WO2021077708A1 - 实现大体积高分辨的时间脉冲光片断层成像方法及*** - Google Patents

实现大体积高分辨的时间脉冲光片断层成像方法及*** Download PDF

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WO2021077708A1
WO2021077708A1 PCT/CN2020/087945 CN2020087945W WO2021077708A1 WO 2021077708 A1 WO2021077708 A1 WO 2021077708A1 CN 2020087945 W CN2020087945 W CN 2020087945W WO 2021077708 A1 WO2021077708 A1 WO 2021077708A1
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imaging
time
light
volume
resolution
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French (fr)
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王平
杨驰
毕亚丽
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华中科技大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0073—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by tomography, i.e. reconstruction of 3D images from 2D projections
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/02—Detecting, measuring or recording pulse, heart rate, blood pressure or blood flow; Combined pulse/heart-rate/blood pressure determination; Evaluating a cardiovascular condition not otherwise provided for, e.g. using combinations of techniques provided for in this group with electrocardiography or electroauscultation; Heart catheters for measuring blood pressure
    • A61B5/026—Measuring blood flow
    • A61B5/0261—Measuring blood flow using optical means, e.g. infrared light
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2576/00—Medical imaging apparatus involving image processing or analysis
    • A61B2576/02—Medical imaging apparatus involving image processing or analysis specially adapted for a particular organ or body part
    • A61B2576/026—Medical imaging apparatus involving image processing or analysis specially adapted for a particular organ or body part for the brain

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  • the present invention relates to the technical field of biomedicine and optical imaging, in particular to a method and system for realizing large-volume and high-resolution three-dimensional tomographic imaging of a time pulse light sheet.
  • optical imaging techniques including confocal fluorescence microscopy, multiphoton fluorescence microscopy and other imaging modes have been widely used to observe the fine structure and functional activities of biological tissues.
  • optical imaging technology has many advantages such as non-toxicity, high resolution, fast imaging speed, and high sensitivity, it is only suitable for imaging the surface layer of biological tissues from a few microns to a few millimeters in depth.
  • Optical imaging generally uses higher-order optical nonlinear effects, higher laser instantaneous power, or thin space illuminating light sheets, and other methods to solve the problem of imaging depth, but for samples larger than 1 mm in size, it still cannot Realize high-resolution optical three-dimensional tomography.
  • tissue optical transparency technology the imaging depth can be increased to the order of centimeters, but it can do nothing for living organisms.
  • MRI magnetic resonance imaging
  • CT computed tomography
  • PET positron emission tomography
  • other technologies can achieve large-volume three-dimensional in vivo imaging, but the spatial resolution is mostly about 1 mm, which cannot be used for smaller tissues.
  • the structure is analyzed.
  • these imaging systems have a certain amount of radiation measurement for living bodies, and the technology is complicated, the system is huge, and the price is expensive.
  • the present invention mainly uses optical means instead of traditional radiographic imaging methods to realize large-volume and high-resolution optical tomography in vivo, obtain three-dimensional images of tissues, organs, lesions, and vascular structures in vivo, and can be combined with optical probes to complete each Kind of targeted functional imaging.
  • the present invention proposes a time-pulse light slice tomographic imaging method and system to achieve high-resolution imaging of large-volume samples or large living organisms to solve the problems of light imaging resolution and imaging depth.
  • the time pulsed optical slice tomography method designed by the present invention to realize large volume and high resolution includes the following steps:
  • the laser outputs two synchronized or phase-locked pulsed light sources ⁇ 1 and ⁇ 2 , where ⁇ 1 and ⁇ 2 represent light pulses with different characteristics, such as different wavelengths and different polarizations;
  • One of the pulsed light sources ⁇ 2 is equipped with a delay device for time delay adjustment
  • the pulsed light ⁇ 1 and ⁇ 2 are completely opposite in the propagation direction, or have an angle opposite, or have an angle and the same direction. By adjusting the relative time delay between the two beams, the two pulsed lights can be realized at the same time. To reach a fault on the sample, the thickness of the fault is determined by the pulse width of the two light pulses;
  • the sample is subjected to labeling processing, and the two pulsed light sources alone cannot excite the signal, and the signal can only be generated when the two pulsed light sources act simultaneously.
  • the sample has not been treated with fluorescent dyes, and when the two light pulses ⁇ 1 and ⁇ 2 coincide in space and time at the same time, they excite endogenous molecules in the sample to emit signals, and the signals include optical, sound (including Ultrasound), thermal, electrical, magnetic, electromagnetic and other signals.
  • the two light pulses ⁇ 1 and ⁇ 2 coincide in space and time at the same time, they excite endogenous molecules in the sample to emit signals, and the signals include optical, sound (including Ultrasound), thermal, electrical, magnetic, electromagnetic and other signals.
  • the sample is subjected to fluorescent, phosphorescent, Raman, photothermal, and photoacoustic probe labeling treatments, and the two pulsed light sources alone cannot excite the labeling group to emit a signal, and only when the two pulsed light sources act at the same time can the signal be generated .
  • the thickness of the time light sheet or the imaging Z-axis resolution is determined by the time pulse width of the two light pulses, and the pulse light source includes attosecond, femtosecond, picosecond, and nanosecond pulsed lasers.
  • the excitation and emission wavelengths of the fluorescent labeling group and the fluorescent group bound with the targeting molecule include visible light, near-infrared first region, near-infrared second region, and far-infrared range.
  • the imaging mode of the time light sheet includes stimulated Raman scattering (SRS) imaging, anti-Stokes coherent Raman (CARS) imaging, pump-probe imaging, transient absorption (TA) imaging, light sheet imaging and photoacoustic imaging.
