WO2021243754A1 - 基于低功率受激发射损耗的超分辨成像方法及成像*** - Google Patents

基于低功率受激发射损耗的超分辨成像方法及成像*** Download PDF

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WO2021243754A1
WO2021243754A1 PCT/CN2020/096779 CN2020096779W WO2021243754A1 WO 2021243754 A1 WO2021243754 A1 WO 2021243754A1 CN 2020096779 W CN2020096779 W CN 2020096779W WO 2021243754 A1 WO2021243754 A1 WO 2021243754A1
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super
resolution
laser
image
signal
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French (fr)
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严伟
王璐玮
屈军乐
王佳林
张佳
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深圳大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells

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  • This application relates to the technical field of super-resolution optical microscopy imaging, in particular to a super-resolution imaging method and imaging system based on low-power stimulated emission loss.
  • Live cell and tissue imaging is very important to the research in the field of biomedicine.
  • advanced imaging methods and imaging systems can guarantee the greatest degree of protection while maintaining the biological characteristics of the observed samples.
  • Optical microscope has the advantages of non-contact, non-damage and specificity. It is an important symbol of the beginning of the development of modern natural science. It can be well applied to imaging of living cells and tissues. However, the diffraction of light limits the resolution capability of optical microscopes, making it impossible to clearly distinguish microscopic biological structures with a size below 200 nm.
  • Super-resolution optical microscopy (SRM) technology inherits the non-contact and specific advantages of optical microscopy.
  • German scientist Stefan W. Hell proposed stimulated emission depletion (STED) microscopy technology based on Einstein's radiation theory. Based on the non-linear relationship between fluorescence saturation and excited fluorescence stimulated emission, STED technology uses a second wavelength red-shifted laser to selectively dissipate excited molecules in advance, and improve imaging by compressing the effective point spread function of the excitation spot Resolution, theoretically, nanometer-level resolution can be achieved in three-dimensional space. As the first far-field super-resolution imaging method proposed theoretically and realized in experiments, STED technology has the advantages of fast imaging and no need for post-image reconstruction.
  • the loss laser wavelength is usually at the tail end of the fluorescent dye emission spectrum. Due to the extremely small stimulated radiation cross section at the tail end of the emission spectrum, STED technology requires extremely high loss energy (usually more than three orders of magnitude higher than the energy of the excitation light) to improve resolution. Excessive laser energy can cause photobleaching and phototoxicity, and cause damage to fluorescent probes and biological tissues, thus limiting the application of this technology in live cell and tissue imaging.
  • Fluorescence lifetime imaging microscopy is a microscope imaging technique that describes the spatial distribution of fluorescence lifetimes. It can be applied to the imaging of fixed cells and living cells, revealing more biological information (such as the microenvironment within the cell). Variety). FLIM technology based on time-correlated single photon counting (TCSPC) divides the pulse period into multiple equally spaced time channels, and converts it into memory based on the time difference of the photon relative to the reference signal reaching the detector. Address, the fluorescence decay curve of the dye is obtained after the accumulation of the photon signal.
  • TCSPC time-correlated single photon counting
  • the intensity and lifetime information of the fluorescence signal can be obtained at the same time, and the dual-mode STED-FLIM super-resolution fluorescence lifetime imaging can be realized.
  • Confocal-FLIM confocal fluorescence lifetime imaging
  • STED-FLIM STED super-resolution fluorescence lifetime imaging
  • STED-FLIM can achieve super-resolution fluorescence lifetime imaging, too high laser energy changes the fluorescence lifetime characteristics of the fluorescent dye itself, so it cannot truly reflect the changes in the microenvironment, resulting in the inability to present fine structural features when imaging the sample , Resulting in poor imaging effects and difficult to obtain high-resolution images.
  • the embodiments of the present application provide a super-resolution imaging method and imaging system based on low-power stimulated emission loss, aiming to solve the high demand for loss laser power in the stimulated radiation loss super-resolution imaging method and super-resolution images under high laser power.
  • the problem of poor quality is a problem of poor quality.
  • an embodiment of the present application provides a super-resolution imaging method based on low-power stimulated emission loss, which includes:
  • Emitting an excitation laser and a loss laser, the excitation laser and the loss laser are both Gaussian pulse lasers, wherein the wavelength of the loss laser is greater than the wavelength of the excitation laser;
  • the fluorescence signal contains the time information and spatial information of the fluorescence photon
  • the confocal image and the initial super-resolution image are analyzed and processed according to preset image processing rules to obtain a higher-resolution target super-resolution image.
  • the embodiments of the present application provide a super-resolution imaging system based on low-power stimulated emission loss, which includes:
  • the signal acquisition device is configured to acquire the excitation light pulse signal as a reference signal, and acquire the fluorescent signal generated after the sample is irradiated;
  • the imaging processing terminal is configured to process the excitation light pulse signal and the fluorescence signal collected by the signal collection device to obtain the target super-resolution image.
  • the embodiments of the present application provide a super-resolution imaging method and imaging system based on low-power stimulated emission loss.
  • the low-power Gaussian loss laser is converted into a ring loss laser and overlaps with the excitation laser to focus and irradiate the sample.
  • Make the collected fluorescence lifetime data include confocal signal and super-resolution signal at the same time.
  • the confocal image and the initial super-resolution image are separated from the fluorescence signal through data processing, and the confocal image and the initial super-resolution image are analyzed and processed according to the image processing rules to obtain the target super-resolution image with further improved resolution.
  • the use of low-power loss lasers reduces the damage to biological samples, reduces the photobleaching effect of fluorescent dyes, and prolongs the effective time of super-resolution imaging.
