CN108742532B - Wide-field chromatographic ultra-spectral microscopic imaging method and device based on space-time focusing - Google Patents

Abstract

The invention discloses a wide-field tomographic hyperspectral microscopic imaging method and device based on space-time focusing, and belongs to the technical field of microspectral imaging and analytical chemistry. In this method, an ultra-short pulse laser light source is used to generate an ultra-short pulse laser, and the focal line is generated in the sample by using spatiotemporal focusing, the excited fluorescence is collected, and the confocal optical slit is used to filter out the stray light and collect the fluorescence spectral information to complete the spectral information of the sample. (x, λ) is obtained, and finally the five-dimensional information of the sample (x, λ, y, z, t) is obtained by three-dimensional space scanning and time-lapse scanning. The device includes an ultra-short pulse laser light source and a beam conversion system, a line scanning system based on space-time focusing, an optical microscope system, and a filtering and synchronizing spectral confocal detection system, and the spectral information acquisition in the filtering and synchronizing spectral confocal detection system Synchronized with line scan trigger signal in line scan system combined with spatiotemporal focusing technology. The invention has the advantages of wide field of view, high spatial resolution, high temporal resolution, high spectral resolution and the like.

Description

Method and device for wide-field tomographic hyperspectral microscopy based on spatiotemporal focusing

technical field

The invention relates to a wide-field tomographic hyperspectral microscopic imaging method and device based on space-time focusing, and belongs to the technical field of microspectral imaging and analytical chemistry.

Background technique

Hyperspectral microscopy (Hyperspectral Microscopy) has important applications in the field of biomedical research, especially in the fields of clinical disease diagnosis, intraoperative image navigation and so on. The use of hyperspectral microscopy imaging technology to obtain spatially resolvable spectral information can provide information on the physiological parameters, morphology and composition of biological tissues for disease diagnosis. At present, the use of hyperspectral microscopy imaging technology has achieved non-invasive detection of various cancers.

Essentially, hyperspectral microscopy imaging technology is a technology to obtain higher-dimensional information (ie, spectral information) based on microscopic imaging. According to the realization method of microscopic imaging technology, the current hyperspectral microscopic imaging technology can be divided into hyperspectral microscopic imaging technology based on ordinary wide-field microscopy, and hyperspectral microscopic imaging technology based on confocal scanning. The former can quickly obtain spectral information in a wide field of view in parallel, but due to the shortcomings of ordinary wide-field microscopy, which does not have the ability to tomography, and is susceptible to signal crosstalk caused by tissue scattering, this technology is only suitable for transparent biological samples. The latter is based on the confocal principle, which suppresses the effect of tissue scattering to a certain extent and achieves axial resolution, but the imaging speed is limited due to the need for point-by-point scanning imaging. In addition, hyperspectral microscopy imaging technology based on light sheet microscopy has emerged in recent years, but unfortunately this technology is also not suitable for imaging of scattering tissue.

In order to overcome the influence of biological tissue scattering and improve the imaging penetration depth, nonlinear optical microscopy was introduced into hyperspectral microscopy imaging, and hyperspectral microscopy based on nonlinear optical effects was developed and widely used in biological Medical Research. Since most common nonlinear optical microscopy techniques still use point scanning to overcome the influence of tissue scattering, the imaging speed and flux are bound to be affected. On the other hand, although the nonlinear optical microscopy using surface excitation eliminates the speed bottleneck caused by point-by-point scanning, the excited signal has serious crosstalk after being scattered by the tissue, which is not suitable for spectral imaging of scattering tissue. Ordinary nonlinear optical microscopy using line scanning can compromise between imaging speed and suppression of scattering effects, but this method is not an ideal choice because of the reduced axial resolution obtained with point scanning.

Therefore, a technical problem that needs to be urgently solved by those skilled in the art is: how to innovatively propose an effective measure to solve the deficiencies in the prior art.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a wide-field tomographic hyperspectral microscopy imaging method and device based on spatiotemporal focusing. The invention is suitable for scattering biological tissue imaging, can improve the imaging speed and flux, and has high axial resolution.