  • SRS stimulated Raman scattering
  • CARS anti-Stokes coherent Raman
  • TA transient absorption
  • the present invention also proposes a tomographic imaging system that realizes a large-volume, high-resolution, time-pulsed light sheet.
  • the system includes a pulsed laser, a power adjustment device, a collimated beam expander, and an optical modulator.
  • Time delay device two-dimensional laser scanning system, photoelectric collection system; the sample is set in the overlapping area of the two opposite beams, the bottom of the sample is provided with an XYZ three-axis movement system, the detector of the photoelectric collection system can also Realize three-dimensional synchronous adjustment, and multi-channel full-angle detection.
  • the lateral resolution of the time light sheet is determined by the beam waist of the light spot;
  • the laser output from the laser includes Gaussian beams, Bessel beams, and beams with adjustable diameters; two-dimensional laser scanning is used to realize the time pulse light sheet.
  • the photoelectric collection system includes a lens and a detector, and the detector adopts a single detector; when the signal background is relatively low, it combines optical modulation and phase-locked amplification technology to amplify the signal to improve the signal-to-background ratio.
  • the detector adopts a one-dimensional array, a circle array or a surface array detector to realize two-dimensional imaging in the light sheet.
  • the beneficial effects of the present invention include:
  • the present invention provides a time-pulse light slice tomographic imaging method scanning along the propagation direction.
  • the thickness of the light sheet can be precisely adjusted.
  • the axial scanning can be realized, and the two-dimensional laser can be combined at the same time.
  • Fast scanning ultimately achieves rapid horizontal imaging of organs, while meeting uniform axial resolution and high signal-to-background ratio, and improving imaging depth while ensuring three-dimensional imaging resolution.
  • the present invention can realize high-resolution tracing imaging of blood vessels in tissues, meeting the needs of clinical medical detection, real-time monitoring of blood circulation and blockage, and analyzing blood oxygen concentration and PH value It can realize optical molecular targeted tumor imaging and expand the biomedical applications of optical probes in humans or animals; it can monitor the neural activity of the deep brain; it can use label-free methods, such as CARS and SRS, to target tissues Observe the metabolites, such as lipids and proteins, complete rapid pathological analysis, identify tumor boundaries, and help doctors perform precise surgical navigation; at the same time, several imaging methods can be combined to achieve multi-mode real-time optical imaging and other physiology Pathological research and clinical application.
  • label-free methods such as CARS and SRS
  • the present invention can be combined with wide-field light sheet illumination to meet the needs of a large field of view, and can also change the shape of the light beam to achieve spatial light illumination, and realize the further application of optical imaging in the medical field.
  • Fig. 1 is a schematic diagram of the time-pulse light slice scan of the present invention.
  • Fig. 2 is a schematic diagram of three-dimensional imaging of a time light sheet with different spot modes of the present invention, 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 wide-field light sheet array Schematic diagram of three-dimensional imaging.
  • FIG. 3 is a schematic diagram of the three-dimensional imaging device of the dual-wavelength time pulse light sheet of the present invention.
  • FIG. 4 is a diagram of the data format and preliminary imaging result of the three-dimensional imaging of the present invention.
  • Figure 5 is a schematic diagram of ICG fluorescently labeled human blood vessel imaging.
  • the present invention proposes a method for realizing large-volume and high-resolution time pulsed optical slice tomography.
  • the method is shown in FIG. 1 and includes the following steps:
  • the laser simultaneously outputs two synchronized or phase-locked pulsed light sources ⁇ 1 and ⁇ 2 , where ⁇ 1 and ⁇ 2 have different characteristics, such as different wavelengths;
  • one of the pulsed light sources ⁇ 2 is equipped with a delay device for time delay adjustment.
  • the delay device can be composed of two mirrors with an angle of 90 degrees installed on a motorized one-dimensional translation stage.
  • the translation stage implements fast tomography;
  • the pulsed light ⁇ 1 and ⁇ 2 are completely opposed or angularly opposed or angularly transmitted in the direction of propagation. By adjusting the relative time delay between the two beams, the two pulsed lights can reach the sample at the same time. For the last fault, the thickness of the fault is determined by the pulse width of the two light pulses;
  • the tomogram where the light pulses overlap can appear signals and produce high-contrast image information.
  • the signal is only generated in signal section 1; adjust the time delay of the beam ⁇ 2 so that the two beams overlap at different positions, as shown in Figure 1b, only generate signal in signal section 2; as shown in Figure 1c ,
  • the distance ⁇ Z 1 moved by the time delay is equal to the distance ⁇ Z 2 moved by the light sheet in the sample.
  • the excitation and emission wavelengths of the fluorescent label group include visible light, near-infrared and far-infrared range.
  • visible light excitation two laser beams with wavelengths of 1040nm & 1130nm can be used for Sulforhodamine 101, Texas red, and quantum dot dyes for two-color two-photon excitation.
  • Pulsed laser has the advantages of ultra-short pulse and high repetition rate.
  • the laser outputs two synchronized light sources.
  • the laser time pulse width is about 100 femtoseconds, and the corresponding optical pulse thickness is 30 ⁇ m, which is light.
  • the thickness of the film; the thickness of the time light film or the Z-axis resolution of the imaging is determined by the time pulse width of the two light pulses.
  • the pulse light source includes attosecond, femtosecond, picosecond, and nanosecond pulse lasers, reducing the time pulse width can be achieved Higher axial resolution.
  • the repetition frequency of the laser is MHz Gaussian mode laser, and the dual-channel with tunable wavelength is used to output femtosecond laser.
