Microscopic imaging system

Abstract

The invention relates to the technical field of optics, and discloses a microscopic imaging system. The system comprises a light source module used for generating a laser beam; the radio frequency drive generation module is used for generating radio frequency drive signals with various frequencies; the heterodyne interference light path module is used for respectively performing optical heterodyne interference processing on the laser beams according to the radio frequency driving signals with various frequencies, respectively forming a light spot array and a reference light source and then combining the light spots and the reference light source, and generating combined light beams to be incident on a sample to be detected; wherein different light spots in the light spot array correspond to different light frequencies; the spectral data receiving module is used for collecting light data generated after the combined light beam is reflected by the to-be-detected sample; wherein the to-be-detected sample is subjected to fluorescence labeling in advance; and the image inversion module is used for receiving the optical data and carrying out image inversion processing on the optical data to generate imaging data of the sample to be detected. According to the scheme, the microscopic imaging response speed is increased, the efficiency is high, and the accuracy is good.

Description

Technical Field

[0001] The 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 often used. 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 capture 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 is composed of N pixels, it is necessary to scan at least N times to scan the entire image. The frame rate increases with the increase in the number of image pixels, resulting in limited frame rate and slow response speed, which makes it 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, the system comprising: A light source module, used for generating a laser beam; A radio frequency drive generation module, used to generate radio frequency drive signals of multiple frequencies; A heterodyne interference optical path module is used to perform optical heterodyne interference processing on the laser beams according to the radio frequency driving signals of the multiple frequencies, respectively, to form a light spot array and a reference light source, and then combine the light beams 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, 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 fluorescently labeled in advance; 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.

[0007] 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 perform optical modulation processing on the first laser beam according to the RF driving signal of the 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.

[0008] In an optional embodiment, the measuring arm comprises a spatial light modulator, and the spatial light modulator is used to introduce a frequency shift into the first laser beam based on the frequency and amplitude of the corresponding radio frequency driving signal to generate a light spot array; The reference arm comprises a beam shaping device for shaping the second laser beam into a reference light source.

[0009] 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; The host computer also receives and displays the imaging data of the sample to be tested.

[0010] In an optional embodiment, the system further comprises an optical path beam shrinking module, and the optical path beam shrinking module is used to scale the combined light beam to a target size and then incident on the sample to be tested.

[0011] 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.

[0012] 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, and 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.

[0013] In an optional implementation, the light source module includes a plurality of lasers; wherein different lasers correspond to different laser bands, laser powers and working times.

[0014] 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 light beam incident on the sample to be tested; The system also includes an imaging camera, which is used to collect a bright field image of the sample to be tested.

[0015] In an optional embodiment, the sample to be tested is a single cell suspension; the system further includes a fluid system pipeline passage; the cells in the single cell suspension flow through the fluid system pipeline passage in sequence for imaging.

[0016] The technical solution provided by the present invention may include the following beneficial effects: The microscopic imaging system provided by the present invention comprises 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, 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 a 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; 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 tested. A radio frequency drive generation module is provided to generate radio frequency drive signals of various frequencies, and optical heterodyne interference processing is performed on the laser light beams respectively according to the radio frequency drive signals of various frequencies through a heterodyne interference optical path module, and radio frequency multiplexing technology is introduced to realize marking the spatial position of the sample to be tested with different radio frequency frequencies. Different spatial positions correspond to different pixels of imaging data, and the speed limitation of fluorescence microscopy imaging in related technologies is eliminated, so that the microscopy imaging is transitioned from point scanning to multi-dimensional scanning, thereby improving the microscopy imaging response speed, and having high efficiency and good accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 is a schematic structural diagram of a microscopic imaging system according to an embodiment of the present invention; Figure 2 is a schematic structural diagram of another microscopic imaging system according to an embodiment of the present invention; Figure 3 is a schematic diagram of an optical beat signal according to an embodiment of the present invention; Figure 4 is a schematic diagram of image inversion according to an embodiment of the present invention.

[0019] Description of reference numerals: 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 measuring 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

[0020] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the 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 creative work are within the scope of protection of the present invention.

[0021] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0022] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0023] 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.

