Resonant scanning sparse structured light illumination based tomographic microscopy apparatus and method

Abstract

The application discloses a kind of based on resonance scanning sparse structured light illumination tomographic microscopic imaging device and method, the device includes illumination unit, scanning unit, microscopic imaging unit and acquisition reconstruction control unit: illumination unit under the control of the control signal generated by acquisition reconstruction control unit generates the illumination light of light intensity with spatial position variation;Scanning unit utilizes the control signal generated by acquisition reconstruction control unit, and the generation and phase shift of sparse scanning stripe structured light are completed by focus scanning;Microscopic imaging unit utilizes the control signal generated by acquisition reconstruction control unit, and the intensity image of different axial slices of sample under the illumination of different phase sparse scanning stripe structured light is collected;Acquisition reconstruction control unit is also used to reconstruct the obtained sample image stack, and obtains the three-dimensional tomographic microscopic image of sample.The application scans and images the same sample using different phase sparse scanning stripe structured light, and three-dimensional tomographic imaging of thick sample can be realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of microscopic imaging technology, and particularly relates to a tomographic microscopic imaging device and method based on resonant scanning sparse structured light illumination. BACKGROUND

[0002] Fluorescence microscopy has the advantages of small sample damage and specific imaging, and is widely used in the fields of biomedicine and material chemistry. However, a common wide-field fluorescence microscope uses parallel light to illuminate a sample, so that the entire fluorescent sample is excited. This situation leads to the loss of longitudinal resolution on the one hand, and the bleaching of fluorescent molecules on the other hand. In addition, out-of-focus noise in wide-field fluorescence microscopic imaging can cause the image of the focal plane position to be blurred, and the signal-to-noise ratio to be reduced. In view of the shortcomings of common wide-field fluorescence microscopy, the currently developed three-dimensional tomographic optical microscopic imaging technology mainly includes light sheet fluorescence microscopy (LSFM), structured light illumination microscopy (OS-SIM), laser scanning confocal microscopy (LSCM), and the like.

[0003] LSFM is a kind of microscopic imaging technology that can quickly perform three-dimensional tomography on a sample. LSFM uses a thin sheet-shaped light beam (referred to as a "light sheet") perpendicular to the imaging direction to illuminate the sample. Only the sample in the thin layer corresponding to the focal plane is excited to emit fluorescence, and other out-of-focus areas are not affected, effectively avoiding the generation of out-of-focus noise. Compared with common wide-field fluorescence microscopy, LSFM has the following advantages: (1) improving the image signal-to-noise ratio and axial resolution: the light sheet illumination technology ensures that the samples above and below the focal plane will not be excited, and has similar optical sectioning function as LSCM; (2) reducing photobleaching and phototoxicity: phototoxicity is reduced by 20-100 times compared with the common wide-field fluorescence excitation mode; (3) improving the imaging speed: based on point scanning imaging, laser three-dimensional tomographic microscopy and two-photon microscopy mostly use photomultiplier tubes (PMT) for point detection of fluorescence signals, while LSFM uses a CCD or sCMOS camera for area imaging, which greatly improves the imaging speed and is particularly suitable for large field imaging. The simplest method for LSFM to generate a light sheet is to introduce a cylindrical lens into the optical path. After the light beam passes through the lens, it remains unchanged in one dimension and is compressed in the other dimension, forming a light sheet on the focal plane. However, the light sheet generated by this method still satisfies the Gaussian distribution law, and the thickness of the light sheet is not uniform in the entire field of view, which easily leads to the uneven contrast of different regions in the same image, limiting its application in large field imaging.

[0004] OS-SIM is the second commonly used three-dimensional tomographic microscopic imaging technology. The principle of OS-SIM was proposed by Professor Neil M.A. of the University of Oxford in 1997. OS-SIM uses sinusoidal fringe field to illuminate the sample, and when the fringe frequency is high enough, only the sample part in the focal plane can load the fringe, and the out-of-focus area cannot. By means of fringe phase shift, the out-of-focus component that remains unchanged during phase shift can be eliminated, so that only the focal plane part forms the so-called "light sheet". OS-SIM has a light cutting ability comparable to LSCM, and overcomes the shortcomings of the latter such as slow imaging speed, complex structure, high cost, etc. However, the traditional OS-SIM uses wide-field fringe structured light, which is easily affected by sample scattering and degraded, resulting in limited imaging depth of OS-SIM, making it difficult to achieve large-depth imaging of thick samples.

[0005] In addition, LSCM is also an important three-dimensional tomographic microscopic imaging technology. LSCM usually uses a focused spot to scan the sample point by point, and the fluorescence generated by the sample is collected by the objective lens and finally imaged by the single-photon detector. A pinhole conjugate to the point source is placed in front of the detector to filter out the out-of-focus noise outside the focal point and only collect the fluorescence signal from the focal point. Compared with ordinary wide-field fluorescence microscopy, three-dimensional tomographic microscopy has higher axial resolution, which makes it have good optical tomographic ability and can detect different depth information of the sample. At present, LSCM has been widely used in industry, materials, and biomedical fields. However, due to the limitation of the scanning rate of the linear scanning galvanometer used in point scanning, LSCM cannot realize fast imaging of dynamic samples. At the same time, due to the existence of the physical pinhole in the imaging system, LSCM is not convenient to combine with other wide-field optical technologies. In addition, LSCM needs to use single-photon detection instruments such as avalanche photodiode (APD), which are generally expensive and easy to damage. The above shortcomings limit the application scenarios of LSCM. SUMMARY

[0006] In order to solve the above problems existing in the prior art, the present application provides a tomographic microscopic imaging device and method based on resonant scanning sparse structured light illumination. The technical problem to be solved by the present application is solved by the following technical scheme:

[0007] One aspect of the present application provides a tomographic microscopic imaging device based on resonant scanning sparse structured light illumination, comprising an illumination unit, a scanning unit, a microscopic imaging unit and a collection and reconstruction control unit, wherein,

[0008] The illumination unit, the scanning unit and the microscopic imaging unit are sequentially coupled and connected to form the overall optical path of the device;

[0009] The illumination unit is connected to the acquisition and reconstruction control unit, and is used for generating illumination light with light intensity varying with spatial position under the control of a control signal generated by the acquisition and reconstruction control unit.

[0010] The scanning unit is connected to the acquisition and reconstruction control unit, and is used for generating and phase-shifting sparse scanning fringe structure light through focal point scanning by using a control signal generated by the acquisition and reconstruction control unit.

[0011] The microscopic imaging unit is connected to the acquisition and reconstruction control unit, and is used for acquiring intensity images of different axial slices of the sample under illumination of sparse scanning fringe structure light at different phases, and synchronously transmitting the acquired intensity images to the acquisition and reconstruction control unit by using a control signal generated by the acquisition and reconstruction control unit.

[0012] The acquisition and reconstruction control unit is further used for reconstructing a tomographic microscopic image of the sample at a current axial slice by selecting a maximum light intensity value of each pixel in an image in an image stack, and finally acquiring a three-dimensional tomographic microscopic image of the sample in real time.