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for wide-field tomographic hyperspectral microscopic imaging based on spatiotemporal focusing proposed by the present invention is characterized in that it includes the following steps:

1) Parameter setting: set the x-axis, y-axis and z-axis along the transverse, longitudinal and axial directions of the sample respectively, set the λ-axis along the laser spectrum direction as the λ-axis, and set the direction along the time dimension as the t-axis; set the sample In the target scanning area XYZ, set the deflection angle step size of the galvanometer to realize the longitudinal line scanning of the sample, set the axial step size of the microscope objective lens to realize the axial scanning of the sample, and set the spectral information according to the size of the target scanning area Acquisition cycle and total scan duration;

2) Utilize ultra-short pulse laser light source to generate ultra-short pulse laser;

3) At the beginning of a scanning period, a focal line focusing in both space and time dimensions is formed in the sample by the spatiotemporal focusing method;

4) The fluorescence signal is excited on the focal line of step 3) through the nonlinear optical effect, the fluorescence signal is collected by the microscope objective lens and then transmitted in the reverse direction, and then the reflected ultra-short pulse laser is filtered out by the filter, and the reflected ultra-short pulse laser is filtered by the confocal optics. The stray light caused by the scattering of the sample is filtered out by the slit, and the stripe-shaped emission fluorescence of the sample is obtained;

5) The obtained stripe-shaped emission fluorescence is spectrally expanded by the dispersive element, and the spectral information is collected by the area array detector to obtain the (x, λ) two-dimensional information of the sample; at the same time, according to the set galvanometer deflection angle step size Change the deflection angle of the line scan to obtain (x, λ, y) three-dimensional information of the sample, until the scanning traverses the XY target area, and completes the acquisition of spectral information at different positions on the two-dimensional plane of the sample; wherein, the detection area of the area array detector The size completely covers the spread of the stripe-shaped emission fluorescence, and the area array detector is synchronized with the trigger signal of the galvanometer;

6) Change the depth of the focal line according to the set axial step size of the microscope objective, and obtain the (x, λ, y, z) four-dimensional information of the sample, until the XYZ target area is scanned and traversed, and the acquisition of spectral information at different depths is completed. Spectral information at different positions in the three-dimensional space in the sample; the current scanning cycle ends, and step 7) is performed;

7) Repeat steps 3) to 6) according to the set spectral information collection cycle to perform time-lapse hyperspectral microscopy imaging to obtain (x, λ, y, z, t) five-dimensional information of the sample until the set value is reached. The total scanning time is used to complete wide-field tomographic hyperspectral microscopy imaging.

The present invention also proposes a device according to the above-mentioned wide-field tomographic hyperspectral microscopy imaging method based on space-time focusing, which is characterized in that it includes an ultra-short pulse laser light source and a beam conversion system, a line scanning system based on space-time focusing, an optical Microscopic systems, and filtering and synchronizing spectral confocal detection systems; wherein,

The ultra-short pulse laser light source and beam conversion system, the ultra-short pulse laser light source is used to provide excitation pulse light for generating nonlinear optical signals, and the beam conversion system is used to adjust the beam size of the excitation pulse light;

The line scanning system based on space-time focusing is placed after the beam conversion system, and includes an optical diffraction element, a lens and an optical scanning element, and the optical diffraction element and the optical scanning element are respectively placed on the object side of the lens. a focal plane and an image-side focal plane; the optical diffraction element is used to introduce the angular dispersion of the excitation pulse beam, and the optical scanning element is used to introduce a variable deflection angle of the excitation pulse beam;

The optical microscope system, placed behind the optical scanning element, includes a lens group and a microscope objective lens. The lens group connects the optical scanning element and the rear entrance pupil surface of the microscope objective lens to form a 4f system. The microscope system is used to form focal lines in the sample that are focused in both spatial and temporal dimensions to excite the tissue sample and generate emission fluorescence based on nonlinear optical effects;

The filtering and synchronizing spectral confocal detection system is placed behind the optical scanning element in the line scanning system based on spatiotemporal focusing that emits fluorescence and is collected by the microscope objective lens and transmitted in reverse, including filters, confocal optical narrow A slit and a spectrometer are used to select the emission fluorescence signal of the sample and collect spectral information; the information acquisition of the spectrometer is synchronized with the trigger signal of the line scanning system based on spatiotemporal focusing.

