CN110755042A - Time pulse light sheet tomography method and system for realizing large-volume high-resolution - Google Patents
Time pulse light sheet tomography method and system for realizing large-volume high-resolution Download PDFInfo
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Abstract
The invention discloses a time pulse light sheet tomography method and a system for realizing large-volume high-resolution, wherein the method comprises the following steps: 1) laser outputs two synchronous or phase-locked pulse light sources lambda1、λ2(ii) a 2) Wherein a beam of pulsed light source lambda2A time delay device is arranged for adjusting time delay; 3) pulsed light lambda1、λ2The two beams of light are completely reflected or emitted in an angle-opposite direction or an angle-same direction in the propagation direction, and the positions of the two beams of light are adjusted to simultaneously realize the overlapping of space and time pulses on an imaging sample so as to form a time light sheet; 4) generating signals by the fault where the time light sheet is located, and providing image information; 5) by changing the relative time delay of the two beams of light or the position of a sample, the tomography imaging of the light sheet is realized. The invention uses the modulated time pulse light sheet, solves the problems of uneven resolution of the mechanical light sheet, large defocused background in depth imaging and signal background ratioLow cost, and can be applied to organ level imaging of large volume.
Description
Technical Field
The invention relates to the technical field of biomedicine and optical imaging, in particular to a method and a system for realizing large-volume and high-resolution time pulse light sheet three-dimensional tomography.
Background
In biomedical research and practical applications, optical imaging techniques including confocal fluorescence microscopy, multiphoton fluorescence microscopy and other imaging modalities have been widely used to observe the fine structure and functional activity of biological tissues. Although the optical imaging technology has many advantages of no toxicity, high resolution, fast imaging speed, high sensitivity, etc., it is only suitable for imaging the depth of the surface layer of biological tissue from several micrometers to several millimeters. For a large-volume biological tissue or other opaque samples (1 mm to 10 cm and above), light scattering and absorption tend to be serious along with the increase of imaging depth, and the traditional optical method is difficult to be applied to large-volume organ level imaging due to the increase of background signals generated by defocusing, so that the traditional optical method is limited by the poor penetration performance of light in the opaque samples, and the optical imaging resolution is sharply reduced along with the imaging depth. Optical imaging generally adopts higher-order optical nonlinear effect, higher laser instantaneous power, or generates thin space illumination light sheet and other methods to solve the problem of imaging depth, but for samples with the size of more than 1 millimeter, high-resolution optical three-dimensional tomography still cannot be realized. The imaging depth can be improved to the centimeter magnitude by combining with the tissue light transparent technology, but the imaging depth is ineffective for living organisms. At present, technologies such as Magnetic Resonance Imaging (MRI), Computed Tomography (CT), Positron Emission Tomography (PET) and the like can realize large-volume three-dimensional living body imaging, but the spatial resolution is mostly about 1 mm, and the finer tissue structure cannot be analyzed. Meanwhile, the imaging systems have certain radiometric capacity on living bodies, and are complex in technology, large in system and high in price. The invention mainly uses optical means to replace the traditional ray imaging method, realizes large-volume and high-resolution optical tomography in vivo, obtains three-dimensional images of tissues, organs, focus, vascular structures and the like in vivo, and can complete various target function imaging by combining with an optical probe.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a time pulse light sheet tomography method and a time pulse light sheet tomography system for realizing high-resolution imaging on a large-volume sample or a large biological living body so as to solve the problems of optical imaging resolution and imaging depth.
In order to achieve the above purpose, the invention provides a method for realizing large-volume high-resolution time pulse optical sheet tomography, which comprises the following steps:
1) laser outputs two synchronous or phase-locked pulse light sources lambda1、λ2Wherein λ is1And λ2Light pulses representing different characteristics, e.g. different wavelengths, different polarizations;
2) wherein a beam of pulsed light source lambda2A time delay device is arranged for adjusting time delay;
3) pulsed light lambda1And λ2The two beams of pulse light reach a fault on a sample simultaneously by adjusting relative time delay between the two beams of light, and the thickness of the fault is determined by the pulse width of the two beams of light;
4) only the time pulse light sheets with the two characteristics can generate signals when overlapped faults occur in space and time at the same time, and image information is provided;
5) by changing the relative time delay of the two beams of light or the position of a sample, the three-dimensional tomography imaging of the light sheet is realized.