[0013] In an embodiment of the present application, the illumination unit comprises, in sequence along an optical path, a pulsed laser, an optical fiber and a beam expander collimator.

[0014] The scanning unit comprises, in sequence along an optical path, a resonant scanning galvanometer, a linear scanning galvanometer, a scanning lens and a first sleeve lens, the resonant scanning galvanometer and the linear scanning galvanometer are arranged in parallel and at a predetermined angle with respect to an optical axis, forming a two-dimensional resonant scanning galvanometer system, the scanning lens and the first sleeve lens form a telescope system, and the incident light beams of the resonant scanning galvanometer and the linear scanning galvanometer are conjugated and imaged to an entrance pupil of an objective lens, so as to ensure that the light intensity of a focused light spot of the sample at different positions is constant.

[0015] In an embodiment of the present application, the microscopic imaging unit comprises an objective lens, a dichroic mirror, a second sleeve lens, a band-pass filter and an sCOMS camera.

[0016] The dichroic mirror is obliquely arranged between the first sleeve lens and the objective lens, and can reflect parallel light from the first sleeve lens to the entrance pupil of the objective lens, and is also used for separating incident laser light from emitted fluorescence from the sample; the second sleeve lens, the band-pass filter and the sCOMS camera are sequentially arranged on the other side of the dichroic mirror away from the objective lens, and are used for collecting light from the sample for imaging.

[0017] In an embodiment of the present application, the acquisition and reconstruction control unit comprises a computer and a data acquisition control card.

[0018] The computer is used for controlling the data acquisition control card to generate control signals to realize control of light intensity of the pulsed laser, scanning process of the two-dimensional resonant scanning mirror system composed of the resonant scanning mirror and the linear scanning mirror, axial movement of the axial displacement table of the objective lens, and external trigger exposure image acquisition of the sCOMS camera, so as to complete real-time image acquisition.

[0019] The computer is further used for reconstructing image stacks of each layer in the thickness direction of the sample to obtain a three-dimensional tomographic microscopic image of the sample in real time.

[0020] In an embodiment of the present application, the data acquisition control card can generate a first analog voltage signal for controlling the resonant scanning mirror to scan the laser beam in a first direction perpendicular to the optical axis, and the scanning displacement function of the resonant scanning mirror is x(t) = A cos(2pft), wherein A is the amplitude, f is the resonant scanning frequency of the resonant scanning mirror, and t is the time; and the data acquisition control card can also generate a second analog voltage signal for controlling the linear scanning mirror to scan the laser beam in a second direction perpendicular to the first direction, and the scanning displacement function of the linear scanning mirror is y(t) = kt, wherein k determines the minimum scanning step length of the linear scanning mirror, and t is the time; the data acquisition control card is further used for completing time sequence line scanning synchronization of the first direction scanning and the second direction scanning by using a resonant mirror synchronous output signal, so as to form a sparse scanning fringe structure light.

[0021] In an embodiment of the present application, the data acquisition control card is further used for controlling the pulsed laser to be turned on only in a central region with uniform scanning light intensity and turned off in a boundary region with non-uniform scanning light intensity by modulating a TTL signal.

[0022] In an embodiment of the present application, the acquisition and reconstruction control unit is specifically used for:

[0023] obtaining image stacks under illumination of sparse scanning fringe structure light of different axial sections and different phases of the sample;

[0024] removing out-of-focus noise by subtracting two images at the same axial section with a distance of N / 2:

[0025]

[0026] wherein I i is an intensity image obtained by the i-th scanning in an image stack, I (i+N / 2) is an intensity image obtained by the i+N / 2-th scanning in an image stack, and I (i-N / 2)is the intensity image obtained in the i-th scan in the image stack, F i is the intensity image obtained in the i-th scan in the image stack, F i is the intensity image obtained in the i-th scan in the image stack, F

[0027] selecting the maximum light intensity value of each pixel in the image stack to reconstruct the tomographic image of the sample at the current axial slice, and finally obtaining the three-dimensional tomographic image of the sample in real time.

[0028] reconstructing the tomographic image of the sample at the current axial slice using the image stack obtained under the illumination of the sparse scanning stripe structured light at different axial slices of the sample and reconstructing the three-dimensional tomographic image of the sample using the tomographic images of different layers.

[0029] Another aspect of the present application provides a tomographic imaging method based on resonance scanning sparse structured light illumination, which is performed by using the tomographic imaging device according to any one of the above embodiments, and the method comprises the following steps:

[0030] S1: dropping an appropriate amount of distilled water on the water immersion objective lens and placing the sample, and moving the sample axially to clearly image it;

[0031] S2: under the control of the reconstruction control unit, collecting the intensity images of the sample at different axial slices under the illumination of the sparse scanning stripe structured light at different phases;

[0032] S3: using the obtained image stack of the sample at different axial slices and under the illumination of the sparse scanning stripe structured light at different phases, selecting the maximum light intensity value of each pixel in the image stack to reconstruct the tomographic image of the sample at the current axial slice, and finally obtaining the three-dimensional tomographic image of the sample in real time.

[0033] In one embodiment of the present application, the S3 comprises:

[0034] S3.1: obtaining the image stack of the sample at different axial slices and under the illumination of the sparse scanning stripe structured light at different phases;

[0035] S3.2: removing the defocus noise by subtracting two images at the same axial slice with a distance of N / 2:

[0036]

[0037] wherein I i is the intensity image obtained in the i-th scan in the image stack, F (i+N / 2) is the intensity image obtained in the i-th scan in the image stack, F (i-N / 2) is the intensity image obtained in the i-th scan in the image stack, F i is the intensity image obtained in the i-th scan in the image stack, F ithe intensity image after subtracting the out-of-focus noise, N is the total phase shift step number, i.e. the number of images in an image stack;

[0038] S3.3: selecting the maximum light intensity value of each pixel in the image stack to reconstruct the tomographic micrograph of the sample at the current axial slice;

[0039] S3.4: reconstructing the tomographic micrograph at the current axial slice using the image stack of different axial slices of the sample and reconstructing the three-dimensional tomographic micrograph of the sample using the tomographic micrographs of different layers.

[0040] In an embodiment of the present application, the S3.3 comprises:

[0041] For an image stack F obtained by scanning the current axial position i , the tomographic image of the sample at the current axial position is obtained using the image stack F i :

[0042] F sec (m,n)=Max{F i (m,n)} i

[0043] Wherein, i=1,2…N, F sec (m,n) represents the light intensity value of the tomographic micrograph F sec at the pixel point (m,n), Max{·} i represents the maximum light intensity value of the pixel at the point (m,n) in the image stack F i .

[0044] Compared with the prior art, the present application has the following beneficial effects:

[0045] 1. The three-dimensional tomographic micrograph imaging device based on resonance scanning and sparse structured light illumination of the present application forms a stripe structured light by scanning a focused spot, which has a greater penetration depth compared with the ordinary wide-field illumination microscopy method. At the same time, different phase sparse scanning stripe structured light is used to scan and image the same sample, which effectively overcomes the influence of the focal sidelobe on the scanning structured light modulation. This method uses the light intensity of each point on the sample under bright stripe illumination to subtract the light intensity under dark stripe illumination to suppress the out-of-focus noise and obtain the three-dimensional tomographic micrograph at the axial position. Compared with other types of structured light, using a focused spot to scan and generate stripe structured light can better overcome the scattering effect of the sample and realize three-dimensional tomographic imaging of thick samples.

