CN115839935A - Tomography microscopic imaging device and method based on resonance scanning sparse structured light illumination - Google Patents
Tomography microscopic imaging device and method based on resonance scanning sparse structured light illumination Download PDFInfo
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Abstract
The invention discloses a tomography microscopic imaging device and method based on resonance scanning sparse structure light illumination, wherein the device comprises an illumination unit, a scanning unit, a microscopic imaging unit and an acquisition reconstruction control unit: the illumination unit generates illumination light with light intensity changing with the space position under the control of the control signal generated by the acquisition and reconstruction control unit; the scanning unit completes the generation and phase shift of sparse scanning stripe structured light through focus scanning by utilizing a control signal generated by the acquisition and reconstruction control unit; the microscopic imaging unit acquires intensity images of different axial slices of the sample under the irradiation of the sparse scanning stripe structure light with different phases by using a control signal generated by the acquisition and reconstruction control unit; the acquisition and reconstruction control unit is also used for reconstructing the obtained sample image stack to obtain a three-dimensional chromatographic microscopic image of the sample. The invention adopts the sparse scanning stripe structure light with different phases to scan and image the same sample, and can realize the three-dimensional tomography of the thick sample.
Description
Technical Field
The invention belongs to the technical field of microscopic imaging, and particularly relates to a tomography microscopic imaging device and method based on resonance scanning sparse structure light illumination.
Background
The fluorescence microscopy has the advantages of small damage to samples, specific imaging and the like, and is widely applied to the fields of biomedicine, material chemistry and the like. However, a common wide-field fluorescence microscope illuminates the sample with parallel light such that the entire fluorescent sample is excited, which results on the one hand in a loss of longitudinal resolving power and on the other hand in bleaching of the fluorescent molecules. In addition, defocusing noise in wide-field fluorescence microscopy can cause image blurring at the focal plane position, and the signal-to-noise ratio is reduced. Aiming at the defects of common wide-field fluorescence microscopy, the currently developed three-dimensional chromatography optical microscopy imaging technology mainly comprises Light Sheet Fluorescence Microscopy (LSFM), structured light illumination microscopy (OS-SIM), laser Scanning Confocal Microscopy (LSCM) and the like.
LSFM is a microscopic imaging technique that can perform rapid three-dimensional tomography on a sample. The LSFM adopts a sheet-shaped light beam (called as a 'light sheet') vertical to the imaging direction to illuminate a sample, only a thin layer of the sample corresponding to a focal plane is excited to emit fluorescence, other defocusing areas are not affected, and the generation of defocusing noise is effectively avoided. Compared with the common wide-field fluorescence microscopy, the LSFM has the following advantages: (1) the image signal-to-noise ratio and the axial resolution are improved: the light sheet illumination technology ensures that samples above and below a focal plane cannot be excited, and has an optical slicing function similar to LSCM; (2) reduced photobleaching and phototoxicity: the phototoxicity is reduced by 20-100 times compared with the common wide-field fluorescence excitation mode; (3) the imaging speed is improved: laser three-dimensional tomography microscopy and two-photon microscopy based on point scanning imaging mostly use a photomultiplier tube (PMT) to detect points of fluorescent signals, LSFM uses a CCD or sCMOS camera to perform area array imaging, the imaging speed is greatly improved, and the laser three-dimensional tomography microscopy and two-photon microscopy are particularly suitable for large-field imaging. The simplest way LSFM produces a light sheet is to introduce a cylindrical lens in the optical path through which the beam passes, remaining unchanged in one dimension, and compressed in the other dimension to form the light sheet in the focal plane. However, the light sheet generated by the method still satisfies the gaussian distribution rule, the thickness of the light sheet in the whole field of view is not uniform, the situation that the contrast of different areas of the same image is not uniform easily occurs, and the application of the light sheet in the aspect of large-field-of-view imaging is limited.
OS-SIM is the second commonly used three-dimensional tomographic microscopy imaging technique. The principle of OS-SIM was proposed in 1997 by professor Neil m.a. of oxford university, uk. The OS-SIM illuminates the sample with a sinusoidal fringe light field, when the fringe frequency is high enough, only the portion of the sample in the focal plane can be loaded with fringes and the out-of-focus region is not. By means of the fringe phase shift, the defocus component, which remains unchanged during the phase shift, can be eliminated, leaving only the focal plane portion forming a so-called "light sheet". The OS-SIM has a light-cutting capability comparable to that of the LSCM, and overcomes the defects of slow imaging speed, complex structure, high manufacturing cost and the like of the LSCM. However, the conventional OS-SIM employs wide-field stripe structured light, which is easily degraded by sample scattering, resulting in a limited imaging depth of the OS-SIM, and it is difficult to realize large-depth imaging of a thick sample.
Furthermore, LSCM is also an important three-dimensional tomographic microscopy imaging technique. LSCM usually scans a sample point by using a focused light spot, fluorescence generated by the sample is collected by an objective lens and finally reaches a single photon detector for imaging, and a pinhole conjugated with a point light source is placed in front of the detector to filter out-of-focus noise in an area outside a focus and only collect a fluorescence signal from the focus. Compared with the common wide-field fluorescence microscopy, the three-dimensional chromatography microscopy has higher axial resolution, and due to the characteristic, the three-dimensional chromatography microscopy has good optical chromatography capability and can detect different depth information of a sample. At present, LSCM is widely used in industrial, material, and biomedical fields. However, LSCM does not allow for rapid imaging of dynamic samples, limited by the scanning rate of the linear scanning galvanometer used for spot scanning. Also, LSCM does not facilitate organic integration with other wide-field optical techniques due to the presence of physical pinholes in the imaging system. Furthermore, LSCM requires the use of single photon detection devices such as Avalanche Photodiodes (APDs), which are generally expensive and easily damaged. The above disadvantages limit the application scenarios of LSCM.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a tomography microscopic imaging device and method based on resonance scanning sparse structured light illumination. The technical problem to be solved by the invention is realized by the following technical scheme:
one aspect of the invention provides a tomography microscopic imaging device based on resonance scanning sparse structured light illumination, which comprises an illumination unit, a scanning unit, a microscopic imaging unit and an acquisition reconstruction control unit, wherein,
the illumination unit, the scanning unit and the microscopic imaging unit are sequentially coupled to form an optical path whole of the device;
the illumination unit is connected with the acquisition and reconstruction control unit and is used for generating illumination light with light intensity changing with the space position under the control of the control signal generated by the acquisition and reconstruction control unit;
the scanning unit is connected with the acquisition and reconstruction control unit and is used for completing the generation and phase shift of sparse scanning stripe structure light through focus scanning by utilizing a control signal generated by the acquisition and reconstruction control unit;
the microscopic imaging unit is connected with the acquisition and reconstruction control unit and is used for acquiring intensity images of different axial slices of the sample under the irradiation of the sparse scanning stripe structure light with different phases by using a control signal generated by the acquisition and reconstruction control unit and synchronously transmitting the acquired intensity images to the acquisition and reconstruction control unit;
the acquisition and reconstruction control unit is also used for reconstructing a chromatographic microscopic image of the sample at the current axial section by using the obtained image stacks irradiated by the sparse scanning stripe structure light with different axial sections and different phases of the sample, and selecting the maximum light intensity value of each pixel in the image in one image stack, so as to finally obtain the three-dimensional chromatographic microscopic image of the sample in real time.