Optionally, the sample is marked, the two pulsed light sources cannot excite the signal when acting alone, and the signal is generated only when the two pulsed light sources act simultaneously.
Optionally, the sample is not treated with a fluorescent dye, the two light pulses λ1、λ2When the two signals are coincided in space and time, the endogenous molecules in the sample are excited to send out signals, wherein the signals comprise optical, sound (including ultrasonic), thermal, electric, magnetic, electromagnetic and other signals.
Optionally, the sample is subjected to fluorescence, phosphorescence, Raman, photothermal and photoacoustic probe labeling treatment, the two pulsed light sources cannot excite the labeling groups to emit signals under the action of single light sources, and the signals are generated only when the two pulsed light sources act simultaneously.
Optionally, the thickness of the temporal slide or the Z-axis resolution of the imaging is determined by the temporal pulse width of two light pulses, and the pulsed light source includes an attosecond, femtosecond, picosecond, nanosecond pulsed laser.
Optionally, the excitation and emission wavelengths of the fluorescent labeling group and the fluorescent group to which the targeting molecule is bound include visible light, near-infrared first region, near-infrared second region, and far-infrared range.
Optionally, the imaging modes of the temporal light sheet include Stimulated Raman Scattering (SRS) imaging, anti-stokes coherent raman (CARS) imaging, Pump-probe imaging, Transient Absorption (TA) imaging, light sheet imaging, and photoacoustic imaging.
Based on the method, the invention also provides a tomography system for realizing the large-volume high-resolution time pulse polished section, which is characterized in that the system comprises a pulse laser, a power adjusting device, a collimation and beam expanding device, an optical modulator, a time delay device, a two-dimensional laser scanning system and a photoelectric collecting system; the sample is arranged in a focus coincidence region of the two types of opposite light, an XYZ three-axis moving system is arranged at the bottom of the sample, and a detector of the photoelectric collecting system can also realize three-dimensional synchronous adjustment and multi-path full-angle detection.
Further, the lateral resolution of the temporal slice is determined by the beam waist of the spot; the laser output by the laser comprises a Gaussian beam, a Bessel beam and a beam with adjustable diameter; the rapid large-range two-dimensional imaging in the time pulse light sheet is realized by adopting two-dimensional laser scanning; the photoelectric collection system comprises a lens and a detector, wherein the detector adopts a single detector; when the signal background is low, the signal is amplified by combining the optical modulation and the phase-locked amplification technology, and the signal-to-back ratio is improved.
Furthermore, the detector adopts a one-dimensional array, a circle array or an area array detector to realize two-dimensional imaging in the light sheet.
Compared with the prior art, the invention has the beneficial effects that:
(1) according to the time pulse optical sheet tomography method for scanning along the propagation direction, the thickness of the optical sheet can be accurately adjusted, axial scanning is realized by changing the time difference of two beams of light, and meanwhile, rapid organ horizontal imaging is finally realized by combining two-dimensional laser rapid scanning, uniform axial resolution and high signal-to-back ratio are simultaneously met, and the imaging depth is improved while the three-dimensional imaging resolution is ensured.
(2) The invention combines various specific fluorescent labeled probes, can realize high-resolution tracing imaging of blood vessels in tissues, meet the requirements of clinical medical detection, monitor the circulation and blockage conditions of blood in real time and analyze the blood oxygen concentration and the PH value; can realize the tumor imaging of optical molecule targeting and expand the biomedical application of the optical probe in human body or animal body; the nerve activity of the deep brain can be monitored; metabolites in tissues, such as oil and protein, can be observed by using a label-free method, such as CARS and SRS, so that rapid pathological analysis is completed, the boundary of a tumor is identified, and a doctor is helped to perform accurate surgical navigation; meanwhile, several imaging modes can be combined to realize multi-mode real-time optical imaging and research and clinical application of other physiopathology.