[0046] 2. The application proposes a simpler light intensity modulation method to correct the non-uniformity of light intensity caused by resonant scanning under constant laser light intensity, that is, by modulating the TTL signal to control the laser to only turn on the laser in the central region of the scanning light intensity uniformity, and finally generate a sparse scanning stripe structure light with uniform light intensity in the field of view. At the same time, turning off the excess laser also helps to improve the signal-to-noise ratio of the fluorescent signal and obtain higher fluorescent image quality. In addition, the light intensity modulation method proposed in the embodiment of the application effectively reduces the signal output flux of the data acquisition / control card and the calculation amount of the computer for the light intensity modulation signal, which can effectively compress the cost of the device system and is more conducive to the popularization and use of industrialization.

[0047] 3. The embodiment of the application uses a surface array detector (sCOMS camera) for imaging, without using expensive APD / PMT point detectors, which is more conducive to industrial development. In addition, this wide-field detection method is convenient for combination with structured light illumination technology, single molecule localization and other wide-field technologies to further improve the lateral resolution.

[0048] The application will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 is a module schematic diagram of a tomographic microscopic imaging device based on resonant scanning sparse structured light illumination provided by the embodiment of the application;

[0050] Figure 2 is a light path principle diagram of a tomographic microscopic imaging device based on resonant scanning sparse structured light illumination provided by the embodiment of the application;

[0051] Figure 3 is a control signal timing diagram of a tomographic microscopic imaging device based on resonant scanning sparse structured light illumination provided by the embodiment of the application;

[0052] Figure 4 is an off-focus noise reduction operation schematic diagram of a tomographic microscopic imaging device based on resonant scanning sparse structured light illumination provided by the embodiment of the application;

[0053] Figure 5 is a sparse scanning stripe structure light period and phase shift measurement diagram of a tomographic microscopic imaging device based on resonant scanning sparse structured light illumination provided by the embodiment of the application;

[0054] Figure 6 is a comparison diagram of the axial resolution of the tomographic microscopic imaging method of the embodiment of the application and the existing three-dimensional scanning wide-field microscopic mode;

[0055] Figure 7 is a comparison diagram of the tomographic microscopic imaging method of the embodiment of the application and the existing three-dimensional scanning wide-field microscopic imaging on a step sample.

[0056] Reference Signs List:

[0057] 1-pulse laser; 2-optical fiber; 3-beam expanding collimator; 4-resonant scanning galvanometer; 5-linear scanning galvanometer; 6-scanning lens; 7-first sleeve lens; 8-sample; 9-objective lens; 10-dichroic mirror; 11-second sleeve lens; 12-band-pass filter; 13-sCOMS camera; 14-computer; 15-data acquisition control card. DETAILED DESCRIPTION

[0058] In order to further clarify the technical means and effects taken by the present application to achieve the predetermined object, the following will be described in detail in combination with the drawings and specific embodiments, and a kind of based on resonant scanning sparse structured light illumination tomographic microscopic imaging device and method according to the present application is described.

[0059] The foregoing and other technical contents, features and effects of the present application can be clearly presented in the specific embodiment detailed description below in combination with the drawings. Through the description of specific embodiments, the technical means and effects taken by the present application to achieve the predetermined object can be more deeply and specifically understood, however, the attached drawings are only provided for reference and description, and are not used to limit the technical solutions of the present application.

[0060] It should be noted that in this paper, such as first and second relationship terms are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between the entities or operations. Moreover, the term "include", "contain" or any other variant is intended to cover non-exclusive inclusion, so that the article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed. Without more limitation, the element defined by the sentence "including a" does not exclude the presence of other identical elements in the article or device including the element.

[0061] Embodiment One

[0062] Please see Figure 1 , Figure 1is a kind of based on resonance scanning sparse structured light illumination tomographic microscopic imaging device module schematic diagram provided in the embodiment of the present application.The tomographic microscopic imaging device includes illumination unit 101, scanning unit 102, microscopic imaging unit 103 and acquisition reconstruction control unit 104, wherein, illumination unit 101, scanning unit 102 and microscopic imaging unit 103 are sequentially coupled and connected to form the overall optical path of the device, and acquisition reconstruction control unit 104 completes the control of each instrument in the optical path, image acquisition and resonance three-dimensional tomographic microscopic image reconstruction processing.Specifically, illumination unit 101 is connected to acquisition reconstruction control unit 104, which uses the generated digital TTL (digital logic level) signal to modulate light intensity, and corrects the non-uniformity of light intensity caused by x-direction resonance scanning to generate illumination light with varying light intensity with spatial position, to realize the illumination of sample;Scanning unit 102 is connected to acquisition reconstruction control unit 104, which uses analog direct current signal (controls the first direction resonance mirror scanning) and analog step signal (controls the second direction linear vibration mirror scanning) generated by acquisition reconstruction control unit 104 to synchronously control the scanning of two directions of two-dimensional vibration mirror system, to complete the generation and phase shift of sparse scanning stripe structured light by focal point scanning;Microscopic imaging unit 103 is connected to acquisition reconstruction control unit 104, which acquires the intensity image of different axial slices of sample under the illumination of sparse scanning stripe structured light at different phases, and synchronously transmits the acquired intensity image to acquisition reconstruction control unit 104, and acquisition reconstruction control unit 104 is also used to use the obtained image stack of different axial slices of sample under the illumination of sparse scanning stripe structured light at different phases, select the light intensity maximum value of each pixel in an image stack to reconstruct the tomographic microscopic image of sample at current axial slice, and finally acquire the three-dimensional tomographic microscopic image of sample in real time.

[0063] In the embodiment, the idea of three-dimensional tomographic microscopic image reconstruction is as follows: by acquiring sparse scanning stripe structured light images at different phases in one direction, an image stack is formed and deblurring processing is performed, then the light intensity maximum value of each pixel in the image stack is selected as the light intensity value of a tomographic microscopic image at the pixel, and the three-dimensional tomographic microscopic image of sample is reconstructed in real time.

[0064] The sparse scanning fringe structured light illumination and imaging process is as follows: the focal point of the objective lens 9 is resonantly scanned in the x direction (f = 12 KHz), and in a complete scanning time period 1 / f = 83.3 μs (corresponding to one round-trip scanning motion in the +x direction), only the central region of the scanning light intensity is turned on to excite light in the +x direction scanning displacement, and the laser is turned off in the boundary region of the scanning light intensity, so that a uniform light intensity scanning bright line can be scanned in the x direction. By setting the linear scanning in the y direction, the focal point is scanned in the y direction into discontinuous and periodic P sparse scanning fringe structured light. In order to separate the sidelobes of the adjacent two bright lines from each other, P is generally 4-10 times the spatial resolution (full width at half maximum of the focal point) of the microscope system. Then, by shifting the sparse scanning fringe structured light along the y direction one by one (each time moving P / 12), a group of sparse scanning structured light images under complete 12-step phase shift are acquired by the sCOMS camera as an image stack.