Compared with the prior art, the present invention has the following advantages: by adopting the line scanning technology based on the spatiotemporal focusing method and proposing the corresponding synchronous spectral confocal detection technology, it can ensure high axial resolution, low scattering signal crosstalk and high-speed spectroscopy. Information acquisition, the realized wide-field tomographic hyperspectral microscopy imaging technology is suitable for deep tissue wide-field high-speed tomographic microscopy imaging.

In summary, the proposed wide-field tomographic hyperspectral microscopy imaging technique can be used for (x, y, z, t, λ) five-dimensional information acquisition of deep biological tissues, with wide field of view, high spatial resolution, It has the advantages of high temporal resolution and high spectral resolution.

Description of drawings

FIG. 1 is a schematic structural diagram of the wide-field tomographic hyperspectral imaging device according to the present invention.

FIG. 2 is a schematic diagram of the principle of the present invention.

FIG. 3 is a schematic structural diagram of Embodiment 1 of the apparatus of the present invention.

FIG. 4 is a schematic structural diagram of Embodiment 2 of the apparatus of the present invention.

Detailed ways

In order to make the above objects, features and advantages of the present invention more clearly understood, the present invention will be described in further detail below with reference to the accompanying drawings and specific embodiments.

A wide-field tomographic hyperspectral microscopic imaging method proposed by the present invention specifically includes the following steps:

1) Parameter setting: set the x-axis, y-axis and z-axis along the transverse, longitudinal and axial directions of the sample respectively, set the direction along the laser spectrum (ie wavelength) as the λ-axis, and set the direction along the time dimension as the t-axis ;Set the target scanning area XYZ in the sample, set the deflection angle step size of the galvanometer to realize the longitudinal line scanning of the sample, set the axial step size of the microscope objective lens to realize the scanning along the sample axis, according to the size of the target scanning area Set the spectral information collection cycle and the total scanning time;

2) Utilize ultra-short pulse laser light source to generate ultra-short pulse laser;

3) At the beginning of a scanning period, a focal line focusing in both space and time dimensions is formed in the sample by the spatiotemporal focusing method (along the x direction, the y-direction scanning of the focal line can be achieved by changing the deflection angle of the galvanometer) ;

4) The fluorescence signal is excited on the focal line of step 3) through the nonlinear optical effect, the fluorescence signal is collected by the microscope objective lens and then transmitted in the reverse direction, and then the reflected ultra-short pulse laser is filtered out by the filter, and the reflected ultra-short pulse laser is filtered by the confocal optics. The stray light caused by the scattering of the sample is filtered out by the slit, and the stripe-shaped emission fluorescence of the sample is obtained;

5) The obtained stripe-shaped emission fluorescence is spectrally expanded by the dispersive element, and the spectral information is collected by the area array detector to obtain the (x, λ) two-dimensional information of the sample; at the same time, according to the set galvanometer deflection angle step size Change the deflection angle of the line scan to obtain the (x, λ, y) three-dimensional information of the sample, until the scanning traverses the XY target area, and completes the acquisition of spectral information at different positions on the two-dimensional plane of the sample; among them, the detection area of the area array detector is completely Covers the spread of the stripe emission fluorescence, and the area array detector is synchronized with the trigger signal of the galvanometer;

6) Change the depth of the focal line according to the set axial step size of the microscope objective, and obtain the (x, λ, y, z) four-dimensional information of the sample, until the XYZ target area is scanned and traversed, and the acquisition of spectral information at different depths is completed. Spectral information at different positions in the three-dimensional space in the sample; the current scanning cycle ends, and step 7) is performed;

7) Repeat steps 3) to 6) according to the set spectral information collection cycle to perform time-lapse hyperspectral microscopy imaging to obtain (x, λ, y, z, t) five-dimensional information of the sample until the set value is reached. The total scanning time is used to complete wide-field tomographic hyperspectral microscopy imaging.