  • the sum or difference frequency of the wavelength of the two lasers meets the conditions of the non-linear absorption of the dye or the vibration of the excited molecule.
  • the optical frequencies ⁇ 1 and ⁇ 2 are not equal, but the sum of their photon energy is equal to the photon energy required for single-photon excitation of the dye; other nonlinear processes include two-color three-photon fluorescence, CARS and SRS, etc. Both need to meet the principle of simultaneous action of dual wavelengths; a single beam of light cannot generate a signal.
  • the laser outputs two pulsed light sources ⁇ 1 , ⁇ 2 with the same repetition frequency, time synchronization and phase lock; the two lasers are directed towards each other.
  • the two laser beams enter the XY two-position scanning system to complete the two-dimensional scanning, or the three-dimensional translation stage can be used to achieve the three-dimensional scanning; when the time pulse coincides in the Z direction, the signal is generated, and multiple detectors are arranged around the sample. It is a ring array or area array; if it is a dual-wavelength imaging system, the lateral resolution of the final system is related to the beam diameter and the focal length of the focusing lens:
  • Dxy is the lateral PSF, ⁇ is the wavelength, and NA is the numerical aperture of the lens;
  • r is the 1/e 2 radius of the spot
  • f is the focal length of the lens
  • the choice of lateral resolution can be achieved by changing the beam diameter and the focal length of the lens. Because the pulse width of the laser does not change, the thickness of the light sheet does not change when passing through the sample.
  • the laser mode used can be Gaussian beam, Bessel beam, and wide-field light source, where the imaging resolution D XY satisfies 0.1-50 ⁇ m, and the depth range Z R is 3-10mm;
  • the pulsed light ⁇ 1 and ⁇ 2 can be completely reflected or angularly reflected in the propagation direction.
  • the moving direction of the light sheet is along the direction of light propagation;
  • the thickness of the light sheet is the thickness of the two beams overlapping in time, which depends on the width of the pulse.
  • the light sheet produced by a 100 femtosecond pulse laser is theoretically 30 ⁇ m, and the thickness of the light sheet The thickness is uniform and does not change with changes in depth.
  • the spot size D xy after the two beams of light is focused is the imaging resolution, and the focal length is the Rayleigh length Z R ; as shown in the side view of Fig. 2a, the imaging signal is collected by a photodetector on the side or other solid angles.
  • the multiplex detector is located on the side of the sample generating signal area. If a fast laser scanning system is not used, it can be combined with an electric scanning translation stage to realize the scanning of two-dimensional images, and control the synchronization of acquisition and scanning to obtain three-dimensional data of the sample; if wide-field illumination is used, a backward detection system can be used, as shown in the figure As shown in 2d, a dichroic mirror is used to reflect the signal into the area array detector.
  • the present invention also provides a large-volume, high-resolution, time-pulse optical slice imaging device.
  • the system includes a pulsed laser and outputs two simultaneous laser pulses ⁇ 1 and ⁇ 2 respectively. Pass through a power adjustment device composed of a half-wave plate and a polarization beam splitting prism.
  • the two beams of light respectively pass through a beam expander collimator composed of lenses, and the spot size after beam expansion is adjusted to meet the requirements of imaging resolution and imaging depth range after focusing;
  • laser ⁇ 2 passes through an optical modulator , Can realize amplitude modulation, polarization and phase modulation, and then pass the modulated beam through the time delay device, and finally enter the two laser beams into the two-dimensional laser scanning system respectively.
  • Two focusing lenses focus the spot on the sample, and finally adopt photoelectric collection System and detection system to obtain the signal; the sample is set at the focal point of the two lenses, the optical path is completely opposite to the beam, the bottom of the sample is provided with an XYZ three-axis moving system, the single or multiple detectors of the photoelectric collection system The side or other azimuth angle of the sample.
  • the laser mode output by the laser includes Gaussian beam and Bessel beam.
  • the collimated beam expander is used to adjust the size of the spot entering the two-dimensional laser scanning system, and the specific spot size is obtained by changing the parameters of the beam expander.
  • the time delay device includes a translation stage and an optical mirror mounted on the platform, and the translation stage is driven by a linear motor or a voice coil.
  • the photoelectric collection system includes photodetectors, which are cameras or photomultiplier tube arrays and other array probes. It can be combined with optical modulation and amplify the signal through phase-locked amplification technology.
  • the detector adopts a one-dimensional array, a circle array or a surface array detector to realize two-dimensional imaging in the light sheet.
  • the collection system includes a lens and a filter.
  • the two-dimensional laser scanning system is a two-dimensional scanning galvanometer, or MEMS and an acousto-optic deflector, which uses two-dimensional laser scanning to achieve fast and large-scale two-dimensional imaging within the optical sheet.
  • the forward detection system includes a dichroic mirror and a filter, and the output end is connected with the data acquisition card of the photoelectric collection system.
  • the electric displacement platform or the laser two-dimensional scanning system is controlled by a computer to synchronize with the data collection.
  • the generated fluorescence enters the photoelectric collection system after passing through the dichroic mirror, and it can also be collected on the side to generate electricity.
  • the signal is transmitted to the lock-in amplifier for demodulation, and the modulated reference signal is connected from the signal source at the same time, and the demodulated signal is transmitted to the data acquisition card, which is displayed and stored by the computer.
  • the three-dimensional stack scanning scanning principle controls the synchronization of the two-dimensional scanning system and data acquisition, and the two scanning systems also need to be synchronized to ensure that the same position is scanned at the same time.
  • the data is collected during the fast axis scanning of the two-dimensional scanning system.