[0065] The process of three-dimensional tomographic image reconstruction is as follows: for each pixel of the plane, the out-of-focus noise can be removed by subtracting the light intensity under dark fringe illumination from the light intensity under bright fringe illumination; then, the tomographic image of the axial plane can be obtained by screening the light intensity maximum value of each pixel using an image stack obtained by scanning each axial position (i.e. thickness direction) of the sample. By performing the above operation on each pixel of the plane, the tomographic image of the sample in the axial plane can be obtained. Then, the objective lens is moved axially, and the above operation is repeated for different axial planes of the sample, so that the three-dimensional tomographic image of the sample can be obtained.

[0066] Specifically, refer to Figure 2 , Figure 2 is a light path principle diagram of a three-dimensional tomographic microscopic imaging device based on resonant scanning and sparse structured light illumination provided by an embodiment of the present application. The illumination unit 101 comprises a laser 1, an optical fiber 2 and a beam expander collimator lens 3 arranged in sequence along the light path direction. The laser 1 of the present embodiment is a pulse laser, which is used as illumination laser, and the laser frequency and light output duty cycle thereof are adjustable, and the laser wavelength is 488 nm, the frequency adjustable range of the digital mode is set to 1-150 MHz, the pulse laser has an external trigger function and can receive a trigger signal from the control unit 102, the laser emitted by the laser 1 is introduced into the scanning light path system by the optical fiber 2, and is expanded and collimated into parallel light by the beam expander collimator lens 3. Preferably, the focal length of the beam expander collimator lens 3 is f3 = 100 mm.

[0067] Further, the scanning unit 102 comprises, in sequence along the optical path, a resonant scanning galvanometer 4, a linear scanning galvanometer 5, a scanning lens 6 and a first sleeve lens 7. The resonant scanning galvanometer 4 and the linear scanning galvanometer 5 are arranged in parallel and at a predetermined angle with the optical axis, forming a two-dimensional resonant scanning galvanometer system. The scanning lens 6 and the first sleeve lens 7 form a telescope system, which images the incident light beam of the resonant scanning galvanometer 4 and the linear scanning galvanometer 5 to the entrance pupil of the objective lens 9, so as to ensure that the light intensity of the focused light spot on the sample at different positions is constant. In this embodiment, the focal length of the scanning lens 6 is f6=100 mm, and the focal length of the first sleeve lens 7 is f7=200 mm.

[0068] The microscopic imaging unit 103 of this embodiment comprises an objective lens 9, a dichroic mirror 10, a second sleeve lens 11, a band-pass filter 12 and an sCOMS camera 13. The microscopic imaging unit 103 is mainly used to realize microscopic imaging of the sample, wherein the sample 8 is arranged at the focal point of the objective lens 9, the dichroic mirror 10 is arranged obliquely between the first sleeve lens 7 and the objective lens 9, and can reflect the parallel light from the first sleeve lens 7 to the entrance pupil of the objective lens 9, which is finally focused on the sample 8 by the objective lens 9. The second sleeve lens 11, the band-pass filter 12 and the sCOMS camera 13 are arranged in sequence on the other side opposite to the dichroic mirror 10 and the objective lens 9, which can receive the emitted fluorescence from the sample 8 and image on the sCOMS camera 13 to form a clear sample image. The band-pass filter 12 is used to filter out noise, and the dichroic mirror 10 is used to separate the incident laser and the emitted fluorescence. Preferably, the focal length of the second sleeve lens 11 is f 11 =400 mm.

[0069] Specifically, in the scanning optical path, the parallel light emitted from the beam expansion collimating lens 3 is scanned by the two-dimensional resonant scanning galvanometer system composed of the resonant scanning galvanometer 4 and the linear scanning galvanometer 5, and imaged by the telescope system composed of the scanning lens 6 and the first sleeve lens 7, and then reflected by the dichroic mirror 10 to the entrance pupil of the objective lens 9, and finally focused on the sample 8 by the objective lens 9.

[0070] In the microscopic imaging optical path, the emitted fluorescence from the sample 8 is finally imaged on the sCOMS camera 13 through the telescope system composed of the objective lens 9 and the sleeve lens 11, forming a clear sample image. The dichroic mirror 10 in between is used to separate the incident laser and the emitted fluorescence, and the band-pass filter 12 in front of the sCOMS camera 13 is used to filter out noise again to further improve the image signal-to-noise ratio.

[0071] Further, the acquisition reconstruction control unit 104 is used for synchronously controlling the illumination unit 101, the scanning unit 102 and the microscopic imaging unit 103. The acquisition reconstruction control unit 104 of the embodiment comprises a computer 14 and a data acquisition control card (DAQ) 15, wherein the computer 14 controls the data acquisition control card 15 through LabVIEW programming to generate the required control signals of the instruments in the optical path, so as to realize the light intensity modulation of the laser 1, the scanning control of the two-dimensional resonant scanning galvanometer system composed of the resonant scanning galvanometer 4 and the linear scanning galvanometer 5, the axial movement control of the axial displacement stage of the objective lens 9, and the external trigger exposure image acquisition control of the sCOMS camera, so as to complete real-time image acquisition; the computer 14 is also used for completing the reconstruction of the image stack at each axial position according to the reconstruction principle of the three-dimensional tomographic microscopic image, and then obtaining the three-dimensional tomographic microscopic image.

[0072] Further, please refer to Figure 3 , Figure 3 is a control signal timing diagram of a three-dimensional tomographic microscopic imaging device based on resonant scanning and sparse structured light illumination provided by the embodiment of the application.

[0073] For each frame of camera trigger signal, the camera trigger TTL signal is generated by the data acquisition card 15 to control the sCOMS camera to start exposure at the rising edge and end exposure at the falling edge. The high level time is equal to the scanning frame time, and the low level time is the image reading time. Wherein, the scanning frame time = the number of sparse scanning stripe periods x single row scanning time (resonant galvanometer synchronous output signal period).

[0074] For the resonant galvanometer scanning displacement (non-electrical signal), the curve function can be expressed as x(t) = A cos(2πft), A is the amplitude of the galvanometer, which determines the scanning range of the resonant scanning galvanometer; f is the resonant scanning frequency of the resonant scanning galvanometer, which is 12KHz; t is time. In the embodiment, only a direct current signal is generated by the data acquisition card 15 as an input voltage to start and control the scanning range (i.e. the input voltage is 0v in static state, and the input voltage is 5v in full-scale scanning). For scanning period and other parameters, they are all inherent parameters of the instrument, that is, for any scanning range, the scanning period (corresponding to one-way round-trip scanning motion in ±x direction) remains unchanged, which is always 1 / 12KHz = 83.3μs.