The present invention also proposes a wide-field tomographic hyperspectral microscopic imaging device based on the above method, the structure of which is shown in Figure 1, including: an ultra-short pulse laser light source and a beam conversion system, a line scanning system based on space-time focusing, an optical Microscopic systems, and filtering and synchronizing spectral confocal detection systems; wherein,

Ultra-short pulse laser light source and beam conversion system, the ultra-short pulse laser light source is used to provide excitation pulse light for generating nonlinear optical signals, and the beam conversion system is used to adjust the beam size of the excitation pulse light;

A line scanning system based on space-time focusing, placed after the above beam conversion system, includes an optical diffractive element, a lens and an optical scanning element, and the optical diffractive element and the optical scanning element are placed on the object focal plane and the image focal plane of the lens respectively; The diffractive element is used to introduce the angular dispersion of the excitation pulse beam, and the optical scanning element is used to introduce the variable deflection angle of the excitation pulse beam;

An optical microscope system, placed after the above-mentioned optical scanning element, includes a lens group and a microscope objective lens, and the lens group connects the above-mentioned optical scanning element and the rear entrance pupil surface of the microscope objective lens, and constitutes a 4f system, which is used to form a simultaneous formation in the sample. Focusing lines focused in both spatial and temporal dimensions to excite tissue samples and generate emitted fluorescence based on nonlinear optical effects;

Filtering and synchronizing spectral confocal detection system, placed in the line scanning system based on space-time focusing, after the optical scanning element that emits fluorescence is collected by the microscope objective lens and transmitted in reverse, including filters, confocal optical slits and spectrometers, used for The emission fluorescence signal of the sample is selected and spectral information acquisition is performed; wherein, the information acquisition of the spectrometer is synchronized with the trigger signal of the line scanning system based on spatiotemporal focusing.

Further, in the ultra-short pulse laser light source and beam conversion system, a dispersion pre-compensation system is also provided before the ultra-short pulse laser is output to pre-compensate the dispersion accumulated by the ultra-short pulse before reaching the focus of the microscope objective lens.

Further, the line scanning system based on the spatiotemporal focusing technology also includes an adaptive optical element placed at the Fourier plane of the optical diffraction element along the propagation direction of the excitation light after the lens is used for spectral phase shaping to further overcome the scattering of biological samples. impact on the final image.

The specific implementation mode of each component in the device of the present invention is as follows:

In the ultrashort pulse laser light source and beam conversion system, the ultrashort pulse laser light source can choose femtosecond pulse laser light source or picosecond pulse laser light source according to the output pulse width; the ultrashort pulse laser light source can be adjusted according to whether the output wavelength is adjustable, and fixed Ultrashort pulse laser light source with wavelength or ultrashort pulse laser light source with tunable wavelength; beam transformation system is Galileo telescope system or Kepler telescope system. The ultrashort pulse laser light source and the beam conversion system provide the nonlinear optical signal in the excitation light to generate the nonlinear optical signal through any one of the two-photon absorption fluorescence effect, the three-photon absorption fluorescence effect or the two-photon excitation-fluorescence resonance energy transfer effect produce.

In the line scanning system based on spatiotemporal focusing technology, gratings, deformable mirrors, spatial light modulators or other optical diffraction elements can be selected as optical diffraction elements; galvanometers, polygon mirrors or acousto-optic modulators can be selected as optical scanning elements.

In the filtering and synchronizing spectral confocal detection system, the filter selects dichroic mirror, band-pass filter, low-pass filter or long-pass filter. The confocal optical slit is placed on the conjugate plane of the biological sample excitation plane, and the width of the confocal optical slit is determined by the design size of the conjugate image of the sample. The spectrometer is composed of a dispersive element, a two-dimensional surface detector and two lenses. The first lens forms an object-image relationship between the confocal optical slit and the dispersive element, and the second lens forms an object-image relationship between the dispersive element and the two-dimensional surface detector; Prisms, gratings or other dispersive elements can be used as dispersive elements; charge-coupled elements (CCD), electron multiplying charge-coupled elements (EMCCD) or scientific-grade complementary metal-oxide semiconductor devices (sCMOS) can be selected for two-dimensional surface detectors.

