0035] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0036] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0037] Figure 1 FIG is a schematic structural diagram of a microscopic imaging system according to an embodiment of the present invention. Figure 1 As shown, the microscopic imaging system includes:

[0038] A light source module, used for generating a laser beam;

[0039] RF drive generation module, used to generate RF drive signals of multiple frequencies;

[0040] A heterodyne interference optical path module is used to perform optical heterodyne interference processing on the laser beams according to the RF drive signals of multiple frequencies, respectively forming a light spot array and a reference light source, and then combining the beams to generate a combined beam that is incident on the sample to be measured; wherein different light spots in the light spot array correspond to different light frequencies;

[0041] A spectral data receiving module is used to collect light data generated by the sample to be tested after reflecting the combined light beam; wherein the sample to be tested is pre-fluorescently labeled;

[0042] The image inversion module is used to receive the light data and perform image inversion processing on the light data to generate imaging data of the sample to be tested.

[0043] Figure 1 The working principle of the microscopic imaging system shown is as follows:

[0044] The sample to be tested can be static, such as being placed on a stage, or dynamic, such as using a flow cytometer to flow the cells in the form of single cells through the detection area in sequence, acting as the scanning axis of the imaging. Among them, the flux of the static sample to be tested can be defined as the speed of replacing the static sample to be tested, and the flux of the dynamic sample to be tested can be defined as the cell flow in the flow cytometer. The sample to be tested is pre-fluorescently marked with a fluorescent probe at a specific position or specific composition of the sample to be tested. The laser beam generated by the light source module can provide the sample to be tested with the excitation light required to generate fluorescence. The laser beam is irradiated on the fluorescent marker in the sample to be tested, which can cause the fluorescent marker to emit a corresponding fluorescent signal. The fluorescent signal carries the optical characteristics of the fluorescent marker, thereby obtaining a fluorescent image of the sample to be tested.

[0045] The RF drive generation module is capable of generating RF drive signals of multiple frequencies. Specifically, the RF drive generation module is capable of generating RF drive signals of any frequency and amplitude according to actual needs. The RF drive signal can be a single-frequency signal or a multi-frequency composite signal to provide the RF drive signal required for the heterodyne interference optical path module to achieve optical modulation. Among them, the image resolution of the microscope imaging system can be adjusted by adjusting the frequency interval and number of frequencies of the RF drive signal. Exemplarily, the RF drive generation module can be composed of various forms of signal generators and high-speed RF signal output devices, such as the signal generator in the related art or an analog signal output module with adjustable frequency, amplitude and power built using an analog-to-digital conversion signal.

[0046] The heterodyne interference optical path module realizes the modulation of the laser beam. Specifically, the heterodyne interference optical path module includes two interference optical paths. The heterodyne interference optical path module can divide the incident laser beam into two polarized lights, and can arbitrarily adjust the light intensity of the two polarized lights, and have a consistent polarization state before beam combining. The two polarized lights are respectively incident on the two interference optical paths. On one of the interference optical paths, the incident laser beam is modulated into a light spot array with different frequency characteristics according to a radio frequency driving signal of multiple frequencies. The optical modulation process spatially disperses the single incident light spot into a light spot array, and introduces a certain optical frequency offset on each light spot, so that each light spot has a different light frequency; on the other interference optical path, the incident laser beam maintains the original light frequency or is adjusted to a certain reference frequency as a reference light source, and then beam combined with the light spot array to generate a difference frequency signal to realize optical heterodyne interference. Among them, the light spot array can be a one-dimensional light spot array or a two-dimensional light spot array. It should be noted that RF tagging technology uses a beat frequency to mark the location of a light spot on the sample under test, i.e., the pixel coordinates. The peak frequency at the beat frequency represents the signal strength at that location, i.e., the pixel signal amplitude. One-dimensional light spot arrays must be combined with flow cytometry cell sorting and require the use of dynamic samples. Two-dimensional light spot arrays can be combined with flow cytometry cell sorting as needed and can be used with either static or dynamic samples.