  • image enhancement processing high resolutions containing fine structural features are obtained. Images have achieved good technical results in the actual application process.
  • FIG. 1 is a schematic flowchart of a super-resolution imaging method based on low-power stimulated emission loss according to an embodiment of the application;
  • FIG. 2 is a schematic diagram of a sub-flow of a super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application;
  • FIG. 3 is a schematic diagram of a sub-process of a super-resolution imaging method based on low-power stimulated emission loss according to an embodiment of the application;
  • FIG. 4 is a schematic diagram of a sub-process of a super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application;
  • FIG. 5 is a schematic diagram of a super-resolution imaging system based on low-power stimulated emission loss according to an embodiment of the application;
  • FIG. 6 is a schematic block diagram of an imaging processing terminal provided by an embodiment of the application.
  • FIG. 7 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss according to an embodiment of the application;
  • FIG. 8 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss according to an embodiment of the application;
  • FIG. 9 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application.
  • FIG. 10 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application.
  • FIG. 1 is a schematic flowchart of a super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of this application
  • FIG. 5 is a low-power stimulated emission loss-based method provided by an embodiment of this application.
  • the super-resolution imaging method based on low-power stimulated emission loss is applied to an imaging system.
  • the imaging system includes a signal acquisition device 10 and an imaging processing terminal 20.
  • the method combines the signal acquisition device 10 with application software installed in the imaging processing terminal 20
  • the imaging system is a system device used to implement a super-resolution imaging method based on low-power stimulated emission loss to achieve high-resolution imaging of the sample
  • the signal acquisition device 10 is used to emit excitation laser and loss
  • the laser detects the sample and collects the excitation light pulse signal and the fluorescence signal.
  • the imaging processing terminal 20 is used to obtain the excitation light pulse signal and the fluorescence signal collected by the signal collection device and perform imaging processing to obtain the target super-resolution image.
  • terminal devices such as workstations, desktop computers, laptops, tablets, or mobile phones.
  • the method includes steps S110 to S160.
  • S110 Place the sample dyed by the fluorescent dye on the stage and adjust the position of the corner reflector in the first light path.
  • the sample can be biological materials such as living cells, viruses or tissues.
  • Fluorescent dyes are dyes that are excited by light and generate fluorescent photons. Adjusting the position of the corner reflector can prolong the loss.
  • FIG. 10 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application. Specifically, as shown in FIG. 10, when the corner reflector is not adjusted, the corner reflector is located in the position in FIG.
  • the optical path length of the lossy laser propagating along the first optical path (the time required for the light to travel a certain distance along a certain path) is ⁇ 1
  • the optical path interval between the excitation laser pulse and the loss laser pulse is ⁇ 1 (excitation The time difference between the laser pulse and the loss laser pulse acting on the sample); adjust the position of the corner reflector to position 2 in Figure 10, and the distance between position 1 and position 2 is S
  • the optical path of the loss laser propagating along the first optical path is ⁇ 1 +2S/c, where c is the speed of light
  • both of the excitation laser and the loss laser are Gaussian pulse lasers, wherein the wavelength of the loss laser is greater than the wavelength of the excitation laser.
  • Both excitation laser and loss laser can be emitted at the same time.
  • Both lasers are pulsed lasers, and the pulse frequency is the same (such as 80MHz).
  • the frequency of the laser is inversely proportional to the pulse period of the laser.
  • the pulse period should include at least a complete autofluorescence process ( Usually on the order of nanoseconds and above). The longer the fluorescence lifetime, the larger the pulse period of the laser, and the smaller the pulse frequency of the laser.
  • the power of the excitation laser is less than the power of the loss laser.
  • the power of the loss laser here is at least an order of magnitude (such as less than 10mW) lower than the traditional stimulated radiation loss super-resolution imaging ( ⁇ 100mW).
  • the power of is related to the spectral characteristics of the luminescent material, usually 0.1-100 ⁇ W.
  • the pulse width of the excitation laser and the pulse width of the loss laser are both on the order of one hundred picoseconds.
  • the pulse width of the excitation laser and the pulse width of the loss laser can both be in the range of 0.1-1 nanoseconds.
  • a spiral phase plate can be installed after the corner reflector of the first optical path, and the loss laser of the Gaussian pulse laser is converted into ring loss laser through the spiral phase plate, and the ring loss laser coincides with the excitation laser propagating along the second optical path.
  • the focal planes of the two laser beams precisely coincide in space and then focus and irradiate the sample. After the dyed sample is irradiated, the fluorescent dye will produce a fluorescent signal.
  • S140 Collect the excitation light pulse signal of the excitation laser and the fluorescence signal generated after the sample is irradiated at the same time, and the fluorescence signal contains time information and spatial information of the fluorescence photon.
  • the excitation light pulse signal of the excitation laser and the fluorescence signal generated after the sample is irradiated are collected.
  • the collected excitation light pulse signal of the excitation laser is used as the starting point of fluorescence lifetime detection; the fluorescent dye is irradiated and spontaneously emits fluorescence photons Signal, the obtained fluorescent photon signal constitutes the above-mentioned fluorescent signal.
  • the fluorescent signal contains the time information and spatial information of the fluorescent photon.
  • the spatial information of the fluorescent photon is the specific position of the radiated fluorescent photon on the two-dimensional plane. Information, the intensity of the fluorescent photon emitted by the fluorescent molecule gradually decreases with time within a single pulse period, and the time information of the fluorescent photon is the time information when the relative reference signal of the collected fluorescent photon reaches the detector.