Referring to FIG. 2, a schematic diagram of the principle of the present invention is shown. Using nonlinear optical effects combined with spatiotemporal focusing technology (US invention patent US20080151238A1), a focal line (x direction) with high axial resolution can be generated on the biological sample, and the excited fluorescence spectral signal is detected by confocal on the second side of the spectrometer. Dimensional photodetection surface (Dx, Dy) imaging, where the Dy direction is the spectral λ dimension. By scanning in the y direction of the sample and performing simultaneous confocal detection, the (x, λ, y) three-dimensional information of the sample in a wide field of view can be obtained. Using the axial tomographic capability of this technology to perform axial scanning (that is, changing the relative position of the moving sample and the microscope objective), the (x, λ, y, z) four-dimensional information of the sample can be obtained; further, using this technology The high-speed spectroscopic imaging capability performs time-lapse information acquisition (that is, collecting four-dimensional information of the sample (x, λ, y, z) at different times), and obtains the (x, λ, y, z, t) five-dimensional information of the sample. It can be seen that the wide-field tomographic hyperspectral microscopy imaging method and device thereof proposed in the present invention have the advantages of wide field of view, high spatial resolution, high temporal resolution, high spectral resolution, etc., and can be used for the study of biological dynamic processes, Provides rich information on the basis of disease diagnosis.

Example 1:

3 , the wide-field tomographic hyperspectral microscopy imaging device of the present embodiment will be described in detail. The device includes an ultra-short pulse laser light source and a beam conversion system, a line scanning system based on space-time focusing, an optical microscope system, a filter With the simultaneous spectral confocal detection system, the biological sample is placed on the sample stage 319 . Among them, the ultrashort pulse laser light source 301 in the ultrashort pulse laser light source and the beam transformation system adopts a femtosecond laser (such as the Coherent Chameleon Discovery series), and the beam transformation system adopts a Kepler telescope system composed of a lens 302 and a cylindrical lens 303 ( is a 4f system); the line scanning system based on spatiotemporal focusing includes a transmission grating 304, a lens 305 and a scanning galvanometer 307; the optical microscope system includes two lenses 308, 309 and a microscope objective 310; filtering and synchronizing spectral confocal detection system It includes a dichroic mirror 306, a low-pass filter 311, a lens 312, a confocal optical slit 313 (the width of the optical slit is determined by the design size of the conjugate image of the biological sample), and two lenses 314, 316, a reflection grating 315 and a two-dimensional surface detector (using sCMOS or EMCCD) 317 to form a spectrometer. The relative positional relationship of the above components is: the lens 302 and the cylindrical lens 303 form a 4f system for beam expansion, the transmission grating 304 is placed at the image surface of the cylindrical lens 303, the lens 305 images the transmission grating 304 at the scanning galvanometer 307, and the lens 308 and 309 form a 4f system, so that the scanning galvanometer 307 and the entrance pupil plane of the microscope objective 310 are conjugated, and the lens 312 and the lens 308 form a 4f system so that the object surface is imaged at the confocal optical slit 313, and the low-pass filter 311 is tight. Before the lens 312 , the lens 314 images the confocal optical slit 313 on the reflection grating 315 , and the lens 316 images the reflection grating 315 on the two-dimensional surface detector 317 . FIG. 3 also shows a computer 318 for controlling the deflection angle of the scanning galvanometer 307 and performing conventional image reconstruction and data processing on the spectral information collected by the two-dimensional surface detector 317 .