[0047] In an optional embodiment, the heterodyne interferometer optical circuit is based on the Mach-Zehnder Interferometer (MZI) interferometer optical circuit. At the front end of the heterodyne interferometer, the input laser beam is split into two beams, a measuring arm beam and a reference arm beam, with perpendicular polarization states and adjustable optical power, to ensure the spatial light modulator's requirements for the polarization state of the incident light. The adjustable optical power of the two beams can maximize the efficiency of heterodyne interferometry. By using one or more spatial light modulators in each of the reference arm and the measuring arm, a light spot array is generated on the measuring arm. Each light spot in the light spot array has an optical frequency shift and deflection angle that is positively correlated with the frequency of the corresponding RF drive signal. A fixed optical frequency offset is applied to the light beam on the reference arm, and the offset can be 0. At the same time, a beam shaping device is used in the reference arm to form a reference light source. The output beams of the reference arm and the measuring arm are combined to achieve heterodyne interferometry. Specifically, the heterodyne interferometer optical circuit module includes a beam splitter, a measuring arm, and a reference arm. The beam splitter is used to split the laser beam into a first laser beam and a second laser beam. The measurement arm is used to optically modulate the first laser beam according to the multiple-frequency RF drive signals to generate a light spot array. The reference arm is used to shape the second laser beam into a reference light source. The reference light source can be a one-dimensional line light source or a two-dimensional surface light source. The shape of the two-dimensional surface light source can be any two-dimensional shape, such as a circle or a rectangle. The reference light source covers the light spot array to ensure that each light spot can achieve an optical beat frequency after the first and second laser beams are combined.

[0048] In an optional embodiment, the measurement arm includes a spatial light modulator (SLM), a device that modulates the spatial distribution of light waves. Under the control of an electrical drive signal, it changes the amplitude or intensity, phase, polarization state, and other aspects of the spatial light distribution. It is a key component in modern optical fields such as real-time optical information processing, adaptive optics, and optical computing. The SLM is used to introduce a frequency offset into the first laser beam based on the frequency and amplitude of the corresponding RF drive signal to produce an array of light spots. One or more SLMs can be used as needed. The reference arm includes a beam shaping device that shapes the second laser beam into a reference light source. Optionally, the beam shaping device can employ a beam shaper known in related art, or be composed of one or more optical components. This beam shaping device can shape the original linear laser beam into a flat-top beam with a uniform, slowly varying profile and no high-frequency noise. A Powell prism, cylindrical lens, or the like can be used to generate a one-dimensional line light source. A two-dimensional surface light source can be used to expand the laser spot and then feed it into a beam transforming mirror to achieve a line or surface light source with uniform light intensity distribution. An aperture can be added to the output end of the light source to achieve line or surface light sources of various shapes. The reference arm can also employ a spatial light modulator to introduce a reference frequency shift into the second laser beam, as required. Alternatively, the reference arm can directly utilize a second laser beam without a frequency shift, without employing any spatial light modulator. It should be noted that the frequency range and output power of the RF drive signal must meet the input requirements of the spatial light modulator.