  • the excitation light pulse signal and the fluorescence signal are respectively transmitted to the imaging processing terminal, and the excitation light pulse signal and the fluorescence signal are analyzed and processed through the imaging processing terminal to obtain a super-resolution image for high-resolution imaging of the sample.
  • the fluorescence signal is segmented according to the segmentation rule and the excitation light pulse signal to obtain the confocal image and the initial super-resolution image.
  • the confocal image is the fluorescence lifetime imaging generated by the excitation laser irradiating the sample
  • the initial super-resolution image is For STED (Stimulated Emission Depletion) super-resolution fluorescence lifetime imaging produced by irradiating the sample with the excitation laser and ring loss laser at the same time.
  • step S150 includes sub-steps S151, S152, S153, and S154.
  • the time at which the excitation light pulse signal is detected is regarded as the start time of fluorescence lifetime detection, that is, as the zero point of the time channel, and the intensity change of the fluorescent photon on the time channel is obtained according to the start time, that is, the time is taken as the horizontal
  • the coordinate is to obtain the intensity change of the fluorescent photon through the accumulation of the number of photons.
  • a time channel is a unit time (for example, a time channel can be set to 0.05 nanoseconds).
  • the ordinate is the intensity value of the fluorescent photon, and the intensity of the fluorescent photon can be It is reflected by the number of fluorescent photons collected by time accumulation in each time channel. The greater the number of fluorescent photons in a certain time channel, the higher the intensity of fluorescent photons, and finally the fluorescence decay curve of the fluorescent signal is obtained.
  • FIG. 7 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application.
  • a sample of fluorescent beads with a diameter of 23nm was used for the experiment.
  • the wavelength of the excitation laser was 635nm
  • the power was 35 ⁇ W
  • the wavelength of the loss laser was 730nm
  • the power was 10mW.
  • the frequency of the excitation laser and the frequency of the loss laser were both 80MHz.
  • the pulse width of the wide and depleted laser is about 0.3 nanoseconds (ns), and the optical path interval ⁇ 2 between the excitation laser pulse and the depleted laser pulse is 2 nanoseconds.
  • the fluorescence decay curve of the obtained fluorescence signal is shown in Figure 7 .
  • the time channel position of the intensity mutation in the fluorescence decay curve according to the segmentation rule as the segmentation point.
  • the signal intensity of the fluorescence signal will have a sudden change, and the sudden change of signal intensity can be reflected in the obtained fluorescence decay curve.
  • step S152 includes sub-steps S1521, S1522, and S1523.
  • S1521 calculate the slope value of each point in the fluorescence decay curve according to the slope value calculation formula to obtain the corresponding slope curve; S1522, calculate the slope change value of each point in the slope curve according to the slope change value calculation formula S1523. Obtain a time channel position in the fluorescence decay curve corresponding to the slope change value with the largest value as the segmentation point.
  • the fluorescence decay curve is composed of multiple points, and the slope calculation formula can calculate the slope value of each point in the fluorescence decay curve to obtain the slope curve.
  • the slope value of y r+2 is the ordinate of the point on the right side of the rth point 2 pixels apart from it
  • y r-2 is the ordinate of the point on the left side of the rth point 2 pixels apart from it
  • x r+2 is the abscissa of the point 2 pixels away from the right of the rth point
  • x r+2 is the abscissa of the point 2 pixels away from the left of the rth point
  • x r+2 is the abscissa of the point 2 pixels away from the left of the r
  • the slope calculation formula can also be a calculation formula for deriving points in the fluorescence attenuation curve.
  • the slope change value calculation formula is used to calculate the slope change value of each point in the slope curve.
  • the calculation method of the slope change value is the same as the calculation method of the above slope value.
  • a non-negative value corresponding to a slope change value is obtained, and the time channel position corresponding to the time channel position in the fluorescence decay curve of the slope change value with the largest value among the non-negative values is obtained as the division point, that is, the slope change value with the largest value is obtained.
  • the corresponding abscissa value in the fluorescence decay curve is used as the dividing point.
  • the spatial information of the fluorescent photons from the start time to the segmentation point in the fluorescence signal is obtained to form a confocal image
  • the spatial information of the fluorescence photons after the segmentation point in the fluorescence signal is obtained to form an initial Super-resolution image.
  • FIG. 8 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application. For example, after segmenting the obtained fluorescent signal, a confocal image of the sample is obtained as shown in Fig. 8(a), and a corresponding initial super-resolution image is obtained as shown in Fig. 8(b).
  • the confocal image and the initial super-resolution image are analyzed and processed through the image processing rules, which can greatly improve the resolution of imaging the sample and obtain the target super-resolution image of the sample.
  • step S160 includes sub-steps S161, S162, and S163.
  • the field of view of the confocal image, the initial super-resolution image, and the resulting ring image is the same (the image size is the same). Specifically, the pixel value of a pixel in the confocal image is subtracted from the pixel value corresponding to the pixel in the initial super-resolution image to obtain the pixel difference value of the pixel, and the pixel difference value of each pixel in the confocal image is obtained and combined You can get a corresponding ring image.
  • the focused image in Figure 8 is named image A
  • the initial super-resolution image is named image B
  • the resulting ring image is named image C
  • the pixel value of each pixel in the ring image is multiplied by the enhancement coefficient to obtain the corresponding enhanced ring image.
  • the enhancement coefficient is a coefficient value preset by the user, the value of the enhancement coefficient is greater than 1, and the enhancement coefficient may be an integer or a decimal number.
  • FIG. 9 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application.