In one embodiment of the present invention, the illumination unit comprises a pulse laser, an optical fiber and a beam expanding and collimating lens, which are sequentially arranged along an optical path direction, wherein,
the scanning unit includes along resonance scanning galvanometer, linear scanning galvanometer, scanning lens and the first sleeve lens that set gradually of light path direction, resonance scanning galvanometer with linear scanning galvanometer parallel arrangement just is predetermined angle with the optical axis, constitutes two-dimentional resonance scanning galvanometer system that shakes, scanning lens with the telescope system is constituteed to first sleeve lens, will resonance scanning galvanometer with the incident beam conjugate imaging of linear scanning galvanometer is to the income pupil department of objective to guarantee that the light intensity of sample focus facula is unchangeable in different positions department.
In one embodiment of the invention, the microscopic imaging unit comprises an objective lens, a dichroic mirror, a second sleeve lens, a band pass filter and an sscmos camera, wherein,
the dichroic mirror is obliquely arranged between the first sleeve lens and the objective lens, can reflect and transmit parallel light from the first sleeve lens to the entrance pupil of the objective lens, and is also used for realizing the separation of incident laser light and emitted fluorescence light from a sample; the second sleeve lens, the band-pass filter and the sCOMS camera are sequentially arranged on the other side, away from the objective lens, of the dichroic mirror and used for collecting light from a sample to perform imaging.
In one embodiment of the invention, the acquisition reconstruction control unit comprises a computer and a data acquisition control card, wherein,
the computer is used for controlling the data acquisition control card to generate a control signal, so as to realize the scanning process of a two-dimensional resonance scanning galvanometer system consisting of the resonance scanning galvanometer and the linear scanning galvanometer, the axial movement of an axial displacement table of the objective lens and the acquisition of an external trigger exposure image of the sCOMS camera, thereby completing the acquisition of a real-time image;
and the computer is also used for reconstructing the image stack of each layer in the thickness direction of the sample and acquiring the three-dimensional chromatographic microscopic image of the sample in real time.
In an embodiment of the present invention, the data acquisition control card is capable of generating a first analog voltage signal for controlling the resonant scanning galvanometer to scan the laser beam along a first direction perpendicular to an optical axis, where a is amplitude, f is resonant scanning frequency of the resonant scanning galvanometer, and t is time, and a scanning displacement function of the resonant scanning galvanometer is x (t) = Acos (2 pi ft); the data acquisition control card can also generate a second analog voltage signal for controlling the linear scanning galvanometer to scan the laser beam along a second direction perpendicular to the first direction, and the scanning displacement function of the linear scanning galvanometer is y (t) = kt, wherein k determines the minimum step length of scanning of the linear scanning galvanometer, and t is time; the data acquisition control card is also used for completing the time sequence line scanning synchronization of the first direction scanning and the second direction scanning by utilizing the synchronous output signal of the resonance galvanometer, thereby forming sparse scanning stripe structured light.
In an embodiment of the invention, the data acquisition control card is further configured to control the pulse laser to turn on the laser only in a central region where the scanning intensity is uniform and turn off the laser only in a boundary region where the scanning intensity is non-uniform by modulating the TTL signal.
In an embodiment of the present invention, the acquisition reconstruction control unit is specifically configured to:
obtaining image stacks of samples which are sliced in different axial directions and are irradiated by sparse scanning stripe structure light with different phases;
the two images at the same axial slice with a distance of N/2 are subtracted to remove the out-of-focus noise:
wherein, I i Intensity images obtained for the ith scan in an image stack, I (i+N/2) Intensity images obtained for the I + N/2 th scan of an image stack, I (i-N/2) Intensity images obtained for the i-N/2 th scan in an image stack, F i Is I i Intensity image after defocus noise subtraction, N being totalThe number of phase shift steps, i.e. the number of images in an image stack;
selecting the maximum light intensity value of each pixel in the collected image in an image stack, and reconstructing a chromatographic microscopic image of the sample at the current axial slice;
and reconstructing a tomography microscopic image at the current axial section by using the image stack at different axial sections of the sample and reconstructing three-dimensional tomography microscopic imaging of the sample by using the tomography microscopic images of different layers.
Another aspect of the present invention provides a tomography microscopic imaging method based on resonance scanning sparse structured light illumination, which is performed by using the tomography microscopic imaging apparatus described in any one of the above embodiments, and the method includes:
s1: dripping a proper amount of distilled water on the water immersion objective lens, placing a sample, and axially moving the sample to enable the sample to be imaged clearly;
s2: under the control of the acquisition and reconstruction control unit, acquiring intensity images of different axial slices of the sample under the irradiation of sparse scanning stripe structure light of different phases;
s3: and (3) utilizing image stacks of the obtained samples under the irradiation of the sparse scanning stripe structure light with different axial slices and different phases, selecting the maximum light intensity value of each pixel in the image in one image stack to reconstruct the chromatographic microscopic image of the sample at the current axial slice, and finally obtaining the three-dimensional chromatographic microscopic image of the sample in real time.
In one embodiment of the present invention, the S3 includes:
s3.1: obtaining image stacks of samples which are sliced in different axial directions and are irradiated by sparse scanning stripe structure light with different phases;
s3.2: the two images at the same axial slice with a distance of N/2 are subtracted to remove the out-of-focus noise:
wherein, I i Intensity images obtained for the ith scan in an image stack, I (i+N/2) For an image pileIntensity image, I, from the I + N/2 th scan in the stack (i-N/2) Intensity images obtained for the i-N/2 th scan of an image stack, F i Is I i Subtracting the intensity image after the defocusing noise, wherein N is the total phase shift step number, namely the number of images in one image stack;
s3.3: selecting the maximum light intensity value of each pixel in the collected image in an image stack, and reconstructing a chromatographic microscopic image of the sample at the current axial slice;
s3.4: and reconstructing a tomography microscopic image at the current axial slice by using the image stack of different axial slices of the sample and reconstructing three-dimensional tomography microscopic imaging of the sample by using the tomography microscopic images of different layers.
In one embodiment of the present invention, said S3.3 comprises:
one image stack F obtained for the current axial position scan i Using said image stack F i Obtaining a tomographic image of the sample at the current axial position:
F sec (m,n)＝Max{F i (m,n)} i
wherein i =1,2 \ 8230n, F sec (m, n) represents a tomographic microscopic image F sec Light intensity value at a pixel point (m, n), max {. The i Is shown in image stack F i The maximum intensity value of the pixel at the (m, n) point is selected.