[0049] Optionally, the spatial light modulator includes acousto-optic deflectors (AODs) and acousto-optic frequency shifters (AOFSs). Using one or more AODs and AOFSs, the second laser beam is given a reference fixed frequency offset to reduce the frequency of the beat frequency peak after beam combining. This shifts the beat frequency signal from the original shifted frequency of the first laser beam to the difference between the first and second shifted frequencies. This reduces the sampling frequency range requirements for the data acquisition card, facilitating card selection and reducing costs. The AOD utilizes the acousto-optic effect to control and deflect the propagation of the laser beam, while also changing the frequency of the laser beam. The AOD can achieve laser beam scanning by varying the RF drive frequency. The scanning position can be random, continuous line scanning, or sequential point deflection. Depending on the crystal, wavelength, and beam size, scan rates exceeding 200 MHz and precise position control in nanoradians (nRad) can be achieved. This acousto-optic frequency shifter changes the frequency of a light beam based on the momentum transfer between acoustic waves (phonons) and interacting photons in an acousto-optic device. When light scatters from the diffraction grating generated by the acoustic wave, it undergoes a Doppler shift. If the light propagates through the crystal in the same direction as the acoustic wave, the frequency of the diffracted beam increases due to the RF drive frequency. If the light and acoustic waves propagate in opposite directions, the frequency of the diffracted beam decreases due to the RF drive frequency. The optical frequency shift of the laser beam can be altered by changing the RF drive frequency. For example, a single acousto-optic deflector is used to generate a one-dimensional light spot array, while two orthogonally placed acousto-optic deflectors are used to generate a two-dimensional light spot array. Generating a two-dimensional light spot array can increase imaging speed by more than an order of magnitude, improving microscopic imaging efficiency. In the one-dimensional and two-dimensional light spot arrays, the spacing between the individual light spots can be equally spaced or unequally spaced. The spacing between the light spots is determined by the frequency spacing between the frequency components of the applied RF drive signal. The number of one-dimensional light spots is determined by the number of frequency components of the RF drive signal applied to the acousto-optic deflector. The number of rows and columns of the two-dimensional light spots is determined by the number of frequency components of the RF drive signal applied to each acousto-optic deflector. The spacing between rows and columns can be equally spaced or unequally spaced. The one-dimensional light spot illuminates one line of the sample at a time, while the two-dimensional light spot illuminates one surface of the sample at a time. Each light spot illuminates a specific spatial location on the sample, and each light spot has a different beat signal frequency. Therefore, the light signal at each spatial location is modulated by a specific beat frequency, achieving the goal of marking the spatial location with the beat frequency, thereby completing RF tagging.Exemplarily, using a single acousto-optic deflector (AOD) can diffract an incident laser beam into a one-dimensional array of parallel light spots. The spacing and number of the spots in the one-dimensional array are determined by the frequency interval and number of the RF drive signal applied to the AOD. Using two AODs can diffract an incident laser beam into a two-dimensional array of light spots arranged in a two-dimensional space. The spacing and number of the spots in the two-dimensional array are determined by the frequency interval and number of the RF drive signal applied to the two AODs. The amount of optical frequency shift introduced in each light spot is related to the frequency of the RF drive signal applied to the AOD or AOF shifter. A single-frequency RF drive signal applied to the AOD can diffract a single light spot, while a composite RF drive signal with multiple frequencies applied to the AOD can simultaneously diffract multiple light spots, each with a different diffraction angle and optical frequency shift, each with a unique beat frequency envelope in the time domain. Exemplarily, an AOF shifter is used to apply a fixed optical frequency shift f to the reference arm laser beam. ref The optical frequency shift of each spot on the measuring arm is f i , then the envelope frequency of the beat signal of each light spot after heterodyne interference is f i -f ref Optionally, the spatial light modulator may also be an acousto-optic modulator (AOM), an electro-optic modulator (EOM), etc., which may also achieve a modulation effect.

[0050] In an optional embodiment, the system also includes a host computer that can run on a variety of software and hardware platforms. The host computer is used to send signal adjustment instructions to the RF drive generation module so that the RF drive generation module can adjust the intensity of the RF drive signal according to the signal adjustment instructions. The RF drive generation module can receive parameters from the host computer to set the number of frequency components, frequency component range, frequency component interval, signal amplitude, signal power, etc. of the RF drive signal. The frequency range and output power of the RF drive signal must meet the input requirements of the spatial light modulator. The signal parameters of the RF drive device are set according to the selected spatial light modulator input requirements. The power, frequency interval, and frequency range of the RF drive signal are related to the intensity of the light spot, the light spot diffraction angle, and the light spot deflection transfer range. The host computer is also used to set various system parameters, issuing setting instructions for one or more parameters including flow rate, frame rate, exposure time, image gain, correction algorithm, and RF signal settings to implement scheduling, detection, and control of system tasks. The host computer also receives and displays the imaging data of the sample to be tested.

[0051] In an optional embodiment, the system also includes an optical path beam shrinking module, which is used to scale the combined light beam to a target size and then incident on the sample to be tested. The optical path beam shrinking module scales the combined light beam after heterodyne interference processing by optical means, and reduces the size of each spot in the spot array. The size of each spot represents the spatial resolution of the imaging. The smaller the spot, the smaller the spatial position corresponding to each pixel of the image after imaging. Finally, the spot size in the spot array is shrunk to an appropriate size. The size of the shrunk spot determines the spatial resolution of the microscope system. The shrunk combined light beam is incident on the sample to be tested, which can excite the fluorescent marker in the sample, so that the fluorescent marker is stimulated to radiate fluorescence, thereby realizing the optical signal collection of certain characteristic parts inside the sample to be tested. The emitted fluorescence modulation frequency and optical power are positively correlated with the incident laser modulation frequency and optical power. For example, depending on the size of the sample to be measured, the combined light beam is reduced to the level of hundreds of nanometers to micrometers. The beam reduction process may involve the enlargement and then reduction of the light spot. The zoom factor needs to be selected based on the actual parameter design, and finally the light spot is scaled to the appropriate size according to the size of the sample to be measured and the required resolution.