[0024] Figure 1 Schematic diagram of the structure of a microscopic imaging system according to an embodiment of the present invention. Figure 1As shown, in the microscopic imaging system, it includes: A light source module, used for generating a laser beam; A radio frequency drive generation module, used to generate radio frequency drive signals of multiple frequencies; A heterodyne interference optical path module is used to perform optical heterodyne interference processing on the laser beam according to the RF driving signals of multiple frequencies, respectively, to form a light spot array and a reference light source, and then combine the light beams 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 is used to collect 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; 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.

[0025] Figure 1 The working principle of the microscopic imaging system shown is as follows: The sample to be tested can be static, such as placed on a stage, or dynamic, such as using a flow cytometer to flow cells in the form of single cells through the detection area in sequence, acting as the scanning axis of 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.

[0026] The RF drive generation module can generate RF drive signals of multiple frequencies. Specifically, the RF drive generation module can generate 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 realize optical modulation. Among them, the image resolution of the microscope system can be adjusted by adjusting the frequency interval and the 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 the analog signal output module with adjustable frequency, amplitude and power built using analog-to-digital conversion signals.

[0027] 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 lines of polarized light, and can arbitrarily adjust the light intensity of the two lines of polarized light, and have a consistent polarization state before beam combination. The two lines of polarized light are respectively incident on 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 disperses the single incident light spot into a light spot array in space, 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 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 the radio frequency tagging technology uses the beat frequency to mark the position of the light spot on the sample to be tested, that is, the pixel coordinates; the frequency peak at the beat frequency represents the strength of the signal at that position, that is, the pixel signal amplitude. The one-dimensional light spot array must be combined with flow cytometry cell sorting, and dynamic samples to be tested must be used; the two-dimensional light spot array can be combined with flow cytometry cell sorting as needed, and static or dynamic samples to be tested can be used.

[0028] In an optional embodiment, the heterodyne interference optical path is based on the Mach-Zehnder Interferometer (MZI) interference optical path. At the front end of the heterodyne interference, the input laser beam is divided into two measuring arm beams and reference arm beams with vertical polarization states and adjustable optical powers to ensure the requirements of the spatial light modulator for the polarization state of the incident light. The adjustable optical powers of the two beams can ensure the maximization of the heterodyne interference efficiency. By using one or more spatial light modulators in 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 a deflection angle that are 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 realize heterodyne interference. Specifically, 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 perform optical modulation processing on the first laser beam according to the RF driving 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, which can be a one-dimensional line light source or a two-dimensional surface light source, and the shape of the two-dimensional surface light source can be any two-dimensional figure such as a circle or a rectangle. The reference light source covers the light spot array to ensure that each light spot can achieve optical beat frequency after the first laser beam and the second laser beam are combined.

[0029] In an optional embodiment, the measuring arm includes a spatial light modulator, which is a device that modulates the spatial distribution of light waves. Under the control of an electrical drive signal, the amplitude or intensity, phase, polarization state, etc. of the light distribution in space are changed. It is a key device in modern optical fields such as real-time optical information processing, adaptive optics and optical computing. The spatial light modulator is used to introduce a frequency offset to the first laser beam based on the frequency and amplitude of the corresponding radio frequency drive signal to generate a spot array. Among them, one or more spatial light modulators can be used as required. The reference arm includes a beam shaping device, which is used to shape the second laser beam into a reference light source. Optionally, the beam shaping device can adopt the beam shaper in the related technology, or be composed of one or more optical components. The beam shaping device can shape the laser beam from the original linear shape into a flat-top beam with a uniform and slowly changing profile and no high-frequency noise. The one-dimensional line light source can be generated by using Powell prisms, cylindrical lenses, etc. The two-dimensional surface light source can expand the laser spot and send it into the beam transforming mirror to achieve a line light source or surface light source with uniform light intensity distribution. An aperture can be added at the output end of the light source to achieve line light sources or surface light sources of various shapes. The reference arm can also use a spatial light modulator to introduce a reference frequency shift offset for the second laser beam according to needs, or it can directly use a second laser beam without frequency shift without using 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.