Compared with the prior art, the invention has the beneficial effects that:
1. the three-dimensional chromatographic microscopic imaging device based on resonance scanning and sparse structured light illumination forms stripe structured light by scanning and focusing light spots, and has larger penetration depth compared with a common wide-field illumination microscopic method. Meanwhile, the same sample is scanned and imaged by adopting sparse scanning stripe structure light with different phases, so that the influence of focus side lobes on a light modulation system of a scanning structure is effectively overcome. According to the method, the defocusing noise is suppressed by subtracting the light intensity of each point on the sample during the illumination of the bright stripes from the light intensity of each point during the illumination of the dark stripes, and the three-dimensional chromatographic microscopic image at the axial position is obtained. Compared with other types of structured light, the method has the advantages that the focusing light spots are used for scanning to generate the stripe structured light, so that the scattering effect of the sample can be better overcome, and the three-dimensional tomography of the thick sample is realized.
2. The invention provides a simpler light intensity modulation mode to correct light intensity nonuniformity caused by resonance scanning under constant laser light intensity, namely, a laser is controlled to be started only in a central area with uniform scanning light intensity by modulating TTL (transistor-transistor logic) signals, and finally sparse scanning stripe structured light with uniform light intensity is generated in a field of view. Meanwhile, the turning off of the redundant laser is also helpful for improving the signal-to-noise ratio of the fluorescence signal and obtaining higher fluorescence image quality. In addition, the light intensity modulation mode provided by the embodiment of the invention effectively reduces the signal output flux of the data acquisition/control card and the calculated amount of the light intensity modulation signal by the computer, can effectively compress the cost of the device system, and is more favorable for industrialized popularization and use.
3. The embodiment of the invention uses an area array detector (sCOMS camera) for imaging, does not need to use expensive APD/PMT (avalanche photo diode/photomultiplier tube) equal-point detectors, and is more beneficial to industrial development. In addition, the wide-field detection mode is convenient to be combined with a structured light illumination technology, a single-molecule positioning technology and other wide-field technologies so as to further improve the transverse resolution.
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Drawings
FIG. 1 is a schematic block diagram of a tomography microscopic imaging apparatus based on resonant scanning sparse structured light illumination according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of an optical path of a tomography microscopic imaging apparatus based on resonant scanning sparse structured light illumination according to an embodiment of the present invention;
fig. 3 is a timing diagram of control signals of a tomography microscopic imaging apparatus based on resonance scanning sparse structured light illumination according to an embodiment of the present invention;
fig. 4 is a schematic diagram of an out-of-focus noise reduction operation of a tomography microscopic imaging apparatus based on resonance scanning sparse structured light illumination according to an embodiment of the present invention;
FIG. 5 is a graph of measurement of the photoperiod and phase shift of a sparse scanning fringe structure of a resonance scanning sparse structure light illumination-based tomographic microscopic imaging apparatus provided by an embodiment of the present invention;
FIG. 6 is a comparison of the axial resolution of a tomographic microscopy imaging method of an embodiment of the present invention compared to a prior art three-dimensional scanning wide-field microscopy mode;
fig. 7 is an image comparison of a tomographic microscopic imaging method and a conventional three-dimensional scanning wide-field microscopic imaging method of an embodiment of the present invention with respect to a step sample.
Description of reference numerals:
1-a pulsed laser; 2-an optical fiber; 3-a beam expanding collimating lens; 4-resonance scanning galvanometer; 5-linear scanning galvanometer; 6-a scanning lens; 7-a first sleeve lens; 8-sample; 9-an objective lens; a 10-dichroic mirror; 11-a second sleeve lens; 12-a band pass filter; 13-sCOMS camera; 14-a computer; 15-data acquisition control card.
Detailed Description
In order to further explain the technical means and effects of the present invention adopted to achieve the predetermined object, a tomographic microscopic imaging apparatus and method based on resonant scanning sparse structured light illumination according to the present invention will be described in detail below with reference to the accompanying drawings and the detailed description.
The foregoing and other technical matters, features and effects of the present invention will be apparent from the following detailed description of the embodiments, which is to be read in connection with the accompanying drawings. The technical means and effects of the present invention adopted to achieve the predetermined purpose can be more deeply and specifically understood through the description of the specific embodiments, however, the attached drawings are provided for reference and description only and are not used for limiting the technical scheme of the present invention.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Furthermore, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that an article or device that comprises a list of elements does not include only those elements but may include other elements not expressly listed. Without further limitation, an element defined by the phrases "comprising one of \8230;" does not exclude the presence of additional like elements in an article or device comprising the element.
Example one
Referring to fig. 1, fig. 1 is a schematic block diagram of a tomography apparatus based on resonant scanning sparse structured light illumination according to an embodiment of the present invention. The tomography microscopic imaging device comprises an illumination unit 101, a scanning unit 102, a microscopic imaging unit 103 and an acquisition and reconstruction control unit 104, wherein the illumination unit 101, the scanning unit 102 and the microscopic imaging unit 103 are sequentially coupled and connected to form a whole light path of the device, and the acquisition and reconstruction control unit 104 completes control of each instrument in the light path, image acquisition and resonance three-dimensional tomography microscopic image reconstruction processing. Specifically, the illumination unit 101 is connected to the acquisition and reconstruction control unit 104, and the acquisition and reconstruction control unit 104 performs light intensity modulation by using the generated digital TTL (digital logic level) signal and corrects non-uniformity of light intensity caused by the x-direction resonance scanning to generate illumination light with light intensity varying with a spatial position, so as to illuminate the sample; the scanning unit 102 is connected with the acquisition and reconstruction control unit 104, and synchronously controls the two-direction scanning of the two-dimensional galvanometer system by using the analog direct-current signal (controlling the first-direction resonance galvanometer scanning) and the analog step signal (controlling the second-direction linear galvanometer scanning) generated by the acquisition and reconstruction control unit 104, and completes the generation and phase shift of the sparse scanning stripe structured light through the focus scanning; the microscopic imaging unit 103 is connected with the acquisition and reconstruction control unit 104, acquires intensity images of different axial slices of the sample under the irradiation of the sparse scanning stripe structure light with different phases, and synchronously transmits the acquired intensity images to the acquisition and reconstruction control unit 104, and the acquisition and reconstruction control unit 104 is further configured to select a maximum light intensity value of each pixel in an image stack to reconstruct a tomographic microscopic image of the sample at the current axial slice by using the acquired image stack of the sample under the irradiation of the sparse scanning stripe structure light with different axial slices and different phases, and finally acquire a three-dimensional tomographic microscopic image of the sample in real time.
In this embodiment, the idea of reconstructing a three-dimensional tomographic microscopic image is as follows: the method comprises the steps of collecting sparse scanning stripe structure light images under different phase shifts in one direction, forming an image stack, conducting defocusing noise reduction and removal processing, screening out the maximum light intensity value of each pixel in the image stack to serve as the light intensity value of a chromatographic microscopic image in the pixel, and reconstructing the three-dimensional chromatographic microscopic image of a sample to be measured in real time.