[0052] In an optional embodiment, the spectral data receiving module includes a multi-channel unit point detector; the multi-channel unit point detector is used to collect optical data from multiple channels generated after the sample to be tested reflects the combined light beam; wherein different channels correspond to optical data of different wavelength bands. The multi-channel unit point detector can be composed of one or more unit point detectors. Each unit point detector has only one photosensitive element and does not have direct imaging capabilities, but has high detection sensitivity and fast response speed, which can meet the application scenarios of high-speed, high-throughput, and weak fluorescence detection. Each unit point detector can simulate a filter of a different wavelength band to achieve multi-channel spectral data collection. Exemplarily, the unit point detector can adopt a photomultiplier tube (PMT), an avalanche photodiode (APD), a photodiode (PD), etc. The spectral data receiving module receives the optical signal emitted by the sample to be tested after being excited and distributes the optical signal to one or more unit point detectors corresponding to the corresponding fluorescence channel through the optical path. An optical filter operating in a different wavelength band can be placed in front of each unit point detector to obtain spectral data of the corresponding wavelength band. The number of fluorescence channels can be one or more, and each fluorescence channel can process information of different wavelength bands. Different wavelength bands can correspond to different fluorescent markers, indicating optical information of multiple specific targets. The optical signal includes forward scattered light and side scattered light. The forward scattered light is in the direction of the laser extension line, reflecting the size of the cell and is used to distinguish target particles from impurities and cell fragments. The side scattered light is in the direction at 90° to the laser beam and reflects the complexity of the cell. The more complex the intracellular structure, the stronger the side scattered light, which is used to reflect the cell information in the sample to be tested.

[0053] In an optional embodiment, the image inversion module includes a data acquisition card and a high-speed logic device; the data acquisition card is used to receive the optical data, convert the optical data into a digital signal, and then scale it; the high-speed logic device is used to convert the scaled digital signal from the time domain to the frequency domain to perform image inversion processing and generate pixel signals as imaging data of the sample to be tested. Optionally, the image inversion module collects the electrical signals (voltage or current signals) output by the multi-channel unit point detector by building an electronic platform, scales the electrical signals, and then sends them to the high-speed logic device after analog-to-digital conversion. The time domain waveform of the electrical signal collected by the multi-channel unit point detector is converted into frequency domain information, and the signal amplitude at multiple frequency shift frequencies is obtained. The image data is then inverted using an imaging algorithm. Among them, in the frequency domain information, the difference between the frequency shift of each light spot in the light spot array of the measuring arm and the frequency shift of the reference light source of the reference arm is the actual frequency shift frequency of each light spot heterodyne interference. The frequency of a certain actual frequency shift frequency peak represents a specific spatial position of the sample, that is, a pixel position of the image. The peak intensity of the actual frequency shift frequency peak is the signal intensity at the image position corresponding to the frequency peak, that is, the signal amplitude of the pixel. Exemplarily, the high-speed logic device can be a data processing platform constructed by combining one or more high-speed logic processors such as a field-programmable gate array FPGA (Field-Programmable Gate Array), a central processing unit CPU (Central Processing Unit), a graphics processing unit GPU (Graphics Processing Unit), an scalable processing platform ZYNQ, a system-on-chip SOC (System on Chip), etc. Fourier transform is run in the high-speed logic device to realize the conversion of time domain signals to frequency domain signals, a data acquisition algorithm is run to obtain the time domain signals of each channel from the detector, an image inversion algorithm is run to realize the inversion from frequency domain data to pixel coordinates and pixel signal values, a data sending algorithm is run to send image data to a host computer, and a variety of image processing and data interaction algorithms are run to realize imaging and data transmission.

[0054] In an optional embodiment, the light source module includes multiple lasers suitable for heterodyne interferometry. Different lasers correspond to different laser wavelengths, laser powers, and operating times, so that the appropriate laser can be selected or switched according to needs to generate the required laser beam. Because the linewidth of the laser will affect the accuracy of the frequency domain extraction performed by the heterodyne interferometry optical path module, a narrow linewidth laser beam is selected, and the theoretical interference length is greater than the actual Mach-Zehnder optical path length to avoid the introduction of frequency noise and ensure accuracy.