[0030] Optionally, the spatial light modulator includes an acousto-optic deflector (AOD) and an acousto-optic frequency shifter (AOFS). One or more acousto-optic deflectors and acousto-optic frequency shifters are used to give the second laser beam a reference fixed frequency offset to reduce the frequency of the beat frequency peak after beam combination, so that the frequency of the beat frequency signal is changed from the original first laser beam frequency shift frequency to the difference between the first laser beam frequency shift frequency and the second laser beam frequency shift frequency, thereby reducing the sampling frequency range requirements for the data acquisition card, facilitating the selection of the data acquisition card, and reducing costs. The acousto-optic deflector uses the acousto-optic effect to control and deflect the propagation of the laser beam, and at the same time changes the frequency of the laser beam. The acousto-optic deflector can realize laser beam scanning by changing the RF drive frequency. The scanning position can realize random position, continuous line scanning and sequential point deflection. According to the crystal, wavelength and beam size, a scanning rate of more than 200 MHz and precise position control of nanoradians (nRad) can be achieved. The acousto-optic frequency shifter changes the frequency of the light beam based on the momentum transfer between the sound wave (phonon) and the interacting photons in the acousto-optic device. When light is scattered from the diffraction grating generated by the sound wave, it will experience a Doppler frequency shift. If the light propagates through the crystal in the same direction as the sound wave, the frequency of the diffracted light beam will increase due to the RF drive frequency. If the light wave and the sound wave propagate in opposite directions, the frequency of the diffracted light beam will decrease due to the RF drive frequency. The optical frequency shift of the laser beam can be changed by changing the RF drive frequency. Exemplarily, an acousto-optic deflector is used to generate a one-dimensional light spot array, and two acousto-optic deflectors are placed orthogonally to generate a two-dimensional light spot array. By generating a two-dimensional light spot array, the imaging speed can be increased by more than an order of magnitude, improving the efficiency of microscopic imaging. In the one-dimensional light spot array and the two-dimensional light spot array, the intervals between the light spots can be arranged at equal intervals or at unequal intervals. The intervals between the light spots are determined by the frequency intervals between the frequency components of the applied radio frequency drive signal. The number of one-dimensional light spots is determined by the number of frequency components of the radio frequency 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 radio frequency drive signal applied to each acousto-optic deflector. The intervals between rows and columns can be equal intervals or unequal intervals. The one-dimensional light spot irradiates one line of the sample each time, and the two-dimensional light spot irradiates one surface of the sample each time. Each light spot irradiates a specific spatial position on the sample, and each light spot has a different beat signal frequency. Therefore, the light signal at each spatial position is modulated by a specific beat frequency, so that the beat frequency is used to mark the spatial position and complete the radio frequency marking.Exemplarily, using an acousto-optic deflector can diffract the incident laser beam into a parallel one-dimensional array of light spots, and the spot spacing and the number of light spots in the one-dimensional array are determined by the frequency interval and the number of frequencies of the radio frequency drive signal applied to the acousto-optic deflector; using two acousto-optic deflectors can diffract the incident laser beam into a two-dimensional array of light spots arranged in a two-dimensional space, and the spot spacing and the number of light spots in the two-dimensional array are determined by the frequency interval and the number of frequencies of the radio frequency drive signal applied to the two acousto-optic deflectors. The frequency shift amount of the optical frequency offset introduced on each light spot is related to the frequency of the radio frequency drive signal applied to the acousto-optic deflector or the acousto-optic frequency shifter. Among them, a single-frequency radio frequency drive signal applied to the acousto-optic deflector can diffract a light spot, and a composite radio frequency drive signal with multiple frequencies applied to the acousto-optic deflector can diffract multiple light spots at the same time, each light spot has a different diffraction angle and optical frequency shift, and each light spot has a unique beat frequency envelope in the time domain. Exemplarily, an acousto-optic frequency shifter is used to apply a fixed optical frequency shift f to the reference arm laser beam. ref , the optical frequency shift imposed by the AOD on 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.