The sparse scan fringe structured light illumination and imaging process is as follows: the focus of the objective lens 9 is scanned in the x direction in a resonant manner (f =12 KHz), and within a complete scanning time period of 1/f =83.3 μ s (corresponding to one round-trip scanning movement in the x direction), the excitation light is turned on only in the central region where the scanning intensity is uniform in the scanning displacement in the + x direction, and the laser is turned off in the boundary region where the scanning intensity is non-uniform, so that a scanning bright line with uniform intensity can be scanned in the x direction. By setting linear scanning in the y direction, the focal point is scanned in the y direction into sparse scanning stripe structured light of a discontinuous, equal period P. In order to separate the side lobes of two adjacent bright lines from each other, P is typically 4 to 10 times the spatial resolution (full width at half maximum of the focal spot) of the microscope system. Then, a complete set of 12-step phase-shifted sparsely scanned structured light images is acquired as one image stack with the sCOMS camera by phase shifting the sparsely scanned structured light in the y-direction one by one (each shift P/12).
The process of three-dimensional tomographic image reconstruction is as follows: for each pixel of the plane, the defocusing noise can be reduced by subtracting the light intensity of the dark fringe illumination from the light intensity of the pixel obtained in the bright fringe illumination; and then, at each axial position (namely the thickness direction) of the sample, screening the maximum value of the light intensity of each pixel by using an image stack obtained by scanning at each axial position to reconstruct a chromatographic image of the axial plane. By performing the above operation for each pixel of the plane, a tomographic image of the sample at the axial plane can be obtained. Then, the objective lens is moved axially, and the operations are repeated on different axial surfaces of the sample, so that a three-dimensional chromatographic image of the sample can be obtained.
Specifically, referring to fig. 2, fig. 2 is a schematic diagram of a resonance-based scanning system according to an embodiment of the present inventionAnd (3) an optical path schematic diagram of the sparse structured light illuminated three-dimensional tomography microscopic imaging device. The illumination unit 101 includes a laser 1, an optical fiber 2, and a beam expanding collimator lens 3, which are sequentially arranged in an optical path direction. The laser 1 of this embodiment is a pulse laser, as an illumination laser, the laser frequency and the light-emitting duty ratio of the pulse laser are adjustable, the laser wavelength is 488nm, the frequency adjustable range in 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, and the laser emitted by the laser 1 is guided into a scanning optical path system by an optical fiber 2, and is expanded and collimated into parallel light by a beam expanding collimating lens 3. Preferably, the focal length of the beam expanding and collimating lens 3 is f 3 ＝100mm。
Further, the scanning unit 102 includes a resonance scanning galvanometer 4, a linear scanning galvanometer 5, a scanning lens 6, and a first sleeve lens 7, which are sequentially arranged in the optical path direction. The resonance scanning galvanometer 4 and the linear scanning galvanometer 5 are arranged in parallel and form a preset angle with an optical axis to form a two-dimensional resonance scanning galvanometer system, the scanning lens 6 and the first sleeve lens 7 form a telescope system, and incident beams of the resonance scanning galvanometer 4 and the linear scanning galvanometer 5 are imaged to an entrance pupil of the objective lens 9 in a conjugate mode to ensure that the light intensity of focused light spots of a sample at different positions is unchanged. In the present embodiment, the focal length of the scanning lens 6 is f 6 =100mm, the focal length of the first sleeve lens 7 is f 7 ＝200mm。
The microscopic imaging unit 103 of the present embodiment includes an objective lens 9, a dichroic mirror 10, a second sleeve lens 11, a band-pass filter 12, and an sscmos camera 13. The microscopic imaging unit 103 is mainly used for realizing microscopic imaging of a sample, wherein the sample 8 is arranged at a focal point of the objective lens 9, the dichroic mirror 10 is obliquely arranged between the first sleeve lens 7 and the objective lens 9, and can reflect and transmit parallel light from the first sleeve lens 7 to an entrance pupil of the objective lens 9, and finally is 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 sequentially arranged on the other side, opposite to the objective lens 9, of the dichroic mirror 10, and can receive 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 for filtering noise, and the dichroic mirror 10 is used for achieving alignmentSeparation of the emitted laser light from the emitted fluorescent light. Preferably, the second sleeve lens 11 has a focal length f 11 ＝400mm。
Specifically, in the scanning optical path, the parallel light emitted from the beam expanding collimator lens 3 is scanned by a two-dimensional resonant scanning galvanometer system composed of a resonant scanning galvanometer 4 and a linear scanning galvanometer 5, is imaged by a telescope system composed of a scanning lens 6 and a first sleeve lens 7, is reflected by a dichroic mirror 10, is transmitted to the entrance pupil of an objective lens 9, and is focused on a sample 8 by the objective lens 9.
In the microscopic imaging optical path, the emitted fluorescence from the sample 8 is finally imaged onto an sCOMS camera 13 via a telescopic system consisting of an objective lens 9 and a tube lens 11, forming a clear sample image. A dichroic mirror 10 is in between to achieve separation of incident laser light from emitted fluorescence, and a band pass filter 12 in front of the sCOMS camera 13 is used to filter out noise again to further improve image signal to noise ratio.
Further, the acquisition reconstruction control unit 104 is used to synchronously control the illumination unit 101, the scanning unit 102, and the microscopic imaging unit 103. The acquisition reconstruction control unit 104 of the present embodiment includes 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, generates a control signal required by an instrument in a light path, and implements light intensity modulation on a laser 1, scanning control on a two-dimensional resonance scanning galvanometer system composed of a resonance scanning galvanometer 4 and a linear scanning galvanometer 5, axial movement control on an axial displacement stage of an objective lens 9, and acquisition control on an external trigger exposure image of an oscmos camera to complete real-time image acquisition; the computer 14 is further configured to complete stack reconstruction of the images at each axial position according to a reconstruction principle of the three-dimensional tomographic microscopic image, and further acquire the three-dimensional tomographic microscopic image.
Further, referring to fig. 3, fig. 3 is a timing diagram of control signals of a three-dimensional tomographic microscopic imaging apparatus based on resonance scanning and sparse structured light illumination according to an embodiment of the present invention.
For the camera trigger signal of each frame, a camera trigger TTL signal is generated through the data acquisition card 15, the sCOMS camera is controlled to start exposure at the rising edge, and the exposure is finished 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. Where scan frame time = sparse scan fringe cycle number × single line scan time (resonant galvanometer synchronous output signal cycle).
For the scanning displacement (non-electric signal) of the resonant galvanometer, a curve function can be expressed as x (t) = Acos (2 pi ft), and A is the amplitude of the galvanometer, so that the scanning range of the resonant scanning galvanometer is determined; f is the resonance scanning frequency of the resonance scanning galvanometer and is 12KHz; t is time. In this embodiment, only one dc signal is generated by the data acquisition card 15 as the input voltage for starting and controlling the scanning range (i.e. the input voltage is 0v in the static state and 5v in the full-scale scanning). The parameters such as the scanning period are intrinsic parameters of the instrument, that is, for any scanning range, the scanning period (corresponding to one reciprocating scanning movement in the ± x direction) remains unchanged and is always 1/12khz =83.3 μ s.