  • the enhancement factor is 1, the ring image is shown in Figure 9(1), and the enhanced ring image obtained at this time is the same as the ring image C; if the enhancement factor is 2, the enhanced ring image obtained at this time is as As shown in Fig. 9(2); taking the enhancement coefficient as 4, the enhanced ring image obtained at this time is as shown in Fig. 9(3).
  • the obtained super-resolution image of the target also has the same field of view as the confocal image. Specifically, the pixel value of a pixel in the initial super-resolution image is subtracted from the pixel value corresponding to the pixel in the ring image to obtain the pixel difference value of the pixel, and the initial super-resolution image and the pixel value of each pixel in the ring image are obtained. The pixel difference can be combined to obtain the corresponding target super-resolution image.
  • the obtained ring image can be expressed as 1 ⁇ C, and the target super-resolution image obtained at this time is shown in Figure 9 (4); if the enhancement factor is 2, the obtained ring image can be expressed as 2 ⁇ C, the target super-resolution image obtained at this time is shown in Figure 9(5); taking the enhancement coefficient as 4, the obtained ring image can be expressed as 4 ⁇ C, and the target super-resolution image obtained at this time is shown in Figure 9(5). Shown in 9(6).
  • the super-resolution imaging method based on low-power stimulated emission loss extends the optical path of the lossy laser propagating in the first optical path by adjusting the position of the corner reflector in the first optical path, and reduces the low-power
  • the Gaussian loss laser is converted into a ring loss laser and coincides with the excitation laser to focus and irradiate the sample, so that the collected fluorescence lifetime data contains both confocal and super-resolution signals.
  • the confocal image and the initial super-resolution image are separated from the fluorescence signal through data processing, and the confocal image and the initial super-resolution image are analyzed and processed according to the image processing rules to obtain the target super-resolution image with further improved resolution.
  • the use of low-power loss lasers reduces the damage to biological samples, reduces the photobleaching effect of fluorescent dyes, and prolongs the effective time of super-resolution imaging.
  • image enhancement processing high resolutions containing fine structural features are obtained. Images have achieved good technical results in the actual application process.
  • the embodiment of the present application also provides a super-resolution imaging system based on low-power stimulated emission loss.
  • the super-resolution imaging system based on low-power stimulated emission loss can be used to realize the aforementioned super-resolution imaging method based on low-power stimulated emission loss. Any embodiment of. Specifically, please refer to FIGS. 5-6.
  • FIG. 5 is a schematic diagram of a super-resolution imaging system based on low-power stimulated emission loss according to an embodiment of the application
  • FIG. 6 is a schematic diagram of an imaging processing terminal provided by an embodiment of the application.
  • the imaging system includes a signal acquisition device 10 and an imaging processing terminal 20.
  • the signal acquisition device 10 is used for acquiring the excitation light pulse signal as a reference signal, and acquiring the fluorescent signal generated after the sample is irradiated.
  • the signal acquisition device includes an excitation laser 101, a loss laser 102, a beam splitter 103, a first dichroic mirror 104, a second dichroic mirror 105, the corner reflector 106, a spiral phase plate 107, a scanning galvanometer 108, The objective lens 109, the stage 110, the preamplifier 111, the first detector 112, the second detector 113, and a time dependent single photon counter (TCSPC) 114.
  • TCSPC time dependent single photon counter
  • the excitation laser 101 is used to emit Gaussian pulse excitation laser; the loss laser 102 is used to emit Gaussian pulse loss laser; the spectroscope is used to split the excitation laser light to A part of the excitation laser light is propagated along the second optical path, and the other part of the excitation laser light is injected into the second detector; The laser light is reflected to propagate to the spiral phase plate; the spiral phase plate 107 is used to convert the incident loss laser light of the Gaussian pulse into ring loss laser light so that it propagates to the second dichroic mirror
  • the first dichroic mirror 104 is used to reflect the excitation laser propagating along the second optical path to make it propagate to the second dichroic mirror, and to transmit the fluorescent signal generated after the sample is irradiated
  • the second dichroic mirror 105 is used to reflect the ring loss laser and transmit the excitation laser propagating along the second optical path so that the two lasers overlap and propagate to the scanning galvanometer, and The fluorescence signal is transmitted; the scanning galvan
  • an electrical connection is made between the excitation laser 101 and the loss laser 102, and the excitation laser 101 is triggered synchronously by the loss laser 102, so that the two laser pulses maintain a stable pulse interval.
  • the imaging processing terminal 20 is configured to process the excitation light pulse signal and the fluorescence signal collected by the signal collection device to obtain the target super-resolution image.
  • the imaging processing terminal 20 is a terminal device used to obtain the excitation light pulse signal and the fluorescence signal collected by the signal acquisition device and then perform imaging processing to obtain the target super-resolution image, such as a workstation, a desktop computer, a notebook computer, a tablet computer, or a mobile phone. Wait.
  • the imaging processing terminal 20 may perform the following steps: separate a confocal image and an initial super-resolution image from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule; The focused image and the initial super-resolution image are analyzed and processed to obtain a high-resolution target super-resolution image.
  • the imaging processing terminal 20 includes a fluorescent signal dividing unit 210 and an image processing unit 220.
  • the fluorescence signal segmentation unit 210 is used to separate the confocal image and the initial super-resolution image from the fluorescence signal according to the excitation light pulse signal and preset segmentation rules; the image processing unit 220 is used to separate the confocal image and the initial super-resolution image according to the preset image
  • the processing rules analyze and process the confocal image and the initial super-resolution image to obtain a higher-resolution target super-resolution image.