[0031] In an optional embodiment, the system also includes a host computer, and the host computer can run on a variety of software and hardware platforms. The host computer is used to send a signal adjustment instruction 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 instruction. The RF drive generation module can receive the parameters of the host computer to realize the setting of 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 input requirements of the selected spatial light modulator. The power, frequency interval, and frequency range of the RF drive signal are related to the intensity of the light spot, the diffraction angle of the light spot, and the deflection transfer range of the light spot. The host computer is also used to set various system parameters, and sends down setting instructions for one or more parameters including flow rate, frame rate, exposure time, image gain, correction algorithm, RF signal setting, etc., to realize the scheduling, detection, and control of system tasks. The host computer also receives the imaging data of the sample to be tested and displays it.

[0032] 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 a suitable 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 and emit 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 tested, 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. Finally, the light spot is scaled to an appropriate size based on the size of the sample to be tested and the required resolution.

[0033] 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, the multi-channel unit point detector can be composed of one or more unit point detectors, the unit point detector has only one photosensitive element, does not have direct imaging capability, but has high detection sensitivity and fast response speed, and can meet the application scenarios of high-speed, high-throughput, and weak fluorescence detection, and each unit point detector can simulate filters of different bands to realize the collection of multi-channel spectral data. 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, and an optical filter working in different bands can be placed in front of each unit point detector to obtain the spectral data of the corresponding band, the number of fluorescence channels can be one or more, each fluorescence channel can process information of different bands, and different bands can correspond to different fluorescent markers, indicating the 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 of 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.

[0034] 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, and 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 the imaging data of the sample to be tested. Optionally, the image inversion module collects the electrical signal (voltage or current signal) output by the multi-channel unit point detector by building an electronic platform, and sends it to the high-speed logic device after scaling and analog-to-digital conversion of the electrical signal, converts the time domain waveform of the electrical signal collected by the multi-channel unit point detector into frequency domain information, obtains the signal amplitude at multiple frequency shift frequencies, and realizes the inversion of the image data through the imaging algorithm. Among them, in the frequency domain information, the difference between the frequency shift amount of each light spot in the light spot array of the measuring arm and the frequency shift amount 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 certain 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 combinations of 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 extensible 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 transformation of time domain signals to frequency domain signals, a data acquisition algorithm is run to obtain 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.

[0035] In an optional embodiment, the light source module includes a plurality of lasers, and the lasers are suitable for heterodyne interference. Different lasers correspond to different laser bands, laser powers and working times, and suitable lasers can be selected or switched according to needs to generate the required laser beams. Since the line width of the laser will affect the accuracy of frequency domain extraction by the heterodyne interference optical path module, a narrow line width 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.

[0036] 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 light beam incident on the sample to be tested. The bright field illumination light source can provide the system with a high uniformity light source required for bright field imaging, and the uniform light source can ensure that the amplitude of the collected signal 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 according to demand. The bright field image can reflect the characteristics of the sample to be tested itself and its internal structure, 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 marker, and plays a role in auxiliary testing.

[0037] In an optional embodiment, the sample to be tested is a single cell suspension; the system further comprises a fluid system pipeline passage; cells in the single cell suspension flow through the fluid system pipeline passage in sequence to be imaged. The sample to be tested flows in the fluid system pipeline passage to achieve linear array and area array push scanning.

[0038] 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, respectively forming a light spot array and a reference light source and then combining them to generate a combined light beam incident on a 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; 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 tested. A radio frequency drive generation module is provided to generate radio frequency drive signals of various frequencies, and optical heterodyne interference processing is performed on the laser light beams respectively according to the radio frequency drive signals of various frequencies through a heterodyne interference optical path module, and radio frequency multiplexing technology is introduced to realize marking the spatial position of the sample to be tested with different radio frequency frequencies. Different spatial positions correspond to different pixels of imaging data, and the speed limitation of fluorescence microscopy imaging in related technologies is eliminated, so that the microscopy imaging is transitioned from point scanning to multi-dimensional scanning, thereby improving the microscopy imaging response speed, and having high efficiency and good accuracy.

[0039] In addition, 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.