[0075] For the resonant scanner synchronous output signal, when the resonant scanner 4 is started by the direct current signal generated by the data acquisition card 15 as the input voltage, a strictly synchronous resonant scanner synchronous output signal (TTL signal) is generated inside the resonant scanner. The frequency of the TTL signal is consistent with the resonant scanner scanning frequency, and the duty cycle is 0.5, that is, it is always 1 / 12KHz = 83.3μs. Among them, in the full cycle, the resonant scanning mirror corresponds to a one-way scanning motion in the +x direction, that is, in the first half cycle, the resonant scanning mirror completes the displacement along the +x direction; in the second half cycle, it returns to the initial position along the -x direction. At the same time, only the first half cycle (1 / 12KHz*0.5 = 41.67μs) of the TTL signal is used as the effective signal in this embodiment, and the rising edge and falling edge of the signal respectively define the starting time and the termination time of the resonant scanner completing a one-way line scanning.

[0076] For the light intensity modulation signal, the signal is a group of TTL signals generated by the data acquisition 15, which is used to correct the non-uniformity of the light intensity caused by the non-uniform speed characteristics of the resonant scanning under the constant laser light intensity, that is, under the uniform light intensity and without modulating the light intensity signal, a non-uniform light intensity scanning line will be formed in the field of view, which is "bright at both ends and dark in the middle". According to the function expression of the resonant scanner scanning displacement (non-electrical signal), it is known that the main body is a cosine function, the entire resonant scanner scanning area can be divided into two parts: a non-uniform light intensity scanning area (scanning two end areas) and a uniform light intensity scanning area (scanning central area). According to experimental determination, the non-uniformity of the light intensity in the uniform light intensity scanning area can be ignored, and the uniform light intensity scanning area accounts for about 0.5. The modulation TTL signal is used as the light intensity modulation signal, and the laser is turned on in the uniform light intensity scanning area and turned off in the non-uniform light intensity scanning area, so that a uniform light intensity single scanning line can be obtained to correct the non-uniformity of the light intensity caused by the resonant scanning under the constant laser light intensity. It should be noted that in order to determine the accurate time of turning on / off the laser, according to the function expression of the resonant scanner scanning displacement (non-electrical signal), the monotonicity of the function in the uniform light intensity scanning area displacement with respect to time is used, and the starting position and the end position of the uniform light intensity scanning area displacement are known, the accurate time of turning on / off the laser corresponding to the uniform light intensity scanning area displacement can be calculated by the inverse function.

[0077] It should be emphasized that for fluorescence imaging, laser is one of the main sources of final noise, so using the light intensity modulation method to turn off the laser in time also helps to improve the signal-to-noise ratio of the fluorescence signal and obtain higher image quality.

[0078] For the linear galvanometer scanning signal, the signal is a step signal generated by the data acquisition card 15 to complete the linear galvanometer scanning in the y direction. Each level signal in the step represents a single scanning line corresponding to a position in the y direction. The number of step signal levels is equal to the number of sparse scanning stripe periods. By ensuring the precise timing synchronization of the camera trigger signal, the resonant galvanometer synchronous output signal, the light intensity modulation signal, and the linear galvanometer scanning signal, a uniform light intensity sparse scanning stripe structured light at a certain phase is finally generated in the field of view by using the light intensity accumulation effect during the camera exposure time. It should be noted that the scanning displacement function of the linear scanning galvanometer 5 can be represented as y(t) = kt, where k determines the minimum step size of the linear scanning galvanometer, and t is the time. In addition, in order to achieve the phase shift operation of the sparse scanning stripe structured light, only a phase shift voltage needs to be added to the amplitude of each level signal of the original linear scanning galvanometer scanning signal, and the amplitude interval of each level signal remains unchanged to ensure that the stripe structured light translates along the y direction. It should be noted that according to the foregoing control, the focal point of the objective lens 9 is scanned in the y direction to form a sparse scanning stripe structured light with discontinuous and equal periods P. For the specific parameter setting of the sparse scanning stripe structured light, in order to separate the sidelobes of adjacent two bright lines, P is generally 4-10 times the spatial resolution (full width at half maximum of the focal point) of the microscope system. Then, by moving the sparse stripe in the y direction by P / 12 each time, the phase shift operation of the sparse scanning stripe structured light is completed.

[0079] After that, the above signal control is repeated only by changing the linear scanning galvanometer scanning signal to complete the phase shift operation of the sparse scanning stripe structured light image, and a group of complete 12-step phase shift sparse scanning structured light images are collected to form an image stack.

[0080] In the specific operation process, the imaging process of the three-dimensional tomographic imaging device is as follows:

[0081] An appropriate amount of distilled water is added to the water immersion objective lens 9, and the sample 8 is placed; the sample 8 is axially moved to be clearly imaged on the plane z = 0 (i.e. the center position in the thickness direction of the sample 8); the axial scanning range is set from z = -10 μm to z = 10 μm, and the number of axial scanning layers is 100; the control program is started; for each axial scanning position, the sample 8 is subjected to x-y focal point scanning imaging, and 12 images under different phase sparse scanning stripe structured light illumination are recorded. The x-y scanning field is 103.2 x 103.2 μm 2 . The x direction is resonant scanning, and the y direction is linear scanning to obtain 43 sparse stripes with equal periods (period P is 2.4 μm). A sCOMS camera sequentially records the intensity images of the sample 8 under different sparse scanning stripe structured light illumination, denoted as I i(x,y),i=1,2…12. Subsequently, computer 14 was used to reconstruct the three-dimensional tomographic micrograph of sample 8.

[0082] In this embodiment, the acquisition and reconstruction control unit 104 is specifically used for:

[0083] Image stacks of sparse scanning stripe structured light illumination were obtained for slices of sample 8 with different axial directions and different phases;

[0084] Defocus noise can be removed by subtracting two images that are N / 2 apart at the same axial slice location:

[0085]

[0086] Among them, I i I is the intensity image obtained from the i-th scan in an image stack. (i-N / 2) Let I be the intensity image obtained from the iN / 2th scan in an image stack. (i+N / 2) F is the intensity image obtained from the (i+N / 2)th scan in an image stack. i For I i After removing out-of-focus noise, the intensity image is N=12, which is the total number of phase shift steps, i.e., the number of images in an image stack.

[0087] Select the maximum light intensity of each pixel in the acquired image in an image stack, and reconstruct the tomographic microscopy image of sample 8 at the current axial slice; reconstruct the tomographic microscopy image of the current axial slice using image stacks of different axial slices of sample 8, and reconstruct the three-dimensional tomographic microscopy imaging of sample 8 using tomographic microscopy images of different layers.