[0055] In an optional embodiment, the light source module also includes a bright field illumination light source, which is used to generate a bright field illumination beam incident on the sample to be tested. The bright field illumination light source can provide the system with the high uniformity light source required for bright field imaging, and the uniform light source can ensure that the collected signal amplitude is consistent. The system also includes an imaging camera, which is used to collect the bright field image of the sample to be tested, and the frame rate of the imaging camera can be adjusted as needed. The bright field image can reflect the characteristics of the sample to be tested itself and its interior, and is used to observe the morphology of cells and stained parts under visible light, laser or fluorescence, and can also be used for observation and positioning of cells under visible light, laser or fluorescence. Optionally, if it is necessary to observe, count or synchronize the sample to be tested, it can also be achieved by using an imaging detector / imaging camera or another unit point detector. The imaging detector or another unit point detector does not need to pass through the optical path of the radio frequency tag and serves as an auxiliary test function.

[0056] In an optional embodiment, the sample to be tested is a single-cell suspension; the system further includes a fluidic system conduit; cells in the single-cell suspension sequentially flow through the fluidic system conduit for imaging. The sample to be tested flows through the fluidic system conduit to achieve linear and area array push scanning.

[0057] The microscopic imaging system provided in this embodiment includes a light source module for generating a laser beam; a radio frequency drive generation module for generating radio frequency drive signals of multiple frequencies; a heterodyne interference optical path module for performing optical heterodyne interference processing on the laser beam according to the radio frequency drive signals of multiple frequencies, forming a light spot array and a reference light source respectively, and then combining the light spots to generate a combined light beam that is incident on a sample to be measured; wherein different light spots in the light spot array correspond to different light frequencies; a spectral data receiving module for collecting light data generated after the sample to be measured reflects the combined light beam; wherein the sample to be measured is pre-fluorescently labeled; and an image inversion module for receiving light data and performing image inversion processing on the light data to generate imaging data of the sample to be measured. By setting up a radio frequency drive generation module to generate radio frequency drive signals of multiple frequencies, and using a heterodyne interference optical path module to perform optical heterodyne interference processing on the laser beam according to the radio frequency drive signals of multiple frequencies, radio frequency multiplexing technology is introduced to achieve marking of the spatial position of the sample to be measured with different radio frequency frequencies. Different spatial positions correspond to different pixels of imaging data, which eliminates the speed limit of fluorescence microscopy imaging in related technologies and enables the transition from point scanning to multi-dimensional scanning during microscopy imaging, thereby improving the response speed of microscopy imaging, high efficiency and good accuracy.

[0058] Moreover, by adopting unit-point detectors instead of imaging detectors in related technologies, the detection sensitivity and response speed are greatly improved, meeting the requirements of weak fluorescence detection for detection sensitivity and response speed. Combining RF multiplexing technology with the sensitivity and speed of unit-point detectors can enable the unit-point detector to transition from serial beam scanning to multi-channel parallel scanning, realizing data collection of all pixels of the entire image within one clock cycle, and the increase or decrease in the number of pixels in the image will not change the acquisition cycle of each image. The number of pixels in the image can be increased or decreased arbitrarily according to demand, achieving data processing and communication speeds of more than 100 MHz, meeting the purpose of high-speed, high-throughput real-time imaging and analysis, and enabling fluorescence microscopy imaging to reach a rate of more than 1,000 frames per second.

[0059] Furthermore, the use of a spatial light modulator as a scanning device enables rapid, mechanical-free, simultaneous deflection of multi-dimensional light spots, avoiding the issues encountered in related technologies where galvanometer mirrors are used for raster scanning of the imaging field of view, which are inflexible and subject to mechanical inertia that limits imaging speed. Both acousto-optic modulators (such as acousto-optic deflectors (AODs) / acousto-optic frequency shifters (AOFSs) / acousto-optic modulators (AOMs)) and electro-optic modulators (such as electro-optic modulators (EOMs)) within spatial light modulators can achieve rapid, mechanical-free, simultaneous deflection of multi-dimensional light spots. For example, an acousto-optic deflector can utilize the acousto-optic effect of an acousto-optic crystal to change the diffraction angle of the emitted light by applying different acoustic wave frequencies, thereby achieving linear deflection of the light beam. This eliminates mechanical inertia issues, resulting in fast scanning speeds and high repeatability. Furthermore, the deflection in two dimensions (X and Y) can be independently controlled, providing a flexible scanning method and enabling rapid, jumpy addressing scanning. Applying a composite RF drive signal at multiple frequencies to the acousto-optic deflector enables simultaneous deflection of beams at multiple deflection angles. Depending on the number and placement of the acousto-optic deflectors, a one-dimensional or two-dimensional array of light spots can be generated on the sample under test. Aligning the beam array with the imaging pixels enables linear or area array imaging of the sample under test. Transitioning from point scanning to multi-dimensional scanning can increase imaging speed by more than an order of magnitude, increasing imaging speed over a wide range. For point scanning imaging, N-pixel one-dimensional linear array imaging, and M×N pixel two-dimensional area array imaging, under the same spatial resolution (M×N pixels) and fluorescence lifetime T, point scanning requires M×N×T time to achieve one frame of image, one-dimensional line scanning requires N×T time to achieve one frame of image, and two-dimensional area array requires T time to achieve one frame of image. Therefore, multi-dimensional scanning enables high-speed, high-throughput imaging.