In this embodiment, the laser beam emitted by the ultra-short pulse laser light source 301 is expanded by the lens 302 and the cylindrical lens 303 (changing the diameter of the laser beam) and then incident on the transmission grating 304, and the ultra-short pulse beam is generated under the action of the transmission grating 304. Angular dispersion (the purpose is to make the angularly dispersed beam fill the back focal plane of the objective lens after passing through the subsequent optical elements), collimated by the lens 305 and projected onto the scanning galvanometer 307 through the dichroic mirror 306 and introduce a variable deflection angle (The deflection angle is driven by the galvanometer driving voltage, and the deflection angle is set according to the scanning area), and finally the focal line is generated on the focal plane of the objective lens in the biological sample through the lenses 308 and 309 and the microscope objective lens 310 . The optical signal generated by the nonlinear optical effect is collected by the microscope objective lens 310 and then transmitted in the reverse direction. After that, the signal beam passes through the low-pass filter 311, the lens 312 and the confocal optical slit 313 in sequence, and finally enters the spectrometer for signal collection. It should be noted that the line scan trigger signal of the scanning galvanometer 307 is synchronized with the frame trigger signal of the two-dimensional surface detector 317 . Using the above technical solution, the (x, λ, y) three-dimensional information of the biological sample can be obtained. By moving the microscope objective 310 to perform axial scanning, the (x, λ, y, z) four-dimensional information of the sample can be obtained. If the time delay information collection is performed, the (x, λ, y, z, t) five-dimensional information of the sample can be obtained.

Example 2:

4 , the wide-field tomographic hyperspectral imaging apparatus of this embodiment will be described in detail. The difference between this embodiment and Embodiment 1 is that an adaptive optical element is added. The device of this embodiment includes an ultra-short pulse laser light source and a beam conversion system, a line scanning system based on spatiotemporal focusing, an optical microscope system, a filtering and synchronizing spectral confocal detection system, and the biological sample is placed on the sample stage 424; The ultra-short pulse laser light source 401 in the pulsed laser light source and the beam conversion system adopts a femtosecond laser (such as the Coherent Chameleon Discovery series), and the beam conversion system adopts a Kepler telescope system (4f system) composed of a lens 402 and a cylindrical lens 403. ; The line scanning system based on spatiotemporal focusing includes a transmission grating 404, five lenses 405, 407, 408, 410, 411, and an adaptive optical element 406 arranged between the lenses 405 and 407 (this embodiment uses a spatial light modulator) , scanning galvanometer 412; optical microscope system includes two lenses 413, 414, microscope objective lens 415; filtering and synchronizing spectral confocal detection system includes dichroic mirror 409, low-pass filter 416, common lens 417, confocal optics A slit 418 (the width of the optical slit is determined by the design size of the conjugate image of the biological sample), and a spectrometer composed of two lenses 419, 421, a reflection grating 420 and a two-dimensional surface detector (using sCMOS or EMCCD) 422. The relative positional relationship of the above components is: the lens 402 and the cylindrical lens 403 form a 4f system for beam expansion, the transmission grating 404 is placed at the image surface of the cylindrical lens 403, and the lens 405 images the transmission grating 404 at the spatial light modulator 406, Lenses 407 and 408, lenses 410 and 411, and lenses 413 and 414 respectively form three groups of 4f systems in series, so that the spatial light modulator 406 is conjugated with the entrance pupil plane of the scanning galvanometer 412 and the microscope objective lens 415, and the lenses 417 and 410 constitute The 4f system makes the object surface image at the confocal optical slit 418, the low-pass filter 416 is placed immediately before the lens 417, the lens 419 images the confocal optical slit 418 on the reflection grating 420, and the lens 421 images the reflection grating 420 on the reflection grating 420. 2D surface detector 422 . FIG. 4 also shows a computer 423 for controlling the deflection angle of the scanning galvanometer 412 and performing conventional image reconstruction and data processing on the spectral information collected by the two-dimensional surface detector 422 .