(3) The invention can combine wide field light sheet illumination, meet the requirement of large field of view, change the shape of light beam, realize the illumination of space light, and realize the further application of optical imaging in the medical field.
Drawings
FIG. 1 is a schematic view of time-pulse optical sheet tomography according to the present invention.
Fig. 2a-2d are schematic diagrams of three-dimensional imaging of a time light sheet with different light spot modes, wherein fig. 2a is a schematic diagram of three-dimensional scanning imaging, fig. 2b is a schematic diagram of a gaussian beam, fig. 2c is a schematic diagram of a bessel beam, and fig. 2d is a schematic diagram of three-dimensional imaging of a wide-field light sheet area array.
FIG. 3 is a schematic diagram of a two-wave long-time pulse optical sheet three-dimensional imaging device according to the present invention;
fig. 4 is a diagram of the data format and preliminary imaging results of the three-dimensional imaging of the present invention.
FIG. 5 is a schematic diagram of ICG fluorescence labeled human blood vessel imaging.
Detailed Description
The invention is described in further detail below with reference to the figures and specific embodiments. In order to clearly illustrate the practical significance of the present invention, the examples given in the embodiments are common and preferred but not limiting.
The invention provides a time pulse light sheet tomography method for realizing large-volume high-resolution, which is shown in figure 1 and comprises the following steps:
1) laser simultaneously outputs two beams of synchronous or phase-locked pulse light source lambda1、λ2Wherein λ is1And λ2Have different characteristics, such as different wavelengths;
2) shown, wherein a beam of pulsed light sources λ2The time delay device is matched for time delay adjustment, the time delay device can be composed of two reflectors which are arranged on the electric one-dimensional translation table and form an included angle of 90 degrees, and the high-speed electric translation table implements rapid tomography;
3) pulsed light lambda1And λ2The two beams of pulse light reach a fault on a sample simultaneously by adjusting relative time delay between the two beams of light, and the thickness of the fault is determined by the pulse width of the two beams of light;
4) only the superposition of the light pulse generation space and time with the two characteristics appears on the fault where the time pulse light sheet is positioned can generate signals, and image information with high signal-to-background ratio is provided;
5) the tomography imaging of the light sheet is realized by changing the relative time delay of the two beams of light or axially moving the position of the sample.
Therefore, the three-dimensional tomography can only generate signals when the light with the two characteristics is overlapped in time and the fault with overlapped light pulses generates high-contrast image information. As shown in fig. 1a, the signal is generated only at signal slice 1; adjusting the beam lambda2The two beams coincide at different positions, as shown in fig. 1b, and only the signal is generated at the signal fault 2; as shown in FIG. 1c, by continuously changing the relative time delay of the two beams of light or the position of the sample, the tomography imaging of the time pulse light sheet is finally realized. Distance Δ of time delay movementZ1Equal to the distance Δ Z over which the light sheet moves in the sample2。
Example fluorescence labeling imaging
For fluorescence labeling imaging, the excitation and emission wavelengths of the fluorescence labeling groups include the visible, near infrared and far infrared ranges. For example, visible light excitation is taken as an example, the two laser wavelengths are 1040nm &1130nm, and the two-color two-photon excitation of Sulforhodamine101, Texas red and quantum dot dyes can be carried out.