[0040] In addition, a spatial light modulator is used as a scanning device to achieve fast and non-mechanical multi-dimensional spot deflection at the same time, avoiding the problem that the galvanometer used in the related technology is not flexible enough when performing raster scanning on the imaging field of view and the imaging speed is limited by mechanical inertia. The acousto-optic modulation device (such as acousto-optic deflector AOD / acousto-optic frequency shifter AOFS / acousto-optic modulator AOM) and electro-optic modulation device (such as electro-optic modulator EOM) in the spatial light modulator can achieve fast and non-mechanical multi-dimensional spot deflection at the same time. For example, the acousto-optic deflector can utilize the acousto-optic effect of the acousto-optic crystal to change the diffraction angle of the outgoing light by loading different sound wave frequencies, thereby achieving linear deflection of the light beam. There is no mechanical inertia problem, the scanning speed is fast, the repetition accuracy is high, and the deflection of the two dimensions (X dimension and Y dimension) can be independently controlled. The scanning method is flexible and can achieve jumpy fast addressing scanning. Applying a composite RF driving signal of multiple frequencies to the acousto-optic deflector can realize the simultaneous deflection of beams with multiple deflection angles. Depending on the number and placement of the acousto-optic deflectors, a one-dimensional or two-dimensional light spot array can be generated on the sample to be tested. By matching the beam array with the imaging pixel points, the linear array or surface array imaging of the sample to be tested can be realized. The transition from point scanning to multi-dimensional scanning can increase the imaging speed by more than one order of magnitude, and improve the imaging speed to the maximum extent. For point scanning imaging, N-pixel one-dimensional linear array imaging, and M×N-pixel two-dimensional surface array imaging, under the same spatial resolution (M×N pixels) and the same fluorescence lifetime T, it takes M×N×T for point scanning to realize one frame of image, N×T for one-dimensional line scanning, and T for two-dimensional surface array to realize one frame of image. Therefore, multi-dimensional scanning can realize high-speed and high-throughput imaging.

[0041] As one or more specific application embodiments of the embodiments of the present invention, the optimal implementation scheme or the scheme that the inventor most wants to embody is described below in combination with specific application scenarios.

[0042] Figure 2 Schematic diagram of the structure of a microscopic imaging system according to an embodiment of the present invention. Figure 2 As shown, in the microscopic imaging system, it includes: Light source module 1, used to generate bright field illumination light source and laser beam; The heterodyne interference optical path module uses a spatial light modulator 7 or multiple spatial light modulators 8 to realize optical frequency shift and light spot array generation, thereby realizing optical beat frequency between the light spot array and the reference light source, and realizing radio frequency spatial marking in the beam combiner 12; The RF driving generation module 20 is used to generate the RF driving signal required by the spatial light modulator; The light path beam reduction module 15 uses an optical device to adjust the size of the light spot in the light spot array, and converges the light spot array into the sample to be tested flowing through the pipeline passage 16 of the liquid flow system; 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; 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.

[0043] The host computer 22 implements the task scheduling and human-computer interaction functions such as parameter issuance, status monitoring, and image display of the entire microscopic imaging system. The host computer includes a human-computer interaction interface, which can generate corresponding adjustment instructions in response to the parameters set by the user, and issue the adjustment instructions to the light source module to control the band switching logic of the laser, and issue the adjustment instructions to the RF drive generation module to control the RF drive generation module to generate a specific single-frequency or multi-frequency RF signal, which is input to the spatial light modulator. The host computer also receives the imaging data output by the image inversion module, and displays the imaging data to the user through the human-computer interaction interface.

[0044] 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 to have a specific optical frequency shift and deflection angle. Figure 3 Schematic diagram of an optical beat signal according to an embodiment of the present invention. Figure 3 As 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 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 is irradiated at 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 the spatial RF tag counting.

[0045] 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.

[0046] The sample to be tested is excited by the incident laser to produce a fluorescence signal or is directly illuminated by the laser to produce 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 FIG. 1 is a schematic diagram of image inversion according to an embodiment of the present invention. The effect after a set of black and white images with a stripe width of 17.5 μm in the resolution plate are collected and image inverted is as follows: Figure 4 shown.