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Abstract

一种基于低功率受激发射损耗的超分辨成像方法及成像***。通过调整设置于第一光路中角反射器（106）的位置延长损耗激光在第一光路中进行传播的光程，将低功率的高斯型损耗激光转换为环形损耗激光并与激发激光重合后聚焦照射样品，使采集到的荧光寿命数据中同时包含共聚焦信号和超分辨信号。通过数据处理从荧光信号中分离出共聚焦图像及初始超分辨图像，根据图像处理规则对共聚焦图像及初始超分辨图像进行分析处理后得到分辨率进一步提升的目标超分辨图像。通过上述方法，采用低功率的损耗激光减小了对生物样品的破坏，降低了荧光染料的光漂白效应，延长了超分辨成像的有效时间，可得到包含细微结构特征的高分辨率图像。

Description

基于低功率受激发射损耗的超分辨成像方法及成像***
本申请是以申请号为202010500061.X、申请日为2020年6月4日的中国专利申请为基础，并主张其优先权，该申请的全部内容在此作为整体引入本申请中。
技术领域
本申请涉及超分辨光学显微成像的技术领域，尤其涉及一种基于低功率受激发射损耗的超分辨成像方法及成像***。
背景技术
活细胞和组织成像对生物医学领域的研究至关重要，除了正确的细胞培养条件和样品制备方法，先进的成像方法及成像***可以在保持被观测样品自身生物特性的基础上，最大程度地保障获取信息的真实性和有效性。光学显微镜具有非接触、无损伤和特异性的优点，是近代自然科学发展开端的重要标志，它可以很好地应用于活细胞和组织的成像。但是，光的衍射限制了光学显微镜的分辨率能力，使其无法清晰地辨别尺寸在200nm以下的微观生物结构。超分辨光学显微镜(Super-resolution optical microscopy,SRM)技术继承了光学显微镜的非接触和特异性优点，通过物理化学原理将光学显微镜的分辨率提升1～2个数量级，可以在分子水平了解生命的发展变化规律，揭示生物体的抗/耐药性和干预治疗作用的细胞分子机理，是本世纪光学显微成像领域最重大的突破之一。近年来，超分辨光学成像技术的快速发展让光学显微镜与生物医学等领域的联系更加紧密，但是现有的技术对样品(制备)和荧光染料有着极其严格的要求，限制了在活体生物成像中的应用。
1994年，德国科学家Stefan W.Hell根据爱因斯坦辐射理论提出受激发射损耗(Stimulated emission depletion,STED)显微技术。基于荧光饱和与激发态荧光受激辐射之间的非线性关系，STED技术利用第二束波长红移的激光对激发态分子提前进行选择性耗散，通过压缩激发光斑的有效点扩展函数提高成像分辨率，理论上可以实现三维空间上纳米级的分辨率。作为第一个理论上提出和实验中实现的远场超分辨成像方法，STED技术具有快速成像和无需后期图像重构的优势。但是，为了避免再激发效应对超分辨图像质量的影响，损耗激光波长通常位于荧光染料发射光谱尾端。由于发射光谱尾端的受激辐射截面极小，STED技术需要极高的损耗能量(通常比激发光能量高三个数量级以上)实现分辨率的 提升。过高的激光能量会引起光漂白和光毒性，对荧光探针和生物组织产生破坏，因此限制了该技术在活细胞和组织成像中的应用。
荧光寿命成像显微镜(Fluorescence lifetime imaging microscopy,FLIM)是一种描述荧光寿命空间分布的显微镜成像技术，可以应用于固定细胞和活细胞的成像中，揭示更多的生物信息(如细胞内微环境的变化)。基于时间相关单光子计数器(Time-correlated single photon counting,TCSPC)的FLIM技术通过将脉冲周期划分为多个等间距的时间通道，根据光子相对参考信号到达探测器的时间差，将其转换为存储器的地址，光子信号积累后得到染料的荧光衰减曲线。将STED和FLIM两种成像技术相结合，可以同时获取荧光信号的强度和寿命信息，实现双模态STED-FLIM超分辨荧光寿命成像。在共聚焦荧光寿命成像(Confocal-FLIM)中，荧光寿命曲线随时间呈单指数衰减；但是在STED超分辨荧光寿命成像(STED-FLIM)中，由于损耗激光对激发态分子的受激辐射作用，荧光寿命曲线随时间呈多指数衰减。虽然STED-FLIM可以实现超分辨率的荧光寿命成像，但是过高的激光能量改变了荧光染料自身的荧光寿命特性，因此无法真实的反映微环境变化，导致对样品进行成像时无法呈现细微结构特征，造成成像效果较差而难以获取高分辨率的图像。
申请内容
本申请实施例提供了一种基于低功率受激发射损耗的超分辨成像方法及成像***，旨在解决受激辐射损耗超分辨成像方法对损耗激光功率需求高，以及高激光功率下超分辨图像质量差的问题。
第一方面，本申请实施例提供了一种基于低功率受激发射损耗的超分辨成像方法，其包括：
将被荧光染料染色的样品置于载物台上并调整第一光路中角反射器的位置；
发射激发激光及损耗激光，所述激发激光及所述损耗激光均为高斯型脉冲激光，其中，所述损耗激光的波长大于所述激发激光的波长；