For the synchronous output signal of the resonant vibrating mirror, when the resonant vibrating mirror 4 is started by using a direct current signal generated by the data acquisition card 15 as an input voltage, a strictly synchronous resonant vibrating mirror synchronous output signal (TTL signal) is generated inside the resonant vibrating mirror, the frequency of the TTL signal is consistent with the scanning frequency of the resonant vibrating mirror, and the duty ratio is 0.5, namely 1/12khz =83.3 μ s. In the whole period of the TTL signal, the resonant scanning galvanometer is corresponding to one reciprocating scanning motion in the +/-x direction, namely in the first half period, the resonant scanning galvanometer completes displacement along the + x direction; in the second half cycle, return to the initial position in the-x direction. Meanwhile, in this embodiment, only the first half cycle (1/12khz × 0.5=41.67 μ s) of the TTL signal is used as an effective signal, and the rising edge/falling edge of the TTL signal respectively defines the starting time and the ending time of the resonant galvanometer when one unidirectional line scan is completed.
For the light intensity modulation signal, the signal is a group of TTL signals generated by the data acquisition 15, and is used to correct the problem of non-uniformity of light intensity caused by the non-uniform velocity characteristic of resonant scanning under constant laser light intensity, i.e. when the light intensity signal is not modulated under uniform light intensity, a non-uniform light intensity scan line with "bright ends and dark middle" will be formed in the field of view. The main body of the functional expression of the scanning displacement (non-electric signal) of the resonant galvanometer is a cosine function, so that the whole scanning area of the resonant galvanometer can be divided into two parts: a non-uniform light intensity scanning area (scanning both end areas) and a uniform light intensity scanning area (scanning center area). Through experimental determination, the problem of light intensity nonuniformity in the uniform light intensity scanning area can be ignored, and the uniform light intensity scanning area proportion is about 0.5, which is more appropriate. And modulating the TTL signal to serve as a light intensity modulation signal, starting laser in the uniform light intensity scanning area, and closing the laser in the non-uniform light intensity scanning area to obtain a single-row scanning line with uniform light intensity so as to correct the problem of non-uniformity of light intensity caused by resonance scanning under constant laser light intensity. It should be noted that, in order to determine the accurate time of laser on/off, it is necessary to calculate the accurate time of laser on/off corresponding to the displacement of the uniform light intensity scanning area through an inverse function according to a function expression of the scanning displacement (non-electrical signal) of the resonant galvanometer, by using the monotonicity of the displacement of the function in the uniform light intensity scanning area with respect to time, knowing the initial position and the final position of the displacement of the uniform light intensity scanning area.
It is emphasized that for fluorescence imaging, laser is one of the main sources of final noise, so using this method of light intensity modulation to turn off laser in time also helps to improve the signal-to-noise ratio of fluorescence signal and obtain higher image quality.
For the linear galvanometer scanning signal, the signal is a step signal generated by the data acquisition card 15 to complete the scanning of the linear galvanometer in the plane y direction, and each level signal in the step represents a position of a single-row scanning line corresponding to the plane y direction. Where the number of step signal levels = the number of sparse scan fringe periods. The accurate time sequence synchronization of a camera trigger signal, a resonance galvanometer synchronous output signal, a light intensity modulation signal and a linear galvanometer scanning signal is ensured. And finally, generating sparse scanning stripe structure light with uniform light intensity in a phase in a field of view by using the light intensity accumulation effect in the exposure time of the camera. It should be noted that the scan displacement function of the linear scan galvanometer 5 may represent y (t) = kt, where k determines the minimum step size of the linear scan galvanometer scan, and t is time. In addition, in order to implement the phase shift operation on the sparse scanning stripe structure 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 is kept unchanged to ensure that the stripe structure light is translated along the y direction. In addition, according to the above control, the focal point of the objective lens 9 is finally scanned in the y direction as the sparse scan stripe structured light of the discontinuous period P. For specific parameter settings of sparsely scanned structured light, P is typically 4-10 times the spatial resolution (full width half maximum of the focal spot) of the microscope system in order to separate the side lobes of two adjacent bright lines from each other. Then, the phase shift operation of the sparsely scanned fringe-structured light is completed by successively shifting the sparsely stripes in the y-direction (each shift by P/12).
And then, repeating the signal control to only change the scanning signal of the linear scanning galvanometer so as to complete the phase shift operation of the sparse scanning stripe structured light image, and collecting a group of complete sparse scanning structured light images under 12-step phase shift to form an image stack.
In a specific operation process, the imaging process of the three-dimensional tomography microscopic imaging device is as follows:
dripping a proper amount of distilled water on the water immersion objective lens 9, and placing a sample 8; the sample 8 is axially moved to be imaged clearly on a plane z =0 (i.e., the center position in the thickness direction of the sample 8); setting an axial scanning range from z = -10 mu m to z =10 mu m, wherein the number of axial scanning layers is 100; starting a control program; for each axial scan position, x-y focal scan imaging was performed on the sample 8, and 12 images under different phase sparsely scanned fringe structured light illumination were recorded. Wherein the x-y scan field of view is 103.2 x 103.2 μm 2 . The x-direction is a resonance scan and the y-direction is a linear scan to obtain 43 sparse stripes of equal period (period P of 2.4 μm). An sCOMS camera sequentially records intensity images of the sample 8 under different sparse scanning fringe structured light illuminations, and the intensity images are marked as I i (x, y), i =1,2 \823012. Three-dimensional tomographic microscopic image reconstruction of the sample 8 is then accomplished using the computer 14.
In this embodiment, the acquisition reconstruction control unit 104 is specifically configured to:
obtaining image stacks of samples 8 under the irradiation of sparse scanning stripe structure light with different axial slices and different phases;
the out-of-focus noise is removed by subtracting two images at the same axial slice, which are N/2 apart:
wherein, I i An intensity image obtained by the ith scanning in an image stack; i is (i-N/2) Intensity images obtained for the I-N/2 th scan in an image stack, I (i+N/2) Intensity images obtained for the i + N/2 th scan of an image stack, F i Is shown as I i After the intensity image is subjected to defocus noise reduction, N =12 is the total phase shift step number, which is the number of images in one image stack.
Selecting the maximum light intensity value of each pixel in the collected image in an image stack, and reconstructing a chromatographic microscopic image of the sample 8 at the current axial slice; reconstructing a tomographic microscopic image at the current axial slice using the image stack at different axial slices of the sample 8 and reconstructing a three-dimensional tomographic microscopic image of the sample 8 using tomographic microscopic images of different layers.