[0047] When it is necessary to obtain the fluorescence lifetime image of the sample to be tested, the sample to be tested needs to be placed on the sample table or the sample to be tested needs to be passed through the liquid flow system pipeline 16 in the form of a single cell through the liquid flow system. The light source module 1 generates a laser beam, which is incident on the heterodyne interference optical path module. The host computer 22 generates a preset RF drive parameter and sends it 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 sent to each spatial light modulator in the heterodyne interference optical path module through the beam splitter 11. Optionally, the beam splitter 11 can use a beam splitter prism. Two spatial light modulators 8 are used on the measuring arm of the heterodyne interference optical path module. The two spatial light modulators are 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 move the overall optical frequency of the incident laser beam to the vicinity of the lowest or highest optical frequency domain of the measuring arm, and then the light spot of the outgoing laser beam is incident on the beam expander 13 through the first reflector 9, and the light spot of the laser beam is expanded to meet the requirements of the subsequent beam homogenizer 14 for the size of the incident light spot. After the laser beam passes through the homogenizer 14, a reference arm homogenized beam is generated. The two-dimensional light spot array of the measuring arm passes through the second reflector 10 and is combined with the reference arm homogenized beam in the beam combiner 12 to generate an optical beat frequency, giving each light spot in the two-dimensional light spot array a unique signal envelope to achieve spatial radio frequency tagging. The combined light beam is optically reduced by the microscope objective 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 pipeline passage 16 of the liquid flow system to excite the specific position of the sample to be tested and form a light signal. The light signal is distributed to different channels using a dichroic mirror 17. Each channel uses a lens 18 to converge the light signal and then 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.

[0048] Optionally, a three-dimensional light spot array can be generated based on the two-dimensional light spot array. Specifically, a displacement device can be set between the beam combiner 12 and the light path beam shrinking module 15, and the displacement device is used to drive the light path beam shrinking module 15 to move forward and backward to achieve the forward and backward movement of the focus position of the two-dimensional light spot array, obtain a three-dimensional light spot array, and then obtain three-dimensional imaging data of the sample to be tested. The three-dimensional light spot array can also be combined with flow cytometry cell sorting as needed, and static samples to be tested or dynamic samples to be tested can be used.

[0049] 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 by 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 requirements 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 beam contraction, 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 that is spatially marked and incident on the sample to be tested. The light signal contains bright field light information and information marked by radio frequency space, which are all distributed to multiple different channels through the dichroic mirror 17, and 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 the A band is completed, the laser group can be actively or passively switched to the B band laser. After the image data acquisition corresponding to the B band laser is completed, it can be switched to the C band laser again, and switched in sequence until the image data acquisition of all required bands is completed.

[0050] 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 measurement 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.

[0051] Optionally, the beam splitter 11 adopts 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.

[0052] Obviously, the above embodiments are only examples for clear explanation, and are not intended to limit the implementation methods. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from them are still within the protection scope of the 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, used to generate radio frequency drive signals of multiple frequencies; A heterodyne interference optical path module is used to perform optical heterodyne interference processing on the laser beams according to the radio frequency driving signals of the multiple frequencies, respectively, to form a light spot array and a reference light source, and then combine the light beams 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, 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 fluorescently labeled in advance; 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.

2. The system according to claim 1, characterized in that 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 perform optical modulation processing on the first laser beam according to the radio frequency driving 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.

3. The system according to claim 2, characterized in that The measuring arm comprises a spatial light modulator, which is used to introduce a frequency shift to the first laser beam based on the frequency and amplitude of the corresponding radio frequency driving signal to generate a spot array; The reference arm comprises a beam shaping device for shaping the second laser beam into a reference light source.

4. The system according to any one of claims 1 to 3, 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 and displays the imaging data of the sample to be tested.

5. The system according to any one of claims 1 to 3, characterized in that: The system further comprises an optical path beam shrinking module, which is used for scaling the combined light beam to a target size and then incident on the sample to be tested.

6. The system according to any one of claims 1 to 3, characterized in that: 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.

7. The system according to any one of claims 1 to 3, 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, and 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.

8. The system according to any one of claims 1 to 3, 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.

9. The system according to any one of claims 1 to 3, characterized in that: The light source module also includes a bright field illumination light source; the bright field illumination light source is used to generate a bright field illumination light beam incident on the sample to be tested; The system also includes an imaging camera, which is used to collect a bright field image of the sample to be tested.

10. The system according to any one of claims 1 to 3, characterized in that: The sample to be tested is a single cell suspension; the system also includes a fluid system pipeline passage; the cells in the single cell suspension flow through the fluid system pipeline passage in sequence to be imaged.

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