  • Match the two synchronization as shown in the darkened area of the color, the arrow indicates the scanning direction, after scanning one line, move the slow axis, and then complete the next line scanning, and finally realize the two-dimensional scanning, and then control the one-dimensional translation stage to adjust the time delay
  • the corresponding time delay is 6.67 femtoseconds
  • the moving distance in the sample axis is 1 ⁇ m, because 100 femtoseconds corresponds to
  • the thickness of the laser light sheet is 30 ⁇ m, so the accuracy of the movement of the light sheet in the axial direction is controlled to be less than 20 ⁇ m each time, which improves the axial sampling rate.
  • the moving distance of the moving platform is equivalent to the moving distance of the light sheet, move the light sheet to the next layer, and then start the next cycle scan, and finally realize the three-dimensional scan, as shown in Figure 4b.
  • the signal detected by the photodetector can be demodulated with a high signal-to-noise ratio fluorescent signal through phase-locked amplification.
  • the fluorescence signal is detected by photodetectors such as SCOMS, EMCCD camera or photomultiplier tube, and the photocurrent generated by the camera is output to the lock-in amplifier.
  • the reference signal is the modulation frequency of the acousto-optic modulator.
  • the fluorescence signal is demodulated and transmitted to The computer shows that the imaging result of the time light plate is shown in Figure 4b. Fill the resilica capillary with fluorescent dyes.
  • the three-dimensional structure of the quartz tube can be reconstructed by three-dimensional scanning, and the effect of the XY and YZ sections can be analyzed.
  • the scale is 500 ⁇ m.
  • the finer thickness of the optical sheet can reduce the pulse width of the femtosecond laser and realize the adjustment of the optical sheet thickness.
  • the certified ICG dye that can be used on the human body is used. Its absorption peak is at 800nm. It can be excited by ⁇ 1300nm and ⁇ 2100nm in the second region of the near-infrared. The excitation light is deeper. Penetration depth, a single beam of light cannot excite fluorescence, only a time-light slice signal is generated where the two beams of light overlap, to achieve tomography of blood vessels, as shown in Figure 5;
  • the sample is subjected to fluorescence, phosphorescence, Raman, photothermal, and photoacoustic labeling treatments.
  • Two pulsed light sources alone cannot excite the labeling group to emit signals. Signals are only generated when two pulsed light sources act at the same time.
  • the difference between this embodiment and the first embodiment is that the sample is not fluorescently labeled, and when the two light pulses ⁇ 1 and ⁇ 2 coincide in space and time at the same time, they excite endogenous molecules in the sample to emit signals.
  • the signals include optical, sound, and Heat, electricity, and magnetic signals.
  • the laser chooses pump light and Stokes light.
  • the specific implementation process of SRS select the wavelength of pump light and Stokes light to excite specific analytical chemical bonds, modulate the Stokes light through the acousto-optic modulator, and output the reference signal of the modulator to the lock-in amplifier ; After a suitable time delay, and then coincide with the pump light on the sample, the high-pass and low-reflection dichroic mirror is used on the light path of the pump light to propagate in the forward direction to reflect the pump light to the photodiode for detection, and then pass
  • the lock-in amplifier demodulates the stimulated Raman signal; for example, the pump light is 800nm, the Stokes light is 1040nm (or use other wavelengths including ⁇ 1300nm and ⁇ 2080nm), and the Raman shift is 2800 ⁇ 3100cm -1 Specific observation of oil and protein components in tissues, to achieve unlabeled organ chemical composition analysis and imaging; select Raman translation at 960cm -1 , which can specifically observe bone signals.
  • the specific implementation process of CARS The previous optical path system is the same as the SRS. The difference is in the detection part, using photomultiplier tubes and other photodetectors to detect scattered anti-Stokes signals.
  • the device is the same as the SRS imaging system, but the difference lies in the different wavelengths used.
  • Embodiment 4 Photoacoustic and photothermal imaging with dual-wavelength excitation
  • the laser uses an excitation wavelength far from the absorption peak of blood or dye, but only two beams of light can be absorbed by the two colors when they act at the same time, and then emit sound or heat at the place where the two beams of light overlap; device
  • the above is similar to the first embodiment, but the difference lies in the detection and collection part.
  • two pulsed light beams or angled beams are used to recombine inside the sample to generate a light sheet signal.
  • a one-dimensional array of photoacoustic transducers or photoacoustic transducers are arranged around the sample. The ring array detects the sound signal and realizes the time-light-sheet photoacoustic and photothermal imaging of dual-wavelength excitation.