[0060] As one or more specific application examples of the embodiments of the present invention, the optimal implementation scheme or the solution that the inventor most wants to embody is described below in combination with specific application scenarios.

[0061] Figure 2 FIG is a schematic structural diagram of a microscopic imaging system according to an embodiment of the present invention. Figure 2 As shown, the microscopic imaging system includes:

[0062] Light source module 1, used to generate bright field illumination light source and laser beam;

[0063] The heterodyne interference optical path module uses a spatial light modulator 7 or multiple spatial light modulators 8 to achieve optical frequency shift and spot array generation, thereby achieving optical beat frequency between the spot array and the reference light source, and realizing radio frequency spatial labeling in the beam combiner 12;

[0064] The RF drive generating module 20 is used to generate the RF drive signal required by the spatial light modulator;

[0065] The optical path beam reduction module 15 uses optical devices to adjust the size of the light spots in the light spot array and converge the light spot array on the sample to be tested flowing through the liquid flow system pipeline 16;

[0066] The spectral data receiving module distributes and collects the fluorescence signal and bright field light signal of the sample to be tested respectively, and finally passes through different filters 23 to be incident on the multi-channel unit point detector 19;

[0067] The image inversion module 21 is used to collect the time domain signal of the unit point detector and convert the time domain signal into a frequency domain signal based on the software and hardware data processing platform to invert the spatial information of the sample to be tested, generate image data and send it to the host computer 22.

[0068] The host computer 22 performs tasks such as parameter issuance, status monitoring, and image display, as well as human-computer interaction functions for the entire microscopic imaging system. The host computer includes a human-computer interaction interface (HMI) that generates adjustment commands based on user-set parameters. These commands are then sent to the light source module to control the laser's band switching logic and to the RF driver generation module to generate specific single-frequency or multi-frequency RF signals for input to the spatial light modulator. The host computer also receives imaging data output by the image inversion module and displays this data to the user through the HMI.

[0069] The heterodyne interference optical path generates an optical beat signal, and the reference arm beam and the measurement arm beam are modulated by the spatial light modulator with a specific optical frequency shift and deflection angle. Figure 3 FIG. 1 is a schematic diagram of an optical beat signal according to an embodiment of the present invention. Figure 3As shown, signal a is the modulated reference arm beam, signal b is the modulated measurement arm beam, and signal a and signal b are combined to form signal c with an optical beat frequency. The envelope frequency of signal c is the optical frequency difference between signal a and signal b. Each light spot in the light spot array has a unique envelope frequency, and each light spot has a specific deflection angle, which illuminates different spatial positions on the sample to be measured. That is, the signal envelope can be used to uniquely mark the spatial position of the measured signal, which is spatial RF tag counting.

[0070] The optical path beam reduction module is used to adjust the spot size in the combined spot array to adjust the spatial resolution. The spot size can reach the diffraction limit to achieve super-resolution microscopic imaging.

[0071] The sample to be tested is excited by the incident laser to produce a fluorescence signal or is directly illuminated by the laser, generating forward scattering and side scattering. The spectral data receiving module collects light signals from multiple different spectral channels and converts them into electrical signals and sends them to the image inversion module to convert the time domain information into the frequency domain. Image inversion and data processing have been performed, and the processing results are sent to the human-computer interaction interface of the host computer for image display. Figure 4 This is a schematic diagram of image inversion according to an embodiment of the present invention. The effect after collecting a set of black and white images with a stripe width of 17.5 μm in the resolution plate and performing image inversion is as follows: Figure 4 shown.