The pulse laser has the advantages of ultrashort pulse and high repetition frequency, taking a femtosecond light source as an example, the laser outputs two beams of synchronous light sources, the laser time pulse width is about 100 femtoseconds, and the thickness of the corresponding light pulse is 30 mu m, namely the thickness of an optical sheet; the thickness of the time light sheet or the imaging Z-axis resolution is determined by the time pulse width of two light pulses, the pulse light source comprises attosecond, femtosecond, picosecond and nanosecond pulse lasers, and higher axial resolution can be realized by reducing the time pulse width. The repetition frequency of the laser is MHz Gaussian mode laser, the femtosecond laser is output by adopting double channels with tunable wavelength, the sum frequency or the difference frequency of the wavelengths of the two beams of laser meets the condition of nonlinear absorption of dye or excited molecule vibration, taking two photons with bicolor wavelength as an example, the optical frequency omega is1And ω2Not equal, but their sum of photon energies is equal to the photon energy required for single photon excitation of the dye; other nonlinear processes include two-color three-photon fluorescence, and the CARS, the SRS and the like all need to meet the principle of simultaneous action of two wavelengths; a single beam of light cannot generate a signal.
The procedure was carried out as shown in fig. 2a for imaging with laser scanning:
1) two beams of pulse light source Lambda with same repetition frequency, time synchronization and phase locking output by laser1、λ2(ii) a Two beams of laser are oppositely shot.
2) Two beams of laser respectively enter an X-Y two-position scanning correlation system to complete two-dimensional scanning, and a three-dimensional translation table can also be adopted to realize three-dimensional scanning; when the time pulses are overlapped in the Z direction, a signal is generated, and a multi-channel detector which can be a circle array or an area array is arranged around the sample; in the case of a dual wavelength imaging system, the lateral resolution and beam diameter of the final system are related to the focal length of the focusing lens:
dxy is the lateral PSF, λ is the wavelength, and NA is the numerical aperture of the lens;
r is light spot 1/e2Radius, f is the focal length of the lens
The selection of the lateral resolution can be achieved by changing the beam diameter and the focal length of the lens. Since the pulse width of the laser does not change, the sheet thickness does not change as it passes through the sample. As shown in FIGS. 2b, 2c and 2D, the laser mode can be Gaussian beam, Bessel beam, wide-field area light source, wherein the imaging resolution D isXYSatisfies the depth range Z of 0.1-50 μmR3-10 mm;
3) one beam of femtosecond laser lambda is emitted2Generating a sine modulation waveform through a signal source by an acousto-optic or electro-optic polarization modulator, wherein the modulation frequency is f1 which is 0.5-2 MHz;
4) will be lambda2The light beam passes through a time delay device, and a linear motor or a voice coil drives and controls a moving platform, wherein the adjustment precision of the motor is 0.1-10 mu m;
5) after two beams of light respectively complete two-dimensional laser scanning, the two beams of light are focused on a sample through two lenses. Pulsed light lambda1And λ2The two beams of light can be completely or angularly collimated in the propagation direction, and the positions of the two beams of light are adjusted to simultaneously realize the superposition of space and time pulses on one fault of an imaging sample so as to generate a fluorescence or scattering signal; the moving direction of the light sheet is along the propagation direction of the light; the thickness of the light sheet is the thickness of the two beams of light which are overlapped in time, the light sheet generated by the 100 femtosecond pulse laser is 30 μm theoretically, and the thickness of the light sheet is uniform and does not change along with the change of the depth. Size D of light spot focused by two beams of lightxyI.e. the imaging resolution, the focal length, i.e. the rayleigh length ZR(ii) a As shown in the side view of fig. 2a, the imaging signals are passed through a photodetector at the side or other stereoscopic positionThe angle realizes the synchronous acquisition of signals, and the multipath detector is positioned on the side of a signal generation area of the sample. If a rapid laser scanning system is not used, the scanning of a two-dimensional image can be realized by combining an electric scanning translation table, and the three-dimensional data of a sample can be acquired by controlling the synchronization of acquisition and scanning; if wide field illumination is used, a backward detection system may be used, as shown in fig. 2d, using a dichroic mirror to reflect the signal into an area array detector.