  • the present invention can also have other embodiments. Any changes, modifications, substitutions, combinations, and simplifications that deviate from the spirit and principle of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

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Abstract

一种实现大体积高分辨的时间脉冲光片断层成像方法及***，实现大体积高分辨的时间脉冲光片断层成像方法包括：1)激光器输出两束同步或相位锁定的脉冲光源λ1、λ2；2)其中一束脉冲光源λ2配有延时装置进行时间延迟调节；3)脉冲光源λ1、λ2在传播方向上完全对射或有角度对射或有角度同向发射，通过调节两束光的位置在成像样本上同时实现空间和时间脉冲上的重叠，形成时间脉冲光片；4)时间脉冲光片所在的断层产生信号，提供图像信息；5)通过改变两束光的相对时间延迟或样本的位置，实现光片的断层扫描成像。使用调制的时间脉冲光片，解决了机械式光片分辨率不均匀，深度成像中离焦的背景大，信号背景比低的问题，可以应用于大体积的器官水平成像。

Description

实现大体积高分辨的时间脉冲光片断层成像方法及*** [技术领域]
本发明涉及生物医学及光学成像技术领域，具体地是指一种实现大体积且高分辨的时间脉冲光片三维断层成像方法及***。
[背景技术]
在生物医学研究和实际应用中，包括共聚焦荧光显微镜、多光子荧光显微镜和其它成像模式在内的光学成像技术已被广泛用于观测生物组织的细微结构和功能活动。虽然光学成像技术具有无毒性、高分辨、成像速度快，灵敏度高等众多优点，但其只适用于生物组织表层从数微米到数毫米的深度的成像。对于体积较大的生物组织或其它不透明样品(1毫米到10厘米及以上)，随着成像深度的增加，光散射和吸收趋于严重，由于离焦产生的背景信号增大，传统光学方法很难应用于大体积器官水平成像，因此受限于光在不透明样品中较差的穿透性能，光学成像分辨率随着成像深度急剧下降。光学成像一般采用更高阶的光学非线形效应，更高的激光瞬时功率，或者产生薄的空间照明光片等其它方法来解决成像深度的问题，但对于大小在1毫米以上的样品，仍然不能实现高分辨的光学三维断层成像。结合组织光透明技术，可以将成像深度提高到厘米量级，但对于活体生物却无能为力。目前，核磁共振成像(MRI)，计算断层成像(CT)，正电子发射断层成像(PET)等技术可以实现大体积三维活体成像，但空间分辨率大都在1毫米左右，无法对更细小的组织结构进行解析。同时，这些成像***对活体都有一定的辐射计量，且技术复杂，***庞大，价格昂贵。本发明主要应用光学手段代替传统射线成像方法，在活体内实现大体积且高分辨的光学断层成像，获取活体内组织、器官、病灶、血管结构等三维图像，并可结合光学探针，完成各种靶向功能成像。
[发明内容]
针对上述背景技术存在的不足，本发明提出一种时间脉冲光片断层成像方法及***对大体积样品或大的生物活体实现高分辨成像，以解决光的成像分辨和成像深度的问题。
为实现上述目的，本发明所设计的实现大体积高分辨的时间脉冲光片断层成像方法，所述方法包括以下步骤：
1)激光器输出两束同步或相位锁定的脉冲光源λ 1、λ 2，其中λ 1和λ 2代表不同特性的光脉冲，例如不同波长，不同偏振；
2)其中一束脉冲光源λ 2配有延时装置进行时间延迟调节；
3)脉冲光λ 1和λ 2在传播方向上完全对射，或有角度对射，或有角度同向发射，通过调节两束光之间的相对时间延时，实现这两束脉冲光同时到达样本上一个断层，断层的厚度由两束光脉冲的脉宽决定；
4)只有这两种特性的时间脉冲光片在空间和时间同时出现重叠的断层才能产生信号，提供图像信息；
5)通过改变两束光的相对时间延迟或样本的位置，实现光片的三维断层扫描成像。
可选地，所述样本经过标记处理，两束脉冲光源单独作用不能激发信号，只有两束脉冲光源同时作用时才产生信号。
可选地，所述样本未经过荧光染料处理，所述两束光脉冲λ 1、λ 2在空间和时间上同时重合时激发样本中内源分子发出信号，所述信号包括光学、声音(包括超声)、热学、电学、磁学、电磁等信号。
可选地，所述样本经过荧光、磷光、拉曼、光热、光声探针标记处理，两束脉冲光源单独作用不能激发标记基团发出信号，只有两束脉冲光源同时作用时才产生信号。
可选地，所述时间光片的厚度或者成像的Z轴分辨率由两束光脉冲的时间脉宽决定，脉冲光源包括阿秒、飞秒、皮秒、纳秒脉冲激光器。
可选地，荧光标记基团以及结合有靶向分子的荧光基团的激发和 发射波长包括可见光、近红外一区、近红外二区和远红外范围。
可选地，所述时间光片的成像模式包括受激拉曼散射(SRS)成像、反斯托克斯相干拉曼(CARS)成像、泵浦探测(Pump-probe)成像、瞬态吸收(TA)成像、光片成像和光声成像。
基于上述方法，本发明还提出一种实现大体积高分辨的时间脉冲光片的断层成像***，其特殊之处，所述***包括脉冲激光器、功率调节装置、准直扩束装置、光学调制器、时间延迟装置、二维激光扫描***、光电收集***；样本设置于所述两种对射光的焦点重合区域，样本的底部设置有XYZ三轴移动***，所述光电收集***的探测器也可以实现三维同步调节，以及多路全角度探测。