In this embodiment, the laser beam emitted by the ultra-short pulse laser light source 401 is expanded by the lens 402 and the cylindrical lens 403 and then incident on the transmission grating 404 . 405 is collimated and projected to the spatial light modulator 406 for spectral phase shaping to further overcome the influence of tissue scattering, and then is introduced on the scanning galvanometer 412 through lens 407, lens 408, dichroic mirror 409, lens 410, and lens 411 in sequence The deflection angle is variable, and finally the focal line is generated on the focal plane of the objective lens in the biological sample through the lenses 413, 414 and the microscope objective lens 415. The fluorescent signal generated by the nonlinear optical effect is collected by the microscope objective lens 415 and then transmitted in reverse, passes through the lens 414 , the lens 413 , the scanning galvanometer 412 , the lens 411 , and the lens 410 in sequence, and is reflected by the dichroic mirror 409 . After that, the signal beam passes through the low-pass filter 416, the lens 417 and the confocal optical slit 418 in sequence, and finally enters the spectrometer for signal collection. It should be noted that the line scan trigger signal of the scanning galvanometer 412 is synchronized with the frame trigger signal of the 2D surface detector 422; the spatial light modulator 406 uses adaptive optics to measure the wavefront distortion and apply a compensation wavefront (in the spectral before imaging). Using the above technical solution, the (x, λ, y) three-dimensional information of the biological sample can be obtained. By moving the microscope objective 415 to perform axial scanning, the (x, λ, y, z) four-dimensional information of the biological sample can be obtained. If delayed information collection (ie, multiple collections) is performed, five-dimensional information of (x, λ, y, z, t) of the biological sample can be obtained.

In the actual experiment, considering that the required excitation light power may be greater than the light damage threshold of the spatial light modulator, it may also be necessary to add a lens group before the spatial light modulator 406, so that the beam expands to the light intensity in the direction perpendicular to the spectral expansion. Below and below the photodamage threshold of the spatial light modulator.

In summary, the present invention performs line scanning technology by combining spatiotemporal focusing technology, and proposes a corresponding synchronous spectral confocal detection technology, which ensures high axial resolution and low scattering signal crosstalk, and is suitable for deep tissue tomographic spectroscopic microscopy imaging. ; Improve the speed of spectral information acquisition, and can realize high-speed spectral microscopy imaging with wide field of view.

The wide-field tomographic hyperspectral microscopy imaging method and device proposed by the present invention have been described above in detail, and specific examples are used in this paper to illustrate the principles and implementations of the present invention. The descriptions of the above embodiments are only used for Help to understand the method and core idea of the present invention; at the same time, for those skilled in the art, according to the idea of the present invention, there will be changes in the specific implementation and application scope, and these changes should belong to the attached document of the present invention. the scope of protection of the claims. In conclusion, the contents of this specification should not be construed as limiting the present invention.

Claims (6)

1. A wide-field chromatography hyper-spectral microscopic imaging method based on space-time focusing is characterized by comprising the following steps:

1) setting parameters: setting an x axis, a y axis and a z axis respectively along the transverse direction, the longitudinal direction and the axial direction of a sample, setting a lambda axis along the laser spectrum direction, and setting a t axis along the time dimension direction; setting a target scanning area XYZ in a sample, setting a galvanometer deflection angle step length for realizing scanning along a longitudinal line of the sample, setting a microscope objective axial step length for realizing axial scanning along the sample, and setting a spectrum information acquisition period and total scanning duration according to the size of the target scanning area;

2) generating ultrashort pulse laser by using an ultrashort pulse laser light source;

3) forming a focusing line which is focused in two dimensions of space and time simultaneously in a sample by a space-time focusing method at the starting moment of a scanning period;

4) exciting a fluorescence signal on the focusing line in the step 3) through a nonlinear optical effect, collecting the fluorescence signal through a microscope objective lens, transmitting the fluorescence signal in a reverse direction, filtering reflected ultrashort pulse laser by a filter plate, and filtering stray light caused by sample scattering by a confocal optical slit to obtain strip-shaped emission fluorescence of the sample;

5) performing spectrum expansion on the obtained bar-shaped emission fluorescence through a dispersion element, and acquiring spectral information through an area array detector to obtain (x, lambda) two-dimensional information of a sample; meanwhile, changing the deflection angle of line scanning according to the set step length of the deflection angle of the galvanometer to obtain (x, lambda, y) three-dimensional information of the sample until the XY target area is traversed by scanning, and acquiring spectral information of different positions of a two-dimensional plane of the sample; the size of a detection area of the area array detector completely covers the unfolding degree of the strip-shaped emission fluorescence, and the area array detector is synchronous with a trigger signal of the galvanometer;

6) changing the depth of a focal line according to the set axial step length of the microscope objective to obtain (x, lambda, y, z) four-dimensional information of the sample until the XYZ target area is scanned and traversed, and finishing the acquisition of spectral information of different depths to obtain spectral information of different positions of a three-dimensional space in the sample; when the current scanning period is finished, executing step 7);

7) and (3) repeating the steps 3) to 6) according to a set spectrum information acquisition period to perform time-delay hyperspectral microimaging, and obtaining (x, lambda, y, z, t) five-dimensional information of the sample until a set total scanning time is reached, thereby completing the wide-field chromatography hyperspectral microimaging.