Based on the method, the invention also provides an imaging device for realizing the large-volume high-resolution time pulse optical sheet fault, as shown in fig. 3, the system comprises a pulse laser and outputs two paths of synchronous laser pulses lambda1、λ2Respectively pass through a power regulating device consisting of a half-wave plate and a polarization beam splitter prism. In order to realize proper resolution, the two beams of light respectively pass through a beam expanding and collimating device consisting of lenses, and the size of a light spot after beam expansion is adjusted to meet the requirements of imaging resolution and imaging depth range after focusing; laser lambda2The method comprises the steps that amplitude modulation, polarization and phase modulation can be achieved through an optical modulator, modulated light beams pass through a time delay device, two laser beams enter a two-dimensional laser scanning system respectively, light spots are focused on a sample through two focusing lenses, and signals are obtained through a photoelectric collection system and a detection system; the sample is arranged at the focus of the two lenses, the light paths are completely opposite to the combined beam, the bottom of the sample is provided with an XYZ three-axis moving system, and the single or multiple detectors of the photoelectric collecting system are arranged on the side or other azimuth angles of the sample.
The laser mode output by the laser comprises a Gaussian beam and a Bessel beam, the collimation and beam expansion device is used for adjusting the size of a light spot entering the two-dimensional laser scanning system, and the specific size of the light spot is obtained by changing the parameters of the beam expansion device. The time delay device comprises a translation stage and an optical reflector mounted on a platform, wherein the translation stage is driven by a linear motor or a voice coil. The photoelectric collection system comprises a photoelectric detector which is a camera or a photomultiplier array and other array probes. Optical modulation may be combined and the signal amplified by a phase-locked amplification technique. The detector adopts a one-dimensional array, a ring array or an area array detector to realize two-dimensional imaging in the light sheet. The collecting system comprises a lens and an optical filter, the background light is filtered, the optical signal is transmitted to a probe of a photoelectric detector, the collected photoelectric signal is input to a phase-locked amplifier for demodulation, the demodulated signal is transmitted to a data acquisition card and is displayed and stored by a computer. The two-dimensional laser scanning system is a two-dimensional scanning galvanometer or an MEMS and acousto-optic deflector, and the two-dimensional laser scanning is adopted to realize the rapid large-range two-dimensional imaging in the optical sheet. The forward detection system comprises a dichroic mirror and an optical filter, and the output end of the forward detection system is connected with a data acquisition card of the photoelectric collection system.
The computer controls the electric displacement platform or the laser two-dimensional scanning system to be synchronous with data acquisition, generated fluorescence enters the photoelectric collection system after passing through the dichroic mirror in the back image detection system and can also be collected on the side surface, the generated electric signal is transmitted to the phase-locked amplifier for demodulation, a modulated reference signal is accessed from a signal source, and the demodulated signal is transmitted to the data acquisition card and is displayed and stored by the computer. As shown in fig. 4a, the three-dimensional stack scanning principle controls the two-dimensional scanning system and the data acquisition to be synchronous, and the two scanning systems also need to be synchronous to ensure that the same position is scanned simultaneously, the data is acquired during the fast axis scanning of the two-dimensional scanning system and is matched with the two synchronous systems, for example, the deepened area of the image color, the arrow indicates the scanning direction, after one line is scanned, the slow axis is moved, the next line is scanned, and finally the two-dimensional scanning is realized, and then the one-dimensional translation stage is controlled to adjust the time delay, for example, the time delay t1,t2,..tnFor example, 1 μm at the time of the moving precision of the platform, the corresponding time delay is 6.67 femtoseconds, and the moving distance in the axial direction of the sample is 1 μm, because the thickness of the laser light sheet corresponding to 100 femtoseconds is 30 μm, the moving precision of the control light sheet in the axial direction each time is less than 20 μm, and the axial sampling rate is improved. After one layer is acquired, the time delay is adjusted, the moving distance of the moving platform is equivalent to the moving distance of the optical sheet, the optical sheet is moved to the next layer, the next cyclic scanning is started, and finally the three-dimensional scanning is realized, as shown in fig. 4 b.