将沿所述第一光路传播的所述损耗激光转换为环形损耗激光并与沿第二光路传播的所述激发激光重合后，聚焦照射所述样品；
同时采集所述激发激光的激发光脉冲信号以及所述样品被照射后产生的荧光信号，所述荧光信号中包含荧光光子的时间信息和空间信息；
根据所述激发光脉冲信号及预置的分割规则从所述荧光信号中分离出共聚焦图像及初始超分辨图像；
根据预置的图像处理规则对所述共聚焦图像及所述初始超分辨图像进行分析处理，以得到更高分辨率的目标超分辨图像。
第二方面，本申请实施例提供了一种基于低功率受激发射损耗的超分辨成像***，其包括：
信号采集装置及成像处理终端；
所述信号采集装置，用于采集得到所述激发光脉冲信号作为参考信号，并采集所述样品被照射后产生的荧光信号；
所述成像处理终端，用于对所述信号采集装置所采集的所述激发光脉冲信号及所述荧光信号进行处理以得到所述目标超分辨图像。
本申请实施例提供了一种基于低功率受激发射损耗的超分辨成像方法及成像***。通过调整设置于第一光路中角反射器的位置延长损耗激光在第一光路中进行传播的光程，将低功率的高斯型损耗激光转换为环形损耗激光并与激发激光重合后聚焦照射样品，使采集到的荧光寿命数据中同时包含共聚焦信号和超分辨信号。通过数据处理从荧光信号中分离出共聚焦图像及初始超分辨图像，根据图像处理规则对共聚焦图像及初始超分辨图像进行分析处理后得到分辨率进一步提升的目标超分辨图像。通过上述方法，采用低功率的损耗激光减小了对生物样品的破坏，降低了荧光染料的光漂白效应，延长了超分辨成像的有效时间，结合图像增强处理得到包含细微结构特征的高分辨率图像，在实际应用过程中取得了良好的技术效果。
附图说明
为了更清楚地说明本申请实施例技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本申请实施例提供的基于低功率受激发射损耗的超分辨成像方法的流程示意图；
图2为本申请实施例提供的基于低功率受激发射损耗的超分辨成像方法的子流程示意图；
图3为本申请实施例提供的基于低功率受激发射损耗的超分辨成像方法的子流程示意图；
图4为本申请实施例提供的基于低功率受激发射损耗的超分辨成像方法的 子流程示意图；
图5为本申请实施例提供的基于低功率受激发射损耗的超分辨成像***的示意图；
图6为本申请实施例提供的成像处理终端的示意性框图；
图7为本申请实施例提供的基于低功率受激发射损耗的超分辨成像方法的使用效果示意图；
图8为本申请实施例提供的基于低功率受激发射损耗的超分辨成像方法的使用效果示意图；
图9为本申请实施例提供的基于低功率受激发射损耗的超分辨成像方法的使用效果示意图；
图10为本申请实施例提供的基于低功率受激发射损耗的超分辨成像方法的使用效果示意图。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
应当理解，当在本说明书和所附权利要求书中使用时，术语“包括”和“包含”指示所描述特征、整体、步骤、操作、元素和/或组件的存在，但并不排除一个或多个其它特征、整体、步骤、操作、元素、组件和/或其集合的存在或添加。
还应当理解，在此本申请说明书中所使用的术语仅仅是出于描述特定实施例的目的而并不意在限制本申请。如在本申请说明书和所附权利要求书中所使用的那样，除非上下文清楚地指明其它情况，否则单数形式的“一”、“一个”及“该”意在包括复数形式。
还应当进一步理解，在本申请说明书和所附权利要求书中使用的术语“和/或”是指相关联列出的项中的一个或多个的任何组合以及所有可能组合，并且包括这些组合。
请参阅图1及图5，图1是为本申请实施例提供的基于低功率受激发射损耗的超分辨成像方法的流程示意图，图5为本申请实施例提供的基于低功率受激发射损耗的超分辨成像***的示意图。该基于低功率受激发射损耗的超分辨成像方法应用于成像***中，成像***包括信号采集装置10及成像处理终端20， 该方法通过信号采集装置10结合安装于成像处理终端20中的应用软件进行执行，成像***即是用于执行基于低功率受激发射损耗的超分辨成像方法以实现对所述样品进行高分辨率成像的***装置，信号采集装置10即是用于发射激发激光及损耗激光对所述样品进行探测并采集激发光脉冲信号和荧光信号的装置，成像处理终端20即是用于获取信号采集装置所采集的激发光脉冲信及荧光信号后进行成像处理得到目标超分辨图像的终端设备，例如工作站、台式电脑、笔记本电脑、平板电脑或手机等。
如图1所示，该方法包括步骤S110～S160。
S110、将被荧光染料染色的样品置于载物台上并调整第一光路中角反射器的位置。
将被荧光染料染色的样品置于载物台上并调整第一光路中角反射器的位置。首先采用荧光染料对样品进行染色，具体的，样品可以是活细胞、病毒或组织等生物材料，荧光染料即为被光照后受激并产生荧光光子的染料，调节角反射器的位置可延长损耗激光在第一光路中进行传播的光程。