In particular, one image stack F obtained for a particular axial position scan i (i =1,2 \8230n), the intensity of light reaches a maximum when it is present at the same position on sample 8 and scanned only by the sparse bright stripes. Thus in a tomographic image at one axial position, the intensity value of each pixel is the maximum value of that pixel in the phase-shifted image stack (i =1,2 \8230n), thus obtaining:
F sec (m, n) ＝ Max{F i (m, n)} i (2)
wherein, F sec (m, n) represents a tomographic microscopic image F sec Light intensity value at a pixel point (m, n), max {. The i Is shown in image stack F i (i =1,2 \8230N) the maximum intensity value of the pixel at the (m, N) point is selected. Repeating the same operation by all pixels in the imageFinally, an image stack F at the axial position is utilized i (i =1,2 \ 8230N) A tomographic microscopic image F of the sample 8 at the axial position is obtained sec . And finally, axially moving the objective lens, sequentially recording 12 intensity images of the sample 8 under different-phase sparse fringe illumination at the current axial position by using an sCOMS camera, repeating the calculation of the formulas (1) to (2) to obtain a chromatographic microscopic image at the current axial position, then continuously axially moving the objective lens, repeating the steps to obtain a plurality of chromatographic microscopic images at different axial positions, and finally reconstructing the three-dimensional chromatographic microscopic imaging of the sample.
The embodiment of the invention adopts sparse scanning stripe structure light with different phases to carry out scanning imaging on the same sample, and utilizes the light intensity of each point on the sample during the illumination of the bright stripe to subtract the light intensity during the illumination of the dark stripe to inhibit defocusing noise, thereby obtaining the three-dimensional chromatography microscopic image at the axial position. Compared with other structured light modes, the method has the advantages that the focused light spots are used for scanning to generate the stripe structured light, so that the scattering effect of a sample can be better overcome, and the three-dimensional tomography of the thick sample is realized.
Example two
On the basis of the above embodiment, the present embodiment provides a three-dimensional tomographic microscopy imaging method based on resonance scanning and sparse structured light illumination, the method including:
s1: and dropping a proper amount of distilled water on a water immersion objective lens, placing a sample, and axially moving the sample to clearly image the sample on a z =0 plane, wherein the z =0 plane refers to the central position of the sample in the thickness direction.
In the present embodiment, an axial scanning range from z = -10 μm to z =10 μm is set, and the number of axial scanning layers is 100 layers.
S2: under the control of the acquisition and reconstruction control unit, acquiring intensity images of different axial slices of the sample under the irradiation of sparse scanning stripe structure light with different phases;
specifically, for each axial scan position, x-y focal scan imaging was performed on the sample, and 12 images under different phase sparse fringe illumination were recorded. Wherein the x-y scan field of view is 103.2 x 103.2 μm 2 . The x-direction is a resonance scan and the y-direction is a linear scan to obtain 43 sparse stripes of equal period (period P of 2.4 μm). An sCOMS camera sequentially records intensity images of samples under different-phase sparse fringe illumination, and the intensity images are marked as I i (x,y),i＝1,2…12。
S3: and (3) utilizing image stacks of the obtained samples under the irradiation of sparse scanning stripe structure light with different axial slices and different phases, selecting the maximum light intensity value of each pixel in one image stack in the images to reconstruct a chromatographic microscopic image of the samples at the current axial slice, and finally obtaining the three-dimensional chromatographic microscopic image of the samples in real time.
In one embodiment of the present invention, the S3 includes:
s3.1: obtaining image stacks of samples under the irradiation of sparse scanning stripe structure light with different axial slices and different phases;
s3.2: the two images at the same axial slice with a distance of N/2 are subtracted to remove the out-of-focus noise:
wherein, I i Intensity images obtained for the ith scan in an image stack, I (i-N/2) Intensity images obtained for the I-N/2 th scan in an image stack, I (i+N/2) Intensity images obtained for the i + N/2 th scan of an image stack, F i Is I i Subtracting the intensity image after the defocusing noise, wherein N is the total phase shift step number, namely the number of images in one image stack;
s3.3: and selecting the maximum light intensity value of each pixel in the acquired image in an image stack, and reconstructing a chromatographic microscopic image of the sample at the current axial position.
In particular, one image stack F obtained for the current axial position scan i Using said image stack F i Obtaining a tomographic image of the sample at the current axial position:
F sec (m,n)＝Max i {F i (m,n)} i
wherein i =1,2 \ 8230n, F sec (m, n) denotes a tomographic microscopic image F sec Light intensity value at a pixel point (m, n), max {. The i Is shown in image stack F i The maximum intensity value of the pixel at the (m, n) point is selected.
S3.4: and reconstructing a tomography microscopic image at the current axial slice by using the image stack of different axial slices of the sample and reconstructing three-dimensional tomography microscopic imaging of the sample by using tomography microscopic images of different layers.
The effect of the three-dimensional tomography microscopic imaging device based on resonance scanning and sparse structured light illumination in the embodiment of the invention is verified through experiments. In experiments, the magnification of the three-dimensional tomography microscopic imaging device based on resonance scanning and sparse structured light illumination is 129 times. The pixel array number of the sCMOS camera is 2048 multiplied by 2048, and the pixel size is 6.5 mu m. The sCMOS camera pixel corresponds to a size of about 50nm on the sample plane. Under 488nm laser illumination, the numerical aperture NA of the water immersion objective lens is 1.2, the theoretical resolution of the device is determined to be 207nm (the full width at half maximum of a focused light spot), and the spatial resolution is measured to be 308nm experimentally.
According to the control principle, the generation and phase shift of sparse scanning stripe structured light with the period of 2.4 μm and the total phase shift step number of 12 steps (in order to ensure that the single-step phase shift distance is less than the resolution of the actual measurement system, namely the full width at half maximum of the focus, namely 2.4 μm/12=200nm is less than or equal to 308 nm) in the y direction can be completed.
Referring to fig. 4, fig. 4 is a schematic diagram of the defocus noise reduction operation of the tomography apparatus based on resonant scanning sparse structured light illumination according to the embodiment of the present invention, wherein I i Intensity images obtained for the ith scan in an image stack, I (i-N/2) Intensity images obtained for the I-N/2 th scan in an image stack, I (i+N/2) Intensity images obtained for the i + N/2 th scan of an image stack, F i Is I i And (4) subtracting the out-of-focus noise from the intensity image. In this embodiment, in the light intensity distribution pattern of the sparse scanning stripe structured light (the period is 2.4 μm, the total phase shift step number is 12 steps), the brightness of the region where the stripe is located is high (marked as ON), and the two bright stripesThe brightness between the stripes is low (noted OFF). The ratio of the bright area in one period is about 1/12. According to the final defocus noise subtraction result, the following results are obtained: under sparse scan fringe structured light illumination, the intensity of OFF versus position comes mainly from the defocus noise of the sample. The defocus noise can be effectively suppressed by subtracting the intensity of the sample at the time of ON and OFF illumination of the fringe structure light.