[0088] Specifically, for an image stack F obtained by scanning at a specific axial position. i (i = 1, 2…N), the light intensity reaches its maximum value when the same position on sample 8 is scanned only by sparse bright fringes. Therefore, in a tomographic image at an axial position, the intensity value of each pixel is the maximum value of that pixel in the phase-shifted image stack (i = 1, 2…N), thus obtaining:

[0089] F sec (m, n) = Max{F i (m, n)} i (2)

[0090] Among them, F sec (m,n) represents the tomographic micrograph F sec The light intensity value at pixel (m,n), Max{·} i In the image stack F i(i = 1, 2…N) selects the maximum light intensity value of the pixel of the (m, n) point. Repeat the same operation through all the pixels in the image, and finally use an image stack F i (i = 1, 2…N) obtains a tomographic micrograph F of the sample 8 at the axial position sec . Finally, the objective is axially moved, and 12 intensity images of the sample 8 under different phase sparse fringe illuminations are recorded in sequence by using the sCOMS camera at the current axial position, the calculation of formulas (1)-(2) is repeated, a tomographic micrograph at the current axial position is obtained, and then the objective is continuously axially moved, the above steps are repeated, and a plurality of tomographic micrographs at different axial positions are obtained. Finally, the three-dimensional tomographic micrograph of the sample can be reconstructed.

[0091] The embodiment of the present application scans and images the same sample by using different phase sparse scanning fringe structured light, and subtracts the light intensity of the sample at each point under dark fringe illumination from the light intensity under bright fringe illumination to suppress defocus noise, so as to obtain a three-dimensional tomographic micrograph at the axial position. Compared with other structured light methods, the focused spot is used to scan and generate fringe structured light, which can better overcome the scattering effect of the sample and realize three-dimensional tomographic imaging of the thick sample.

[0092] Embodiment two

[0093] On the basis of the above embodiment, the present embodiment provides a three-dimensional tomographic micrograph imaging method based on resonance scanning and sparse structured light illumination, which comprises the following steps:

[0094] S1: A proper amount of distilled water is added on the water immersion objective, and the sample is placed, the sample is axially moved, and the sample is clearly imaged on the plane of z = 0, where the plane of z = 0 refers to the central position in the thickness direction of the sample.

[0095] In the present embodiment, the axial scanning range is set from z = -10 μm to z = 10 μm, and the number of axial scanning layers is 100.

[0096] S2: Under the control of the reconstruction control unit, the intensity images of the sample at different axial slices under the irradiation of different phase sparse scanning fringe structured light are collected;

[0097] Specifically, for each axial scanning position, the sample is subjected to x-y focal point scanning imaging, and 12 images under different phase sparse fringe illuminations are recorded. The x-y scanning field is 103.2 x 103.2 μm 2 . The x direction is resonance scanning, and the y direction is linear scanning, so as to obtain 43 sparse fringes with equal periods (the period P is 2.4 μm). A sCOMS camera records the intensity images of the sample under different phase sparse fringe illuminations in sequence, which are denoted as I i (x, y), i = 1, 2…12.

[0098] S3: using the obtained image stacks of the sample at different axial slices and under different phase of the sparse scanning fringe structured light, selecting the maximum light intensity value of each pixel in the image in one image stack to reconstruct the tomographic micro-image of the sample at the current axial slice, and finally obtaining the three-dimensional tomographic micro-image of the sample in real time.

[0099] In one embodiment of the present application, the S3 comprises:

[0100] S3.1: obtaining the image stacks of the sample at different axial slices and under different phase of the sparse scanning fringe structured light;

[0101] S3.2: removing the out-of-focus noise by subtracting two images at the same axial slice with a distance of N / 2:

[0102]

[0103] wherein, I i is the intensity image obtained by the i-th scanning in one image stack, I (i-N / 2) is the intensity image obtained by the i-N / 2-th scanning in one image stack, I (i+N / 2) is the intensity image obtained by the i+N / 2-th scanning in one image stack, F i is the intensity image after removing the out-of-focus noise, and N is the total phase shift step number, i.e. the number of images in one image stack. i

[0104] S3.3: selecting the maximum light intensity value of each pixel in the image in one image stack to reconstruct the tomographic micro-image of the sample at the current axial position.

[0105] Specifically, for one image stack F i obtained by the scanning at the current axial position, the tomographic image of the sample at the current axial position is obtained by using the image stack F i :

[0106] F sec (m,n)=Max i {F i (m,n)} i

[0107] wherein, i=1,2…N, F sec (m,n) represents the light intensity value of the tomographic micro-image F sec at the pixel point (m,n), and Max{·} i represents selecting the maximum light intensity value of the pixel at the point (m,n) in the image stack F i .

[0108] ​S3.4: reconstructing a tomographic micrograph at the current axial slice using the image stack of different axial slices of the sample and reconstructing a three-dimensional tomographic micrograph of the sample using the tomographic micrographs of different layers.

[0109] The effect of the three-dimensional tomographic micrography device based on resonant scanning and sparse structured light illumination of the embodiment of the present application is verified by experiments. In the experiment, the magnification of the three-dimensional tomographic micrography device based on resonant scanning and sparse structured light illumination of the embodiment of the present application is 129 times. The number of pixel arrays of the sCMOS camera is 2048x2048, and the pixel size is 6.5μm. The corresponding size of the sCMOS camera pixel on the sample plane is about 50nm. Under the illumination of the 488nm laser, the numerical aperture NA of the water immersion objective is 1.2, which determines that the theoretical resolution of the device is 207nm (full width at half maximum of the focused spot), and the spatial resolution measured in the experiment is 308nm.

[0110] According to the foregoing control principle, the generation and phase shift of the sparse scanning stripe structured light with a period of 2.4μm and a total phase shift step number of 12 steps (to ensure that the single-step phase shift distance is less than the measured system resolution, that is, the focal point half width, that is, 2.4μm / 12=200nm≤308nm) can be completed.

[0111] Please refer to Figure 4 , Figure 4 is an out-of-focus noise reduction operation schematic diagram of a tomographic micrography device based on resonant scanning sparse structured light illumination provided by the embodiment of the present application, wherein I i is the intensity image obtained by the i-th scanning in an image stack, I (i-N / 2) is the intensity image obtained by the i-N / 2-th scanning in an image stack, I (i+N / 2) is the intensity image obtained by the i+N / 2-th scanning in an image stack, F i is I i is the intensity image after the out-of-focus noise reduction. In the embodiment, on the light intensity distribution mode of the sparse scanning stripe structured light (the period is 2.4μm, and the total phase shift step number is 12 steps), the brightness of the stripe area is higher (marked as ON), and the brightness between the two bright stripes is lower (marked as OFF). The proportion of the bright area in one period is about 1 / 12. According to the final out-of-focus noise reduction result, it can be known that the intensity of OFF for the position of the sample under the illumination of the sparse scanning stripe structured light mainly comes from the out-of-focus noise of the sample. By subtracting the intensity of the sample under the illumination of the ON and OFF of the stripe structured light, the out-of-focus noise can be effectively suppressed.

[0112] Please refer to Figure 5 , Figure 5is a sparse scanning fringe structure light period and phase determination map of a tomographic microscopic imaging device based on resonance scanning and sparse structure light illumination provided by the embodiment of the present application, wherein (a) is a sparse scanning fringe structure light image in each phase state in an image stack, and the sample is a broadband mirror with a reflection wavelength of 400-700 nm; (b) is a determination of the sparse scanning fringe structure light period and phase in an image stack, and the image scale is 20 μm. By 12 steps of phase shift, an image stack I i (x,y), i = 1, 2…12 is obtained. The 12 images in the image stack are superimposed and averaged The corresponding scanning wide-field image of the system can be obtained.