进一步地，时间光片的横向分辨率由光斑的束腰决定；所述激光器输出的激光包括高斯光束、贝塞尔光束、直径大小可调的光束；采用二维激光扫描实现时间脉冲光片内的快速大范围二维成像；光电收集***包括镜头，探测器，所述探测器采用单探测器；当信号背景比较低时，结合光学调制和锁相放大技术放大信号，提高信背比。
更进一步地，所述探测器采用一维阵列、圈阵列或者面阵列探测器实现光片内的二维成像。
与现有技术相比，本发明的有益效果包括：
(1)本发明提供的一种沿传播方向扫描的时间脉冲光片断层成像方法，光片的厚度可以精确的调整，通过改变两束光的时间差，实现轴向扫描，同时结合二维的激光快速扫描，最终实现快速的器官水平成像，同时满足均匀的轴向分辨率和高信背比，在保证三维成像分辨率的同时提高成像深度。
(2)本发明结合各种特异性荧光标记探针，可以实现组织中血管的高分辨示踪成像，满足临床医学检测的需求，实时监测血液的流通和堵塞情况，分析血氧浓度和PH值；可以实现光学分子靶向的肿瘤成像，拓展光学探针在人体或动物体内的生物医学应用；可以对深层大脑的神经活动进行监测；可以利用免标记的方法，例如CARS和 SRS，对组织中的代谢产物，比如油脂和蛋白质进行观测，完成快速的病理分析，鉴定肿瘤的边界，帮助医生进行精确的手术导航；同时可以把几种成像的方式结合，实现多模式的实时光学成像以及其它生理病理的研究和临床应用。
(3)本发明可以结合宽场的光片照明，满足大视场的需求，也可以改变光束的形状，实现空间光的照明，实现了光学成像在医学领域的进一步应用。
[附图说明]
图1为本发明的时间脉冲光片断层扫描示意图。
图2为本发明的不同光斑模式的时间光片三维成像示意图,其中图2a为三维扫描成像示意图，图2b为高斯光束示意图，图2c为贝塞尔光束示意图，图2d为宽场光片面阵三维成像示意图。
图3为本发明的双波长时间脉冲光片三维成像装置示意图；
图4为本发明的三维成像的数据格式与初步成像结果图。
图5为ICG荧光标记的人体血管成像示意图。
[具体实施方式]
以下结合附图和具体实施例对本发明作进一步的详细描述。为了更清楚的说明本发明的实用意义，在具体实施例里，所举的例子为常用且较佳的方案，但是并不局限于此。
本发明提出的一种实现大体积高分辨的时间脉冲光片断层成像方法，所述方法如图1所示，包括以下步骤：
1)激光器同时输出两束同步或相位锁定的脉冲光源λ 1、λ 2，其中λ 1和λ 2具有不同特性，例如波长不同；
2)所示，其中一束脉冲光源λ 2配有延时装置进行时间延迟调节，延时装置可以由安装在电动一维平移台上的两个呈90度夹角的反射镜组成，高速电动平移台实施快速断层扫描；
3)脉冲光λ 1和λ 2在传播方向上完全对射或有角度对射或有角度同向发射，通过调节两束光之间的相对时间延时，实现这两束脉冲 光同时到达样本上一个断层，断层的厚度由两束光脉冲的脉宽决定；
4)只有时间脉冲光片所在的断层上出现这两种特性的光脉冲发生空间和时间的重叠才能产生信号，提供高信号背景比的图像信息；
5)通过改变两束光的相对时间延迟或轴向移动样本的位置，实现光片的断层扫描成像。
因此三维断层扫描成像只有当这两种特性的光在时间上重合，光脉冲发生重叠的断层才能出现信号，产生高对比度的图像信息。如图1a所示，只在信号断层1产生信号；调节光束λ 2的时间延迟，两束光在不同的位置重合，如图1b所示，只在信号断层2产生信号；如图1c所示，通过连续改变两束光的相对时间延迟或样本的位置，最终实现时间脉冲光片的断层扫描成像。时间延迟移动的距离ΔZ 1等于光片在样本中移动的距离ΔZ 2。
实施例一 荧光标记成像
对于荧光标记成像，荧光标记基团的激发和发射波长包括可见光、近红外和远红外范围。以可见光激发为例，两束激光波长为1040nm & 1130nm，可以做Sulforhodamine 101、Texas red、量子点染料的双色双光子激发。
脉冲激光具有超短脉冲，高重复频率的优点，以飞秒光源为例，激光器输出两束同步光源，其激光时间脉冲宽度为100飞秒左右，对应的光脉冲的厚度为30μm,也就是光片的厚度；时间光片的厚度或者成像的Z轴分辨率由两束光脉冲的时间脉宽决定，脉冲光源包括阿秒、飞秒、皮秒、纳秒脉冲激光器，减少时间脉宽可以实现更高的轴向分别率。激光的重复频率为MHz高斯模式激光，采用可调谐波长的双通道输出飞秒激光，两束激光的波长的和频或差频，满足染料的非线性吸收或者受激分子振动的条件，以双色波长的双光子为例，光学频率ω 1和ω 2不相等，但是它们的光子能量之和等于染料单光子激发所需的光子能量；其它的非线性过程包括双色三光子荧光，CARS和SRS等均需满足双波长同时作用的原理；单独的一束光不能产生信 号。
实施步骤如图2a所示采用激光扫描的成像：
1)激光器输出两束相同重复频率，时间同步且相位锁定的脉冲光源λ 1、λ 2；两束激光相向对射。
2)两束激光分别进入X-Y二位扫描对射***完成二维扫描，也可以采用三维平移台实现三维扫描；当时间脉冲在Z方向重合时产生信号，在样本周围布置多路探测器，可以是圈阵列或面阵列；若为双波长成像***，最后的***的横向分辨率和光束直径和聚焦透镜的焦距相关：
Figure PCTCN2020087945-appb-000001
Dxy为横向的PSF，λ为波长，NA为透镜的数值孔径；
r为光斑1/e 2半径，f为透镜的焦距