Referring to fig. 5, fig. 5 is a graph for measuring the period and phase shift of the sparse scanning fringe structure of the resonant scanning sparse structured light illumination-based tomographic microscopy imaging apparatus according to the embodiment of the present invention, wherein (a) the graph is a sparse scanning fringe structure light image in each phase state in an image stack, and a sample is a broadband mirror with a reflection wavelength of 400nm to 700 nm; (b) The figure shows the measurement of the photoperiod and phase of a sparse scan fringe structure in an image stack with an image scale of 20 μm. Obtaining image stack I recorded during different-phase sparse scanning fringe structured light illumination through 12-step phase shift i (x, y), i =1,2 \823012. The 12 images in the image stack are superposed and averaged
Then the scanning wide field image corresponding to the system can be obtained.
In order to determine the axial resolution capability of the three-dimensional tomography microscopic imaging device based on resonance scanning and sparse structured light illumination, in the experiment, a single-layer fluorescent bead sample with the diameter of 250nm, the excitation wavelength of 488nm and the emission wavelength of 520nm is subjected to resonance scanning three-dimensional tomography microscopy and three-dimensional scanning wide-field microscopy imaging. Referring to fig. 6, fig. 6 is an axial resolution comparison diagram of a tomographic microscopic imaging method according to an embodiment of the present invention and a conventional 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) The figure is xz sectional image (upper) of single layer fluorescence small ball three-dimensional tomography image and axial intensity distribution (lower) along the small ball center; (c) The figure is xz sectional image (upper) of single-layer fluorescence small three-dimensional scanning wide-field microscopic image and axial intensity distribution (lower) along the center of the small sphere; the axial scanning step length is 0.2 μm, and the number of scanning layers is 100. Comparing fig. 6 (b) and fig. 6 (c), it can be seen that the image obtained by the method of the embodiment of the present invention has lower background noise. By carrying out quantitative analysis on the axial intensity distribution of the globules on the section, the axial resolution of the three-dimensional tomography micro-imaging method based on resonance scanning and sparse structured light illumination is 1.38 +/-0.25 mu m, and the axial resolution of the three-dimensional scanning wide-field micro-imaging is 1.79 +/-0.15 mu m. The measurement result shows that the method improves the axial resolution ratio by about 1.19 to 1.45 times compared with the axial resolution ratio under a three-dimensional scanning wide-field microscope.
In addition, in the experiment of the embodiment of the invention, a step sample is imaged by using a three-dimensional tomography microscopic imaging method based on resonance scanning and sparse structured light illumination and scanning wide-field imaging respectively, the imaging results are respectively shown in fig. 7, wherein the left side is an image of the sample at different axial positions in the existing three-dimensional scanning wide-field microscopic imaging mode; the right sample is a three-dimensional tomographic microscopic image obtained by using the method provided by the embodiment of the invention; all scales are 20 μm. The experimental results show that: compared with three-dimensional scanning wide-field microscopic imaging, the three-dimensional tomography microscopic imaging method based on resonance scanning and sparse structured light illumination effectively inhibits defocusing noise in imaging and has good tomography capacity.
The embodiment of the invention uses the resonance scanning galvanometer (the scanning frequency can reach 12KHz at most), and the imaging speed of the method is improved by tens of times compared with the traditional confocal microscopic imaging. Taking a conventional confocal microscope as an example, assuming that the spatial resolution of the system is 308nm, and the imaging field of view is 103.2 μm × 103.2 μm, according to the nyquist sampling theorem, the confocal scanning step is generally set to 308nm/2.3=134nm, and the number of unidirectional scanning pixels of the confocal microscope is 103.2 μm/0.134 μm = 770. If the single pixel dwell time is 30 μ s, the imaging frame time of the conventional confocal microscope is (103.2/0.308) 2 ×30μs＝17.79s。
The scanning speed of the method provided by the embodiment of the invention in the x direction is 12 KHz/line, namely the period of scanning one line is about 83.3 mus. Wherein the fringe period is 2.4 μm, the number of fringe periods in the single-step phase-shifted image =103.2 μm/2.4 μm =43, and the number of phase-shifted steps is 12-step phase shift. The time taken for the three-dimensional chromatography method according to the present invention described above is about 12 × 43 × 83.3 μ s =43ms.
Compared with the imaging speed of a confocal microscope, the method provided by the embodiment of the invention has the advantages that the multiplying power is increased by about 40 times: the effective improvement of the imaging speed can greatly widen the application scene of the three-dimensional chromatographic microscope and effectively improve the detection efficiency of the rapid sample detection in the industrial and biological fields.
The embodiment of the invention provides a simpler light intensity modulation mode, namely, the TTL signal is modulated to control the laser to start the laser only in the central area with uniform scanning light intensity and to close the laser in the boundary area with non-uniform scanning light intensity so as to correct the problem of non-uniformity of light intensity caused by resonance scanning under constant laser light intensity and finally generate sparse scanning stripe structure light with uniform light intensity in a field of view. Meanwhile, the turning off of the redundant laser is also helpful for improving the signal-to-noise ratio of the fluorescence signal and obtaining higher fluorescence image quality. In addition, the light intensity modulation mode provided by the embodiment of the invention effectively reduces the signal output flux of the data acquisition/control card and the calculated amount of the light intensity modulation signal by the computer, can effectively compress the cost of the device system, and is more favorable for industrialized popularization and use.
In addition, the embodiment of the invention uses an area array detector (sCOMS camera) for imaging, does not need to use expensive APD/PMT (avalanche photo diode/photomultiplier tube) point detectors and is more beneficial to industrial development. In addition, the wide-field detection mode is convenient to be combined with a structured light illumination technology, a single molecule positioning technology and the like so as to further improve the transverse resolution.
The foregoing is a further detailed description of the invention in connection with specific preferred embodiments and it is not intended to limit the invention to the specific embodiments described. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (10)
1. A tomography microscopic imaging device based on resonance scanning sparse structured light illumination is characterized by comprising an illumination unit (101), a scanning unit (102), a microscopic imaging unit (103) and an acquisition reconstruction control unit (104),
the illumination unit (101), the scanning unit (102) and the microscopic imaging unit (103) are sequentially coupled to form an optical path whole of the device;
the illumination unit (101) is connected with the acquisition and reconstruction control unit (104) and is used for generating illumination light with light intensity changing with the space position under the control of a control signal generated by the acquisition and reconstruction control unit (104);
the scanning unit (102) is connected with the acquisition and reconstruction control unit (104) and is used for completing the generation and phase shift of sparse scanning stripe structure light through focus scanning by utilizing a control signal generated by the acquisition and reconstruction control unit (104);
the microscopic imaging unit (103) is connected with the acquisition and reconstruction control unit (104) and is used for acquiring intensity images of different axial slices of the sample under the irradiation of sparse scanning stripe structure light with different phases by using a control signal generated by the acquisition and reconstruction control unit (104) and synchronously transmitting the acquired intensity images to the acquisition and reconstruction control unit (104);
the acquisition and reconstruction control unit (104) is also used for reconstructing a chromatographic microscopic image of the sample at the current axial slice by using the obtained image stacks of the sample under the irradiation of the sparse scanning stripe structure light with different axial slices and different phases, and selecting the maximum light intensity value of each pixel in the image in one image stack, so as to finally obtain the three-dimensional chromatographic microscopic image of the sample in real time.