[0113] In order to determine the axial resolution of the three-dimensional tomographic microscopic imaging device based on resonance scanning and sparse structure light illumination provided by the embodiment of the present application, a single-layer fluorescent bead sample with a diameter of 250 nm, an excitation wavelength of 488 nm and an emission wavelength of 520 nm is subjected to resonance scanning three-dimensional tomographic microscopy and three-dimensional scanning wide-field microscopic imaging in the experiment. Please refer to Figure 6 , Figure 6 is a comparison diagram of the tomographic microscopic imaging method of the embodiment of the present application and the axial resolution of the existing three-dimensional scanning wide-field microscopic mode, wherein (a) is a resonance scanning three-dimensional tomographic microscopic image of a single-layer fluorescent bead at different axial positions; (b) is an xz cross-sectional image (upper) and an axial intensity distribution along the center of the bead (lower) of a three-dimensional tomographic image of a single-layer fluorescent bead; (c) is an xz cross-sectional image (upper) and an axial intensity distribution along the center of the bead (lower) of a three-dimensional scanning wide-field microscopic image of a single-layer fluorescent bead; the axial scanning step is 0.2 μm, and the number of scanning layers is 100. By comparing Figure 6 (b) and Figure 6 (c), it can be found that the image obtained by the method provided by the embodiment of the present application has a lower background noise. By quantitatively analyzing the axial intensity distribution of the bead on the cross section, it is found that the axial resolution of the three-dimensional tomographic microscopic imaging method based on resonance scanning and sparse structure light illumination is 1.38±0.25 μm, and the axial resolution of the three-dimensional scanning wide-field microscopic imaging is 1.79±0.15 μm. The determination result shows that the axial resolution of the method is about 1.19-1.45 times higher than that of the three-dimensional scanning wide-field microscopy.

[0114] In addition, the experiment of the embodiment of the present application also uses the three-dimensional tomographic microscopic imaging method based on resonance scanning and sparse structure light illumination and the scanning wide-field imaging to image a step sample, and the imaging results are respectively as Figure 7As shown in the figure, the left side is the image of the sample at different axial positions in the existing three-dimensional scanning wide-field microscopic imaging mode; the right side is the three-dimensional tomographic microscopic image of the sample obtained using the method proposed in the embodiment of the present application; the scale is 20 μm. The experimental results show that, compared with three-dimensional scanning wide-field microscopic imaging, the three-dimensional tomographic microscopic imaging method based on resonant scanning and sparse structured light illumination proposed in the embodiment of the present application effectively suppresses the out-of-focus noise in imaging, and has good tomographic capability.

[0115] The embodiment of the present application uses a resonant scanning galvanometer (the scanning frequency of which can be up to 12 KHz), and the imaging speed of the method proposed is tens of times higher than that of the traditional confocal microscopic imaging. Taking a traditional confocal microscope as an example, assuming that the spatial resolution of the system is 308 nm and the imaging field of view is 103.2 μm x 103.2 μm, according to the Nyquist sampling theorem, the confocal scanning step is generally set to 308 nm / 2.3 = 134 nm, and the number of single-direction scanning pixels of the confocal microscope is 103.2 μm / 0.134 μm = 770. 2 If the single-pixel dwell time is 30 μs, the imaging frame time of the traditional confocal microscope is (103.2 / 0.308)

[0116] The scanning speed of the method proposed in the embodiment of the present application in the x direction is 12 KHz / line, that is, the period of scanning a line is about 83.3 μs. Among them, the stripe period is 2.4 μm, the number of stripe periods in a single-step phase shift image is 103.2 μm / 2.4 μm = 43, and the number of phase shift steps is 12 steps of phase shift. According to the foregoing three-dimensional tomographic method of the present application, the time consumption is about 12 x 43 x 83.3 μs = 43 ms.

[0117] The imaging speed of the method proposed in the embodiment of the present application is about 40 times higher than that of the confocal microscope: the effective improvement of the imaging speed will greatly expand the application scenarios of the three-dimensional tomographic microscope, and will effectively improve the detection efficiency of sample rapid detection in the industrial and biological fields.

[0118] The embodiment of the present application proposes a simpler light intensity modulation method, that is, by modulating the TTL signal to control the laser to be turned on only in the central region where the scanning light intensity is uniform, and to be turned off in the boundary region where the scanning light intensity is non-uniform, so as to solve the problem of non-uniform light intensity caused by resonant scanning under constant laser light intensity, and finally generate a uniform light intensity sparse scanning stripe structured light in the field of view. At the same time, turning off the excess laser also helps to improve the signal-to-noise ratio of the fluorescent signal and obtain higher fluorescent image quality. In addition, the light intensity modulation method proposed in the embodiment of the present application effectively reduces the signal output flux of the data acquisition / control card and the calculation amount of the computer for the light intensity modulation signal, can effectively compress the cost of the device system, and is more conducive to the popularization and use of industrialization.

[0119] In addition, the embodiment of the present application uses a surface array detector (sCOMS camera) for imaging, without using expensive point detectors such as APD / PMT, and is more conducive to industrial development. In addition, the wide-field detection method is convenient for being combined with structured light illumination technology and single molecule localization technology to further improve the lateral resolution.

[0120] The above is a further detailed description of the present application in combination with specific preferred embodiments, and the specific implementation of the present application should not be limited to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, a number of simple deductions or substitutions can be made without departing from the concept of the present application, and all of them should be considered as falling within the protection scope of the present application.

Claims

1. A tomographic microscopic imaging apparatus based on resonant scanning sparse structured light illumination, characterized by, The device comprises an illumination unit (101), a scanning unit (102), a microscopic imaging unit (103) and a collection and reconstruction control unit (104), wherein, The illumination unit (101), the scanning unit (102) and the microscopic imaging unit (103) are sequentially coupled to form the whole optical path of the device; The illumination unit (101) is connected to the collection and reconstruction control unit (104) to generate illumination light with spatially-varying light intensity under the control of the control signal generated by the collection and reconstruction control unit (104); The scanning unit (102) is connected to the collection and reconstruction control unit (104) to generate and phase-shift the sparse scanning fringe structure light by focal point scanning under the control of the control signal generated by the collection and reconstruction control unit (104); The microscopic imaging unit (103) is connected to the collection and reconstruction control unit (104) to collect the intensity images of different axial slices of the sample under the illumination of the sparse scanning fringe structure light at different phases and synchronously transmit the collected intensity images to the collection and reconstruction control unit (104) under the control of the control signal generated by the collection and reconstruction control unit (104); The collection and reconstruction control unit (104) is further used to reconstruct the tomographic microscopic image of the sample at the current axial slice by selecting the maximum light intensity of each pixel in the image in one image stack and finally obtain the three-dimensional tomographic microscopic image of the sample in real time. The collection and reconstruction control unit (104) is specifically used to: obtain the image stack of different axial slices of the sample under the illumination of the sparse scanning fringe structure light at different phases; remove the out-of-focus noise by subtracting two images at the same axial slice with a distance of N / 2: wherein, is the intensity image obtained from the first scan of the image stack, i is the intensity image obtained from the Nth scan of the image stack, is the intensity image obtained from the first scan of the image stack, i is the intensity image obtained from the N / 2th scan of the image stack, is the intensity image obtained from the first scan of the image stack, i is the intensity image obtained from the -N / 2th scan of the image stack, is the intensity image obtained from the Nth scan of the image stack, is the intensity image obtained from the Nth scan of the image stack, select the maximum light intensity of each pixel in the collected image in one image stack to reconstruct the tomographic microscopic image of the sample at the current axial slice; reconstruct the tomographic microscopic image at the current axial slice by using the image stack at different axial slices of the sample and reconstruct the three-dimensional tomographic microscopic image of the sample by using the tomographic microscopic images of different layers.