2. The device for the wide-field chromatographic ultra-spectral microscopic imaging method based on the space-time focusing is characterized by comprising an ultra-short pulse laser light source and a light beam transformation system, a line scanning system based on the space-time focusing, an optical microscopic system and a filtering and synchronous spectral confocal detection system; wherein,

the ultra-short pulse laser light source is used for providing excitation pulse light for generating nonlinear optical signals, and the light beam conversion system is used for adjusting the size of the excitation pulse light beam;

the line scanning system based on the space-time focusing is arranged behind the light beam transformation system and comprises an optical diffraction element, a lens and an optical scanning element, wherein the optical diffraction element and the optical scanning element are respectively arranged on an object focal plane and an image focal plane of the lens; the optical diffraction element is used for introducing the angular dispersion of the excitation pulse beam, and the optical scanning element is used for introducing the variable deflection angle of the excitation pulse beam;

the optical microscope system is arranged behind the optical scanning element, comprises a lens group and a microscope objective, and is connected with the optical scanning element and the microscope objective through the lens group to form a 4f system, wherein the optical microscope system is used for forming a focal line which focuses in two dimensions of space and time simultaneously in a sample so as to excite the tissue sample and generate emission fluorescence based on a nonlinear optical effect;

the filtering and synchronous spectrum confocal detection system is arranged in the space-time focusing-based line scanning system and is used for selecting a fluorescence emission signal of a sample and acquiring spectrum information, and comprises a filter, a confocal optical slit and a spectrometer, wherein the optical scanning element is used for collecting and reversely transmitting fluorescence emitted by the line scanning system through the microscope objective; the information acquisition of the spectrometer is synchronized with the trigger signal of the spatio-temporal focusing based line scanning system.

3. The apparatus of claim 2, wherein a dispersion pre-compensation system is further disposed in the ultra-short pulse laser source and the beam transformation system before the ultra-short pulse laser output for pre-compensating the dispersion accumulated by the ultra-short pulse before reaching the focusing surface of the microscope objective.

4. The apparatus of claim 2, wherein the line scanning system based on spatiotemporal focusing technique further comprises an adaptive optical element disposed at the fourier plane of the optical diffraction element after passing through the lens along the propagation direction of the excitation light for performing spectral phase shaping to further overcome the effect of scattering of the biological sample on the final imaging.

5. The device of the wide-field chromatography hyper-spectral microscopic imaging method based on the space-time focusing as claimed in claim 2, wherein in the ultra-short pulse laser light source and the light beam transformation system, the ultra-short pulse laser light source selects a femtosecond pulse laser light source or a picosecond pulse laser light source according to the output pulse width; selecting an ultrashort pulse laser source with fixed wavelength or an ultrashort pulse laser source with tunable wavelength according to whether the output wavelength is tunable or not; the light beam transformation system is a Galileo telescope system or a Keplerian telescope system;

the ultrashort pulse laser source and the light beam conversion system provide the nonlinear optical signal in the exciting light for generating the nonlinear optical signal, and the nonlinear optical signal is generated through any one of a two-photon absorption fluorescence effect, a three-photon absorption fluorescence effect or a two-photon excitation-fluorescence resonance energy transfer effect.

6. The apparatus of the wide-field chromatographic ultra-spectral microscopic imaging method based on space-time focusing according to claim 2, wherein in the filtering and synchronous spectrum confocal detection system, the spectrometer consists of a dispersion element, a two-dimensional plane detector and two lenses; the confocal optical slit is arranged on a conjugate surface of a sample excitation surface, the first lens enables the confocal optical slit and the dispersion element to form an object-image relationship, and the second lens enables the dispersion element and the two-dimensional surface detector to form the object-image relationship.

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