可以通过改变光束直径和透镜焦距实现横向分辨率的选择。因为激光器的脉宽不会改变，在经过样本时，光片厚度也不会改变。如图2b,2c,2d所示，采用的激光模式可以是高斯光束，贝塞尔光束，宽场面光源，其中成像分辨率D XY满足0.1～50μm，深度范围Z R为3～10mm；
3)将其中一束飞秒激光λ 2经过声光或电光偏振调制器，通过信号源产生正弦调制波形，调制频率为f1＝0.5～2MHz；
4)将λ 2这个光束经过一个时间延迟装置，并通过直线电机或者音圈驱动控制移动平台，电机的调节精度为0.1～10μm；
5)两束光分别完成二维激光扫描后，经过两个透镜聚焦在样本上。脉冲光λ 1和λ 2在传播方向上可以完全对射或有角度对射，通过调节两束光的位置在成像样本的一个断层上同时实现空间和时间脉冲的重叠，产生荧光或者散射信号；光片的移动方向沿光的传播方向；光片的厚度为两束光在时间上重合的厚度，取决于脉冲的宽度，100飞秒的脉冲激光产生的光片理论上为30μm，光片的厚度是均匀的，不 随深度的改变而变化。两束光聚焦后的光斑的大小D xy即成像分辨率，焦点长度即瑞利长度Z R；如图2a的侧视图所示，成像信号通过光电探测器在侧面或者其它立体角实现信号同步采集，多路探测器位于样本产生信号区域的侧面。若不使用快速的激光扫描***，可以结合电动扫描平移台实现二维图像的扫描，控制采集和扫描的同步来获取样本的三维数据；若使用宽场照明，可以采用后向探测***，如图2d所示，采用二向色镜讲信号反射进入面阵探测器。
基于上述方法，本发明还提出一种实现大体积高分辨的时间脉冲光片断层的成像装置，如图3所示，所述***包括脉冲激光器，输出两路同步激光脉冲λ 1、λ 2分别经过由半波片和偏振分束棱镜组成的功率调节装置。为了实现合适的分辨率，两束光分别经过由透镜组成的扩束准直装置，调节扩束之后的光斑大小满足聚焦之后，成像分辨率和成像深度范围的要求；激光λ 2经过光学调制器，可以实现幅度调制，偏振和相位调制，然后将调制之后的光束经过时间延迟装置，最后将两束激光分别进入二维激光扫描***，两个聚焦透镜将光斑聚焦到样本上，最终采用光电收集***、探测***来获得信号；样本设置于所述两个透镜的焦点，光路完全对射合束，样本的底部设置有XYZ三轴移动***，所述光电收集***的单个或多个探测器至于样本的侧面或其它方位角。
其中，激光器输出的激光模式包括高斯光束、贝塞尔光束，准直扩束装置用于调节进入二维激光扫描***的光斑大小，通过改变扩束装置参数得到具体的光斑大小。时间延迟装置包括由平移台和安装于平台上的光学反射镜，平移台通过直线电机或者音圈驱动。光电收集***包括光电探测器，光电探测器为相机或者光电倍增管阵列及其它阵列探头。可结合光学调制，并通过锁相放大技术放大信号。探测器采用一维阵列、圈阵列或者面阵列探测器实现光片内的二维成像。收集***包括镜头和滤光片，将背景光滤除后，将光信号传给光电探测器的探头，采集到的光电信号输入至锁相放大器解调，解调后的信号 传输至数据采集卡，并由计算机显示和存储。二维激光扫描***为二维扫描振镜，或者MEMS和声光偏转器，采用二维激光扫描实现光片内的快速大范围二维成像。前向探测***包括二向色镜和滤光片，输出端与光电收集***的数据采集卡连接。
通过计算机控制电动位移平台或者激光二维扫描***，与数据采集同步，在后像探测***中，产生的荧光经过二向色镜之后进入到光电收集***，也可以在侧面进行收集，产生的电信号输给锁相放大器解调，同时从信号源接入调制的参考信号，解调之后的信号传给数据采集卡，并由计算机显示和存储下来。如图4a所示，三维堆栈扫描扫描原理，控制二维扫描***和数据采集同步，并且两个扫描***也需要同步，保证同时扫描相同的位置，二维扫描***的快轴扫描的时候采集数据，匹配两者同步，如图颜色的加深区域，箭头表示扫描方向，扫完一行之后，移动慢轴，再完成下一行扫描，最终实现二维的扫描，然后，控制一维平移台调节时间延迟，例如时间延迟t 1,t 2,..t n，例如平台的移动精度时1μm，对应时间上的延迟为6.67飞秒，在样本轴向上的移动距离为1μm，因为100飞秒对应的激光光片厚度为30μm，所以控制光片每次在轴向上的移动精度小于20μm，提高轴向采样率。采集完一个层面之后，调节时间延迟，移动平台移动的距离相当于光片移动距离，将光片移动到下一个层面，再开始下一个循环扫描，最终实现三维扫描，如图4b所示。
由于一束激光受到调制，光电探测器探测的信号可以通过锁相放大解调出高信噪比的荧光信号。通过SCOMS，EMCCD相机或者光电倍增管等光电探测器探测荧光信号，并将相机产生的光电流输出给锁相放大器，参考信号为声光调制器的调制频率，解调出荧光信号，并传输给电脑显示，时间光片的成像结果如图4b所示，将荧光染料装满再石英毛细管内，通过三维扫描可以重建石英管的三维结构，并可以分析XY，YZ截面的效果,比例尺是500μm。
更精细的光片厚度，可以降低飞秒激光器的脉宽，实现光片厚度 的调节。
实施例二 血管成像
本实施例与实施例一的区别在于采用经过认证的，可以在人体上使用的ICG染料，它的吸收峰在800nm，可以使用近红外二区的～1300nm和～2100nm激发，激发光有更深的穿透深度，单独一束光不能激发荧光，只有在两束光重合的地方产生时间光片信号，实现血管的层析成像，如图5所示；
样本经过荧光、磷光、拉曼、光热、光声标记处理，两束脉冲光源单独作用不能激发标记基团发出信号，只有两束脉冲光源同时作用时才产生信号。