图10为本申请实施例提供的基于低功率受激发射损耗的超分辨成像方法的使用效果示意图，具体的，如图10所示，未调节角反射器时角反射器位于图10中位置①处，损耗激光沿第一光路传播的光程(光沿某一路径传播一定路程所需的时间)为τ 1，此时激发激光脉冲与损耗激光脉冲之间的光程间隔为Δτ 1(激发激光脉冲与损耗激光脉冲作用于样品的时间差)；调节角反射器的位置至图10中位置②处，位置①与位置②的距离为S，则损耗激光沿第一光路传播的光程为τ 1+2S/c，其中c为光速，则此时激发激光脉冲与损耗激光脉冲之间的光程间隔为Δτ 2＝Δτ 1+2S/c。
S120、发射激发激光及损耗激光，所述激发激光及所述损耗激光均为高斯型脉冲激光，其中，所述损耗激光的波长大于所述激发激光的波长。
可同时发射激发激光及损耗激光，两束激光均为脉冲型激光，且脉冲频率相同(如80MHz)，激光的频率与激光的脉冲周期呈反比，脉冲周期应至少包含一个完整的自发荧光过程(通常为纳秒及以上的时间量级)。荧光寿命越长，则激光的脉冲周期就要越大，激光的脉冲频率就越小。其中，所述激发激光的功率小于所述损耗激光的功率，具体的，此处损耗激光的功率比传统受激辐射损耗超分辨成像(～100mW)低至少一个数量级(如小于10mW)，激发激光的功率与发光材料的光谱特性有关，通常为0.1-100μW。损耗激光的功率越大， 所得到的环状图像中环形光斑的中心零强度区域越小，且峰值强度越大。所述激发激光的脉宽与所述损耗激光的脉宽均为百皮秒量级，例如，激发激光的脉宽与损耗激光的脉宽的取值范围均可以是0.1-1纳秒，损耗激光的波长大于激发激光的波长，脉宽即为激发激光脉冲或损耗激光脉冲的宽度，为实现对样品进行超分辨成像，需控制激发激光的脉宽及损耗激光的脉宽均为百皮秒量级(100皮秒＝0.1纳秒)。
S130、将沿所述第一光路传播的所述损耗激光转换为环形损耗激光并与沿第二光路传播的所述激发激光重合后，聚焦照射所述样品。
具体的，可在第一光路的角反射器之后设置螺旋相位板，通过螺旋相位板将高斯型脉冲激光的损耗激光转换为环形损耗激光，环形损耗激光与沿第二光路传播的激发激光重合，两束激光的焦平面在空间上精准重合后进行聚焦并照射样品，已染色的样品被照射后荧光染料会产生荧光信号。
S140、同时采集所述激发激光的激发光脉冲信号以及所述样品被照射后产生的荧光信号，所述荧光信号中包含荧光光子的时间信息和空间信息。
同时采集得到激发激光的激发光脉冲信号，以及样品被照射后产生的荧光信号，所采集到的激发激光的激发光脉冲信号作为荧光寿命探测的起始点；荧光染料被照射后自发辐射产生荧光光子信号，所得到的荧光光子的信号即组成上述荧光信号，荧光信号中包含荧光光子的时间信息和空间信息，荧光光子的空间信息即为所辐射出的该荧光光子在二维平面上的具***置信息，荧光分子所辐射出的荧光光子强度在单个脉冲周期内随时间逐渐减弱，荧光光子的时间信息即为采集到该荧光光子的相对参考信号到达探测器的时间信息。
S150、根据所述激发光脉冲信号及预置的分割规则从所述荧光信号中分离出共聚焦图像及初始超分辨图像。
激发光脉冲信号及荧光信号分别被传输至成像处理终端，通过该成像处理终端对激发光脉冲信号及荧光信号进行解析处理，以得到对所述样品进行高分辨率成像的超分辨图像。具体的，首先根据分割规则及激发光脉冲信号对荧光信号进行分割，得到共聚焦图像及初始超分辨图像，共聚焦图像即为通过激发激光照射样品所产生的荧光寿命成像，初始超分辨图像即为通过激发激光及环形损耗激光同时照射样品所产生的STED(受激发射损耗，Stimulated emission depletion)超分辨荧光寿命成像。
在一实施例中，如图2所示，步骤S150包括子步骤S151、S152、S153和 S154。
S151、将采集到的所述激发光脉冲信号的时间点作为荧光寿命探测的起始时间，获取所述荧光光子在时间通道上的强度变化，以得到所述荧光信号的荧光衰减曲线。
将探测到激发光脉冲信号的时刻作为荧光寿命探测的起始时间，也即是作为时间通道的零点，根据该起始时间获取荧光光子在时间通道上的强度变化，也即是以时间作为横坐标，通过光子数累积获取荧光光子的强度变化，一个时间通道即为一段单位时间(例如，可设置一个时间通道为0.05纳秒)，纵坐标即为荧光光子的强度值，荧光光子的强度可通过每一时间通道内时间积累采集得到的荧光光子的数量进行体现，某一时间通道内荧光光子数量越多则荧光光子的强度越高，最后得到荧光信号的荧光衰减曲线。
图7为本申请实施例提供的基于低功率受激发射损耗的超分辨成像方法的使用效果示意图。采用直径为23nm的荧光珠样品进行试验，激发激光的波长为635nm，功率为35μW，损耗激光的波长为730nm，功率为10mW，激发激光的频率及损耗激光的频率均为80MHz，激发激光的脉宽及损耗激光的脉宽约为0.3纳秒(ns)，激发激光脉冲与损耗激光脉冲之间的光程间隔Δτ 2为2纳秒，所得到的荧光信号的荧光衰减曲线如图7所示。
S152、根据所述分割规则获取所述荧光衰减曲线中强度突变的时间通道位置作为分割点。
根据分割规则获取荧光衰减曲线中强度突变的时间通道位置作为分割点。当激发激光及环形损耗激光同时照射样品时，荧光信号的信号强度会产生突变，信号强度的突变即可在所得到的荧光衰减曲线中得到体现，获取荧光衰减曲线中强度突变的时间通道位置作为分割点，即可对荧光信号进行分割。