Because one laser beam is modulated, the signal detected by the photoelectric detector can be amplified and demodulated into a fluorescent signal with high signal-to-noise ratio through phase locking. The fluorescence signal is detected by a photoelectric detector such as an SCOMS, an EMCCD camera or a photomultiplier, the photocurrent generated by the camera is output to a lock-in amplifier, a reference signal is the modulation frequency of an acousto-optic modulator, the fluorescence signal is demodulated and transmitted to a computer for display, the imaging result of a time light sheet is shown in figure 4b, fluorescent dye is filled in a quartz capillary tube, the three-dimensional structure of the quartz tube can be reconstructed through three-dimensional scanning, the effects of XY and YZ sections can be analyzed, and the scale bar is 500 mu m.
The thickness of the optical sheet is finer, the pulse width of the femtosecond laser can be reduced, and the adjustment of the thickness of the optical sheet is realized.
Example two vessel imaging
The difference between this embodiment and the first embodiment is that the certified ICG dye which can be used on human body is used, its absorption peak is 800nm, it can be excited by near infrared two-region-1300 nm and-2100 nm, the exciting light has deeper penetration depth, one light beam can not excite fluorescence, only the place where the two light beams coincide generates time light sheet signal, realizes the tomography of blood vessel, as shown in fig. 5;
the sample is subjected to fluorescence, phosphorescence, Raman, photo-thermal and photo-acoustic marking treatment, the two pulse light sources cannot excite the marking group to send out signals under the independent action, and the signals are only generated when the two pulse light sources act simultaneously.
Example three lipid, SRS or CARS imaging of proteins
This example differs from the first example in that the sample is not fluorescently labeled and two light pulses λ1、λ2When the space and time coincide, the endogenous molecules in the sample are excited to send out signals, and the signals comprise optical, sound, heat, electric and magnetic signals. The laser selects pump light and stokes light.
The SRS implementing process comprises the following steps: selecting wavelengths of the pump light and the Stokes light to excite specific analytical chemical bonds, modulating the Stokes light by an acousto-optic modulator, and modulating a reference signal of the modulatorThe signal is input to a phase-locked amplifier; after proper time delay, the pump light is coincided with the pump light on the sample in a correlation mode, the pump light is reflected to a photodiode for detection by a high-pass low-reflection dichroic mirror on a forward propagation light path of the pump light, and then a stimulated Raman signal is demodulated by a phase-locked amplifier; for example, the pump light is 800nm, the Stokes light is 1040nm (or other wavelengths including 1300nm and 2080nm) and the Raman shift is 2800-3100 cm-1The oil and protein components in the tissues can be specifically observed, and the unmarked organ chemical component analysis imaging is realized; raman shift was selected at 960cm-1The signals of the bone can be observed specifically.
The specific implementation process of CARS comprises the following steps: the optical path system is consistent with the SRS, and the scattered anti-Stokes signals are detected by adopting a photoelectric detector such as a photomultiplier tube in a detection part.
The Pump-probe (Pump-probe) and the stimulated emission imaging mode are not described in detail, and the device is the same as the SRS imaging system, except that the used wavelength is different.
Example four Dual wavelength excited photoacoustic and photothermal imaging
The difference between the embodiment and the embodiment I is that the laser uses an excitation wavelength far away from an absorption peak of blood or dye, but the laser can be absorbed by two colors when only two beams act simultaneously, and then emits sound or heat at the position where the two beams coincide; the device is similar to the first embodiment, and the difference lies in a detection and collection part, two beams of pulse light are oppositely emitted or irradiated at an angle in the same way, light sheet signals are superposed and generated in a sample, and a photoacoustic transducer one-dimensional array or ring array is arranged around the sample to detect sound signals, so that the time light sheet photoacoustic imaging and the photothermal imaging excited by dual wavelengths are realized.
In addition to the above embodiments, the present invention may have other embodiments. Any changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principles of the invention are intended to be covered by the scope of the invention which is defined by the claims which follow.