[0072] When a fluorescence lifetime image of a sample is required, the sample is placed on a sample stage or passed through the fluidics system as a single cell through the fluidics system pipeline 16. The light source module 1 generates a laser beam, which is incident on the heterodyne interferometer optical path module. The host computer 22 generates preset RF drive parameters and transmits them to the RF drive generation module 20. The RF drive generation module 20 generates an RF drive signal (electrical signal) of corresponding power and frequency domain, which is then transmitted to each spatial light modulator in the heterodyne interferometer optical path module through the beam splitter 11. Optionally, the beam splitter 11 can be a dichroic prism. Two spatial light modulators 8 are used on the measuring arm of the heterodyne interferometer optical path module, arranged orthogonally. The first spatial light modulator generates a one-dimensional light spot array, which is incident on the second spatial light modulator and expanded into a two-dimensional light spot array. A spatial light modulator 7 is used on the reference arm to shift the overall optical frequency of the incident laser beam to near the lowest or highest optical frequency domain of the measuring arm. The spot of the outgoing laser beam is then incident on a beam expander 13 through a first reflector 9, which expands the laser beam spot to meet the incident spot size requirements of the subsequent beam homogenizer 14. After passing through homogenizer 14, the laser beam generates a homogenized reference arm beam. The two-dimensional light spot array of the measuring arm passes through a second reflector 10 and is combined with the homogenized reference arm beam in a beam combiner 12 to generate an optical beat frequency, giving each light spot in the two-dimensional light spot array a unique signal envelope, thus achieving spatial radio frequency tagging. The combined light beam is optically reduced by the microscope objective lens in the optical path reduction module 15, and each light spot can be reduced to the diffraction limit to achieve super-resolution microscopic imaging. The reduced light spot is incident on the sample to be tested in the liquid flow system pipeline 16 to excite the specific position of the sample to be tested and form a light signal. A dichroic mirror 17 is used to distribute the light signal to different channels. Each channel uses a lens 18 to converge the light signal and pass it through a filter 23, and then it is incident on the unit point detector to collect the time domain information of the light signal. The image inversion module 21 reads the time domain signal of the unit point detector and converts it into frequency domain information to obtain the spatial image information of the sample to be tested, and uploads it to the host computer 22 for image display.

[0073] Optionally, a three-dimensional light spot array can be generated based on the two-dimensional light spot array. Specifically, a displacement device can be provided between the beam combiner 12 and the optical path beam reduction module 15. This displacement device is used to drive the optical path beam reduction module 15 to move back and forth, thereby achieving back and forth movement of the focal position of the two-dimensional light spot array, thereby obtaining a three-dimensional light spot array and, in turn, obtaining three-dimensional imaging data of the sample to be tested. The three-dimensional light spot array can also be optionally combined with flow cytometry cell sorting, and can be used with static or dynamic samples to be tested.

[0074] Optionally, the light source module 1 is a laser group, and the wavelength of each laser is determined by the excitation wavelength corresponding to the fluorescent dye pre-marked on the sample to be tested. The laser group is composed of multiple lasers with different wavelengths. When in use, it meets the system's high-throughput demand for laser wavelength switching. Exemplarily, the laser beam emitted by the A-band laser has different light offsets and deflection angles after heterodyne interference, and realizes optical beat frequency in the beam combiner 12. After being beam-contracted, it is incident on the sample to be tested, generating a bright field light signal and a signal with a spatial position mark. The bright field light signal is a forward scattering signal, a side scattering signal or a fluorescence signal generated by the laser itself irradiating the sample to be tested. The signal with a spatial position mark is a light signal generated by the beat frequency laser beam incident on the sample to be tested with a spatial mark. The light signal contains bright field light information and information marked with radio frequency space, and is distributed to multiple different channels by the dichroic mirror 17. It is finally received by the unit point detector, and the light signal is converted into an electrical signal for the image inversion module 21 to perform data processing and inversion imaging. After the image data acquisition of band A is completed, the laser group can be actively or passively switched to the laser of band B. After the image data acquisition corresponding to the laser of band B is completed, it can be switched to the laser of band C again, and switched in sequence until the image data acquisition of all required bands is completed.

[0075] Optionally, the unit point detector 19 can be a detector with a single unit pixel, such as an avalanche photodiode APD or a photodiode PD. The dichroic mirror 17 can be replaced by a semi-transparent and semi-reflective mirror to reflect light in a specific band and refract light in other bands. The spatial light modulator 7 in the reference arm of the heterodyne interference optical path module and the spatial light modulator 8 in the measuring arm can be an acousto-optic deflector AOD, an acousto-optic frequency shifter AOFS, an electro-optic modulator EOM, an acousto-optic modulator AOM, and other optical devices that can change the deflection angle and light frequency of the incident light beam. The second laser beam in the reference arm of the heterodyne interference optical path module can be shaped by a Powell prism or a cylindrical lens to produce a one-dimensional or two-dimensional line or surface light source, instead of using a beam expander 13 plus a homogenizer 14. The lens 18 can be composed of an objective lens or other optical devices to achieve the purpose of converging light.