实施例三 油脂，蛋白质的SRS或者CARS成像
本实施例与实施例一的区别在于样本未经过荧光标记，两束光脉冲λ 1、λ 2在空间和时间上同时重合时激发样本中内源分子发出信号，所述信号包括光学、声音、热量、电学、磁学信号。激光器选择泵浦光和斯托克斯光。
SRS的具体实施过程：选择泵浦光和斯托克斯光的波长激发特定的分析化学键，将斯托克斯光调制经过声光调制器调制，并将调制器的参考信号输给锁相放大器；经过合适的时间延迟，然后与泵浦光对射在样本上重合，在泵浦光前向传播的光路上采用高通低反的二向色镜将泵浦光反射给光电二极管探测，然后通过锁相放大器解调出受激拉曼信号；例如泵浦光为800nm，斯托克斯光为1040nm(或者使用别的波长包括～1300nm和～2080nm)，拉曼平移在2800～3100cm -1可以特异性观测组织中的油脂和蛋白质成分，实现无标记的器官化学成分分析成像；将拉曼平移选择在960cm -1,可以特异性观察骨头的信号。
CARS的具体实施过程：前面的光路***和SRS的一致，区别在探测部分，采用光电倍增管等光电探测器探测散射的反斯托克斯信号。
泵浦探测(Pump-probe)和受激辐射的成像方式不再详述，装置 上和SRS成像***相同，区别在于使用的波长不一样。
实施例四 双波长激发的光声和光热成像
本实施例与实施一的区别在于激光使用远离血液或染料的吸收峰的激发波长，但是只有两束光同时作用的时候可以被双色吸收，然后在两束光重合的地方发出声音或者热量；装置上与实施例一相似，区别在于探测收集部分，同理采用两束脉冲光对射或有角度照射，在样本内部重合并产生光片信号，在样本周围布置光声换能器一维阵列或圈阵列探测声音信号，实现双波长激发的时间光片光声和光热成像。
除上述实施例外，本发明还可以有其他实施方式。凡任何背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化均应为等效的置换方式，都包含在本发明要求的保护范围。

Claims (10)

  1. 实现大体积高分辨的时间脉冲光片断层成像方法，其特征在于：所述方法包括以下步骤：
    1)激光器输出两束同步或相位锁定的脉冲光源λ 1、λ 2，其中λ 1、λ 2具有不同特性；
    2)其中一束脉冲光源λ 2配有延时装置进行时间延迟调节；
    3)脉冲光λ 1、λ 2在传播方向上完全对射或有角度对射或有角度同向发射，通过调节两束光光之间的相对时间延时，实现这两束脉冲光同时到达样本上一个断层，断层的厚度由两束光脉冲的脉宽决定；
    4)只有时间脉冲光片所在的断层上出现这两种特性的光脉冲发生空间和时间的重叠才能产生信号，提供图像信息；
    5)通过改变两束光的相对时间延迟或样本的位置，实现光片的断层扫描成像。
  2. 根据权利要求1所述的实现大体积高分辨的时间脉冲光片断层成像方法，其特征在于：所述样本经过标记处理，两束脉冲光源单独作用不能激发信号，只有两束脉冲光源同时作用时才产生信号。
  3. 根据权利要求1所述的实现大体积高分辨的时间脉冲光片断层成像方法，其特征在于：所述样本未经过荧光染料处理，所述两束光脉冲λ 1、λ 2在空间和时间上同时重合时激发样本中内源分子发出信号，所述信号包括光学、声音、热量、电学、磁学，电磁信号。
  4. 根据权利要求1所述的实现大体积高分辨的时间脉冲光片断层成像方法，其特征在于：所述样本经过荧光、磷光、拉曼、光热、光声标记处理，两束脉冲光源单独作用不能激发标记基团发出信号，只有两束脉冲光源同时作用时才产生信号。
  5. 根据权利要求2所述的实现大体积高分辨的时间脉冲光片断层成像方法，其特征在于：所述时间光片的厚度或者成像的Z轴分辨率由两束光脉冲的时间脉宽决定，脉冲光源包括阿秒、飞秒、皮秒、纳秒脉冲激光器。
  6. 根据权利要求4所述的实现大体积高分辨的时间脉冲光片断层成像方法，其特征在于：荧光标记基团以及结合有靶向分子的荧光基团的激发和和发射波长包括可见光、近红外一区、近红外二区和远红外范围。
  7. 根据权利要求3所述的实现大体积高分辨的时间脉冲光片断层成像方法，其特征在于：所述时间光片的成像模式包括受激拉曼散射成像SRS、反斯托克斯相干拉曼成像CARS、泵浦探测成像Pump-probe、瞬态吸收成像、光片成像和光声成像。
  8. 一种实现权利要求1～7中任一项所述的实现大体积高分辨的时间脉冲光片断层成像方法的成像***，其特征在于：所述***包括激光器、功率调节装置、准直扩束装置、光学调制器、时间延迟装置、二维激光扫描***、两个透镜和光电收集***；样本设置于所述两个透镜的焦点，两个透镜的光路合束，样本的底部设置有XYZ三轴移动***，以及多路全角度探测。
  9. 根据权利要求1所述的实现大体积高分辨的时间脉冲光片断层成像方法的成像***，其特征在于：时间光片的横向分辨率由光斑的束腰决定；所述激光器输出的激光模式包括高斯光束、贝塞尔光束、直径大小可调的光束；采用二维激光扫描实现光片内的快速大范围二维成像；光电收集***包括探测器，所述探测器采用单探测器；结合光学调制，并通过锁相放大器解调信号。
  10. 根据权利要求1所述的实现大体积高分辨的时间脉冲光片断层成像方法的成像***，其特征在于：所述探测器采用一维阵列、圈阵列或者面阵列探测器实现光片内的二维成像。
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