Claims (10)
1. The time pulse light sheet tomography method for realizing large-volume high-resolution is characterized by comprising the following steps of: the method comprises the following steps:
1) laser outputs two synchronous or phase-locked pulse light sources lambda1、λ2Wherein λ is1、λ2Have different characteristics;
2) wherein a beam of pulsed light source lambda2A time delay device is arranged for adjusting time delay;
3) pulsed light lambda1、λ2The two beams of pulse light reach a fault on a sample simultaneously by adjusting the relative time delay between the two beams of light, and the thickness of the fault is determined by the pulse width of the two beams of light;
4) only the superposition of the space and time of the two characteristics of light pulse generation appears on the fault where the time pulse light sheet is positioned can generate signals and provide image information;
5) by changing the relative time delay of the two beams of light or the position of a sample, the tomography imaging of the light sheet is realized.
2. The method for realizing large-volume high-resolution time pulse optical sheet tomography according to claim 1, wherein: the sample is marked, the two pulse light sources can not excite signals under the independent action, and only when the two pulse light sources act simultaneously, signals are generated.
3. The method for realizing large-volume high-resolution time pulse optical sheet tomography according to claim 1, wherein: the sample is not treated with a fluorescent dye, and the two light pulses lambda1、λ2When the space and time coincide, the endogenous molecules in the sample are excited to send out signals, and the signals comprise optical, sound, heat, electricity, magnetism and electromagnetic signals.
4. The method for realizing large-volume high-resolution time pulse optical sheet tomography according to claim 1, wherein: the sample is subjected to fluorescence, phosphorescence, Raman, photo-thermal and photo-acoustic labeling treatment, the two pulse light sources cannot excite the labeling groups to send signals under the independent action, and the signals are only generated when the two pulse light sources act simultaneously.
5. The method for realizing large-volume high-resolution time pulse optical sheet tomography according to claim 2, wherein: the thickness of the time light sheet or the imaging Z-axis resolution is determined by the time pulse width of two light pulses, and the pulse light source comprises attosecond, femtosecond, picosecond and nanosecond pulse lasers.
6. The method for realizing large-volume high-resolution time pulse optical sheet tomography according to claim 4, wherein: the excitation and emission wavelengths of the fluorescent marker group and the fluorescent group combined with the targeting molecule include visible light, a near-infrared region I, a near-infrared region II and a far-infrared range.
7. The method for realizing large-volume high-resolution time pulse optical sheet tomography according to claim 3, wherein: the imaging modes of the time light sheet comprise stimulated Raman scattering imaging (SRS), anti-Stokes coherent Raman imaging (CARS), Pump detection imaging (Pump-probe), transient absorption imaging, light sheet imaging and photoacoustic imaging.
8. An imaging system for realizing the method for realizing the large-volume high-resolution time pulse light sheet tomography according to any one of claims 1 to 7, wherein the imaging system comprises: the system comprises a laser, a power adjusting device, a collimation and beam expanding device, an optical modulator, a time delay device, a two-dimensional laser scanning system, two lenses and a photoelectric collecting system; the sample is arranged at the focus of the two lenses, and an XYZ three-axis moving system and a multi-path full-angle detection system are arranged at the bottom of the sample.
9. The imaging system for implementing the large-volume high-resolution temporal pulsed light sheet tomography method according to claim 1, wherein: the transverse resolution of the time light sheet is determined by the beam waist of the light spot; the laser mode output by the laser comprises a Gaussian beam, a Bessel beam and a beam with adjustable diameter; the rapid large-range two-dimensional imaging in the optical sheet is realized by adopting two-dimensional laser scanning; the photoelectric collection system comprises a detector, wherein the detector adopts a single detector; the optical modulation is combined and the signal is demodulated by a lock-in amplifier.
10. The imaging system for implementing the large-volume high-resolution temporal pulsed light sheet tomography method according to claim 1, wherein: the detector adopts a one-dimensional array, a ring array or an area array detector to realize two-dimensional imaging in the light sheet.
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