[0076] Optionally, the beam splitter 11 uses a polarization beam splitter prism, and a first glass slide 2 is used in front of the beam splitter 11 to adjust the polarization state of the incident laser beam. The polarization beam splitter prism splits the incident laser beam into two beams of light with perpendicular polarization states. A second glass slide 3 is placed in front of the spatial light modulator 7 of the reference arm, and a third glass slide 4 is placed in front of the spatial light modulator 8 of the measuring arm to ensure that the light incident on the spatial light modulator has a suitable polarization state. A fourth glass slide 5 and a fifth glass slide 6 are placed in front of the beam combiner 12 to ensure that the optical beat frequency efficiency of the combined beam is maximized.

[0077] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A microscopic imaging system, characterized in that: The system comprises: A light source module, used for generating a laser beam; A radio frequency drive generation module is used to generate radio frequency drive signals of multiple frequencies; wherein the frequency interval and number of frequencies of the radio frequency drive signals are adjusted to adjust the imaging resolution; A heterodyne interference optical path module is used to perform optical heterodyne interference processing on the laser beams according to the RF drive signals of the multiple frequencies, respectively, to form a light spot array and a reference light source, and then combine the beams to generate a combined beam that is incident on the sample to be measured; wherein different light spots in the light spot array correspond to different light frequencies; and the pixel coordinates of the light spots on the sample to be measured are marked using the beat frequency; a spectral data receiving module, configured to collect light data generated by the sample to be tested after the sample to be tested reflects the combined light beam; wherein the sample to be tested is fluorescently labeled in advance; an image inversion module, configured to receive the light data and perform image inversion processing on the light data to generate imaging data of the sample to be tested; The heterodyne interferometer optical path module includes a beam splitter, a measuring arm, and a reference arm; the beam splitter is used to split the laser beam into a first laser beam and a second laser beam; the measuring arm is used to perform optical modulation processing on the first laser beam according to the radio frequency drive signals of multiple frequencies to generate a light spot array; the reference arm is used to shape the second laser beam into a reference light source; wherein the reference light source covers the light spot array; The spectral data receiving module includes a multi-channel unit point detector; the multi-channel unit point detector is used to collect optical data of multiple channels generated after the sample to be tested reflects the combined light beam; wherein different channels correspond to optical data of different bands.

2. The system according to claim 1, wherein: The measurement arm includes a spatial light modulator, which is used to introduce a frequency offset into the first laser beam based on the frequency and amplitude of the corresponding radio frequency drive signal to generate a light spot array; The reference arm includes a beam shaping device, which is used to shape the second laser beam into a reference light source.

3. The system according to claim 1 or 2, characterized in that The system further includes a host computer; the host computer is used to send a signal adjustment instruction to the RF drive generation module, so that the RF drive generation module adjusts the intensity of the RF drive signal according to the signal adjustment instruction; The host computer also receives the imaging data of the sample to be tested and displays it.

4. The system according to claim 1 or 2, characterized in that The system further comprises an optical path beam reduction module, which is used to scale the combined light beam to a target size and then incident on the sample to be measured.

5. The system according to claim 1 or 2, characterized in that The image inversion module includes a data acquisition card and a high-speed logic device; the data acquisition card is used to receive the optical data, convert the optical data into a digital signal, and then scale it; The high-speed logic device is used to convert the scaled digital signal from the time domain to the frequency domain to perform image inversion processing and generate pixel signals as imaging data of the sample to be measured.

6. The system according to claim 1 or 2, characterized in that The light source module includes a plurality of lasers, wherein different lasers correspond to different laser bands, laser powers and working times.

7. The system according to claim 1 or 2, characterized in that The light source module further includes a bright field illumination light source; the bright field illumination light source is used to generate a bright field illumination beam incident on the sample to be tested; The system further comprises an imaging camera, which is used to acquire a bright field image of the sample to be tested.

8. The system according to claim 1 or 2, characterized in that The sample to be tested is a single cell suspension; the system further comprises a fluid flow system pipeline; the cells in the single cell suspension flow through the fluid flow system pipeline in sequence for imaging.

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