2. The tomographic microscopic imaging apparatus based on resonant scanning sparse structured light illumination according to claim 1, wherein, The illumination unit (101) comprises a pulsed laser (1), an optical fiber (2) and a beam expanding and collimating lens (3) arranged in sequence along the optical path direction, wherein, The scanning unit (102) comprises a resonant scanning galvanometer (4), a linear scanning galvanometer (5), a scanning lens (6) and a first sleeve lens (7) arranged in sequence along the optical path direction, the resonant scanning galvanometer (4) and the linear scanning galvanometer (5) are arranged in parallel and at a predetermined angle with the optical axis to form a two-dimensional resonant scanning galvanometer system, the scanning lens (6) and the first sleeve lens (7) form a telescope system to image the incident light beams of the resonant scanning galvanometer (4) and the linear scanning galvanometer (5) to the entrance pupil of the objective lens (9) to ensure that the light intensity of the focused light spot of the sample at different positions is constant.

3. The tomographic microscopic imaging apparatus based on resonant scanning sparse structured light illumination according to claim 2, wherein, The microscopic imaging unit (103) comprises an objective lens (9), a dichroic mirror (10), a second sleeve lens (11), a band-pass filter (12) and an sCOMS camera (13), wherein, The dichroic mirror (10) is obliquely arranged between the first sleeve lens (7) and the objective lens (9), can reflect the parallel light from the first sleeve lens (7) to the entrance pupil of the objective lens (9), and is also used for separating the incident laser light from the emitted fluorescence from the sample; the second sleeve lens (11), the band-pass filter (12) and the sCOMS camera (13) are sequentially arranged on the other side of the dichroic mirror (10) away from the objective lens (9), and are used for collecting the light from the sample for imaging.

4. The tomographic microscopic imaging apparatus based on resonant scanning sparse structured light illumination according to claim 3, wherein, The acquisition and reconstruction control unit (104) comprises a computer (14) and a data acquisition control card (15), wherein, The computer (14) is used for controlling the data acquisition control card (15) to generate a control signal, and controlling the light intensity of the pulsed laser (1), the scanning process of the two-dimensional resonant scanning galvanometer system composed of the resonant scanning galvanometer (4) and the linear scanning galvanometer (5), the axial movement of the axial displacement stage of the objective lens (9), and the external trigger exposure image acquisition of the sCOMS camera (13), so as to complete real-time image acquisition; The computer (14) is also used for reconstructing the image stack of each layer in the thickness direction of the sample, and acquiring the three-dimensional tomographic microscopic image of the sample in real time.

5. The tomographic microscopic imaging apparatus based on resonant scanning sparse structured light illumination according to claim 4, wherein, The data acquisition control card (15) can generate a first analog voltage signal for controlling the resonant scanning galvanometer (4) to scan the laser beam along a first direction perpendicular to the optical axis, and the scanning displacement function of the resonant scanning galvanometer (4) is x ( t ) = A cos(2π ft ), wherein, A is the amplitude, f is the resonant scanning frequency of the resonant scanning galvanometer, t is the time; and the data acquisition control card (15) can also generate a second analog voltage signal for controlling the linear scanning galvanometer (5) to scan the laser beam along a second direction perpendicular to the first direction, and the scanning displacement function of the linear scanning galvanometer (5) is y ( t ) = kt, wherein, k determines the minimum step length of the linear scanning galvanometer scanning, t is the time; the data acquisition control card (15) is also used for completing the timing line scanning synchronization of the first direction scanning and the second direction scanning by using the resonant mirror synchronous output signal, so as to form the sparse scanning fringe structure light.

6. The tomographic microscopic imaging apparatus based on resonant scanning sparse structured light illumination according to claim 4, wherein, The data acquisition control card (15) is also used for controlling the pulsed laser (1) to start the laser only in the central region with uniform scanning light intensity and to stop the laser in the boundary region with non-uniform scanning light intensity by modulating the TTL signal.

7. A method of tomographic microscopic imaging based on resonant scanning sparse structured light illumination, characterized in that, The method is performed by using the tomographic microscopic imaging device of any one of claims 1 to 6, and the method comprises: S1: dropping a proper amount of distilled water on the water immersion objective lens and placing the sample, and axially moving the sample to clearly image it; S2: under the control of the acquisition and reconstruction control unit, collecting the intensity images of different axial sections of the sample under the irradiation of sparse scanning stripe structured light at different phases; S3: using the obtained image stack of the sample at different axial sections and under the irradiation of sparse scanning stripe structured light at different phases, selecting the maximum light intensity of each pixel in an image stack to reconstruct the tomographic microscopic image of the sample at the current axial section, and finally acquiring the three-dimensional tomographic microscopic image of the sample in real time.

8. The method of tomographic microscopic imaging based on resonant scanning sparse structured illumination of claim 7, wherein, The S3 comprises: S3.1: obtaining the image stack of the sample at different axial sections and under the irradiation of sparse scanning stripe structured light at different phases; S3.2: removing the out-of-focus noise by subtracting two images at the same axial section with a distance of N / 2: wherein is the intensity image obtained at the i is the intensity image obtained at the is the intensity image obtained at the i is the intensity image obtained at the is the intensity image obtained at the i is the intensity image obtained at the F i is the intensity image obtained at the is the intensity image obtained at the S3.3: selecting the maximum light intensity of each pixel in an image stack to reconstruct the tomographic microscopic image of the sample at the current axial section; S3.4: reconstructing the tomographic microscopic image at the current axial section by using the image stack of the sample at different axial sections, and reconstructing the three-dimensional tomographic microscopic image of the sample by using the tomographic microscopic images of different layers.

9. The tomographic microscopic imaging method based on resonant scanning sparse structured light illumination of claim 8, wherein, The S3.3 comprises: for the current axial position scan F i obtaining a tomographic image of the sample at the current axial position using said image stack F i for the current axial position scan F sec ( m , n ) = Max{ i ( m , n )} i wherein, i = 1, 2… N , F sec ( m , n ) denotes a tomographic microscopic image F sec the light intensity value at pixel point ( m , n ), Max{·} i denotes the maximum light intensity value of the pixels of the image stack F i selecting the maximum light intensity value of the pixels of the points ( m , n ).

Images