2. The tomography and microscopy imaging device based on resonance scanning sparse structure light illumination is characterized in that the illumination unit (101) comprises a pulse laser (1), an optical fiber (2) and a beam expanding and collimating lens (3) which are arranged in sequence along the direction of an optical path,
scanning unit (102) including the resonance scanning mirror that shakes (4), the linear scanning mirror that shakes (5), scanning lens (6) and first sleeve lens (7) that set gradually along the light path direction, resonance scanning mirror that shakes (4) with linear scanning mirror that shakes (5) parallel arrangement just is predetermined angle with the optical axis, constitutes two-dimensional resonance scanning mirror system that shakes, scanning lens (6) with telescope system is constituteed in first sleeve lens (7), will resonance scanning mirror that shakes (4) with the incident beam conjugate imaging of linear scanning mirror that shakes (5) is to the entrance pupil department of objective (9) to guarantee that the light intensity of sample focus facula is unchangeable in different positions department.
3. The tomographic microscopy imaging apparatus based on resonance scanning sparse structured light illumination as defined in claim 1, wherein the microscopy 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 and transmit parallel light from the first sleeve lens (7) to the entrance pupil of the objective lens (9), and is also used for realizing separation of incident laser light and emitted fluorescence from a sample; the second sleeve lens (11), the band-pass filter (12) and the sCOMS camera (13) are sequentially arranged on the other side, away from the objective lens (9), of the dichroic mirror (10) and used for collecting light from a sample to image.
4. The resonance scanning sparse structured light illumination based tomographic microscopy imaging device as recited in claim 1, wherein said acquisition 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, realizing the scanning process of a two-dimensional resonance scanning galvanometer system consisting of the resonance scanning galvanometer (4) and the linear scanning galvanometer (5) on the light intensity of the pulse laser (1), the axial movement of an axial displacement table of the objective lens (9), and the acquisition of an external trigger exposure image of the sCOMS camera (13) so as to finish the real-time image acquisition;
and the computer (14) is also used for reconstructing the image stack of each layer in the thickness direction of the sample and acquiring a three-dimensional chromatographic microscopic image of the sample in real time.
5. The tomographic microscopy imaging apparatus as claimed in claim 1, wherein the data acquisition control card (15) is capable of generating 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, the scan displacement function of the resonant scanning galvanometer (4) is x (t) = Acos (2 pi ft), where a is amplitude, f is resonant scanning frequency of the resonant scanning galvanometer, and t is time; 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 a scanning displacement function of the linear scanning galvanometer (5) is y (t) = kt, where k determines a minimum step length of scanning of the linear scanning galvanometer and t is time; the data acquisition control card (15) is also used for completing the time sequence line scanning synchronization of the first direction scanning and the second direction scanning by utilizing the resonance galvanometer synchronous output signal, thereby forming sparse scanning stripe structured light.
6. The tomographic microscopy imaging device as claimed in claim 1, wherein the data acquisition control card (15) is further configured to control the pulsed laser (1) to turn on the laser only in the central region where the scanning intensity is uniform and turn off the laser in the boundary region where the scanning intensity is non-uniform by modulating the TTL signal.
7. The resonance scanning sparse structured light illumination based tomographic microscopy imaging apparatus as claimed in claim 1, wherein the acquisition reconstruction control unit (104) is specifically configured to:
obtaining image stacks of samples which are sliced in different axial directions and are irradiated by sparse scanning stripe structure light with different phases;
the two images at the same axial slice with a distance of N/2 are subtracted to remove the out-of-focus noise:
wherein, I i Intensity images obtained for the ith scan in an image stack, I (i+N/2) Intensity images obtained for the I + N/2 th scan of an image stack, I (i-N/2) Intensity images obtained for the i-N/2 th scan of an image stack, F i Is I i Subtracting the intensity image after the defocusing noise, wherein N is the total phase shift step number, namely the number of images in one image stack;
selecting the maximum light intensity value of each pixel in the collected image in an image stack, and reconstructing a chromatographic microscopic image of the sample at the current axial slice;
and reconstructing a tomography microscopic image at the current axial section by using the image stack at different axial sections of the sample and reconstructing three-dimensional tomography microscopic imaging of the sample by using the tomography microscopic images of different layers.
8. A tomography microscopic imaging method based on resonance scanning sparse structured light illumination, characterized in that it is performed with the tomography microscopic imaging apparatus of any of claims 1 to 7, the method comprising:
s1: dripping a proper amount of distilled water on the water immersion objective lens, placing a sample, and axially moving the sample to enable the sample to be clearly imaged;
s2: under the control of the acquisition and reconstruction control unit, acquiring intensity images of different axial slices of the sample under the irradiation of sparse scanning stripe structure light with different phases;
s3: and (3) utilizing image stacks of the obtained samples under the irradiation of sparse scanning stripe structure light with different axial slices and different phases, selecting the maximum light intensity value of each pixel in one image stack in the images to reconstruct a chromatographic microscopic image of the samples at the current axial slice, and finally obtaining the three-dimensional chromatographic microscopic image of the samples in real time.
9. The resonance scanning sparse structured light illumination based tomographic microscopy imaging method as recited in claim 8, wherein the S3 comprises:
s3.1: obtaining image stacks of samples which are sliced in different axial directions and are irradiated by sparse scanning stripe structure light with different phases;
s3.2: the two images at the same axial slice with a distance of N/2 are subtracted to remove the out-of-focus noise:
wherein, I i For intensity images from the ith scan in an image stack, I (i+N/2) Intensity images obtained for the I + N/2 th scan of an image stack, I (i-N/2) Intensity images obtained for the i-N/2 th scan of an image stack, F i Is shown as I i Subtracting the intensity image after the defocusing noise, wherein N is the total phase shift step number, namely the number of images in one image stack;
s3.3: selecting the maximum light intensity value of each pixel in the collected image in an image stack, and reconstructing a chromatographic microscopic image of the sample at the current axial slice;
s3.4: and reconstructing a tomography microscopic image at the current axial slice by using the image stack of different axial slices of the sample and reconstructing three-dimensional tomography microscopic imaging of the sample by using tomography microscopic images of different layers.
10. The resonance scanning sparse structured light illumination based tomographic microscopy imaging device as recited in claim 9, wherein said S3.3 comprises:
one image stack F obtained for the current axial position scan i Using said image stack F i Obtaining a tomographic image of the sample at the current axial position:
F sec (m,n)＝Max{F i (m,n)} i
wherein i =1,2 \ 8230n, F sec (m, n) denotes a tomographic microscopic image F sec The intensity value at a pixel point (m, n), max {. The i Is shown in image stack F i SelectingAnd the maximum light intensity value of the pixel at the (m, n) point.
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