在一实施例中，如图3所示，步骤S152包括子步骤S1521、S1522和S1523。
S1521、根据所述斜率值计算公式计算所述荧光衰减曲线中每一点的斜率值以得到对应的斜率曲线；S1522、根据所述斜率变化值计算公式计算所述斜率曲线中每一点的斜率变化值；S1523、获取数值最大的一个所述斜率变化值在所述荧光衰减曲线中对应的时间通道位置作为所述分割点。
具体的，荧光衰减曲线由多个点组成，斜率计算公式即可对荧光衰减曲线中每一点的斜率值进行计算，得到斜率曲线。例如，斜率计算公式可以是A r＝(y r+2-y r-2)/(x r+2-x r-2)，其中A r为计算所得到的荧光衰减曲线中第r个点的斜率 值，y r+2为第r个点右侧与之间隔2个像素的点的纵坐标，y r-2为第r个点左侧与之间隔2个像素的点的纵坐标，x r+2为第r个点右侧与之间隔2个像素的点的横坐标，x r+2为第r个点左侧与之间隔2个像素的点的横坐标，在r为任意点的情况下，x r+2-x r-2恒等于5。若第r个点左侧没有点，则y r-2＝y r、x r-2＝x r；若第r个点右侧没有点，则y r+2＝y r、x r+2＝x r。斜率计算公式还可以是对荧光衰减曲线中的点进行求导的计算公式。通过斜率变化值计算公式对斜率曲线中的每一点的斜率变化值进行计算，斜率变化值的计算方法同上述的斜率值的计算方法，对所计算得到的斜率变化值取绝对值，得到与每一斜率变化值对应的一个非负数值，获取非负数值中数值最大的一个斜率变化值在荧光衰减曲线中对应的时间通道位置作为所述分割点，也即是获取数值最大的一个斜率变化值在荧光衰减曲线中对应的横坐标值作为分割点。
例如，对图7中所示的荧光衰减曲线采用上述方法得到的分割点的横坐标值为τ x＝3ns，则可以3ns作为分割点对荧光信号进行分割。
S153、根据所述荧光信号中位于所述分割点之前的荧光光子的空间信息组成所述共聚焦图像。
S154、根据所述荧光信号中位于所述分割点之后的荧光光子的空间信息组成所述初始超分辨图像。
以分割点为基准，获取荧光信号中从起始时间至该分割点的荧光光子的空间信息组成一张共聚焦图像，获取荧光信号中从该分割点之后的荧光光子的空间信息组成一张初始超分辨图像。
图8为本申请实施例提供的基于低功率受激发射损耗的超分辨成像方法的使用效果示意图。例如，对所得到的荧光信号进行分割后，得到样品的一张共聚焦图像如图8(a)所示，得到对应的一张初始超分辨图像如图8(b)所示。
S160、根据预置的图像处理规则对所述共聚焦图像及所述初始超分辨图像进行分析处理，以得到高分辨率的目标超分辨图像。
通过图像处理规则对共聚焦图像及初始超分辨图像进行分析处理，可大幅提升对样品进行成像的分辨率，得到样品的目标超分辨图像。
在一实施例中，如图4所示，步骤S160包括子步骤S161、S162和S163。
S161、将所述共聚焦图像的强度值减去所述初始超分辨图像的强度值得到环状图像。
共聚焦图像、初始超分辨图像以及所得到的环状图像的视场相同(图像尺 寸相同)。具体的，将共聚焦图像中一个像素的像素值减去初始超分辨图像中与该像素对应的像素值，得到该像素的像素差值，获取共聚焦图像中每一像素的像素差值进行组合即可得到对应的一张环状图像。
例如，图8中的聚焦图像命名为图像A，初始超分辨图像命名为图像B，所得到的环状图像命名为图像C，则C＝A-B，所得到的一张环状图像如图8(c)所示。
S162、将所述环状图像与所述图像处理规则中的增强系数相乘以得到增强环状图像。
具体的，将环状图像中每一像素的像素值与增强系数相乘，得到对应的增强环状图像。其中，所述增强系数为用户预设的一个系数值，增强系数的取值大于1，增强系数可以为整数或者小数。
图9为本申请实施例提供的基于低功率受激发射损耗的超分辨成像方法的使用效果示意图。例如，取增强系数为1，环状图像如图9(1)所示，此时得到的增强环状图像与环状图像C相同；取增强系数为2，此时得到的增强环状图像如图9(2)所示；取增强系数为4，此时得到的增强环状图像如图9(3)所示。
S163、将所述初始超分辨图像的强度值减去所述增强环状图像的强度值得到所述目标超分辨图像。
所得到的目标超分辨图像也与共聚焦图像的视场相同。具体的，将初始超分辨图像中一个像素的像素值减去环状图像中与该像素对应的像素值，得到该像素的像素差值，获取初始超分辨图像和环状图像中每一像素的像素差值进行组合即可得到对应的目标超分辨图像。
例如，取增强系数为1，得到的环状图像可表示为1×C，此时得到的目标超分辨图像如图9(4)所示；取增强系数为2，得到的环状图像可表示为2×C，此时得到的目标超分辨图像如图9(5)所示；取增强系数为4，得到的环状图像可表示为4×C，此时得到的目标超分辨图像如图9(6)所示。
本申请实施例所提供的基于低功率受激发射损耗的超分辨成像方法，通过调整设置于第一光路中角反射器的位置延长损耗激光在第一光路中进行传播的光程，将低功率的高斯型损耗激光转换为环形损耗激光并与激发激光重合后聚焦照射样品，使采集到的荧光寿命数据中同时包含共聚焦信号和超分辨信号。通过数据处理从荧光信号中分离出共聚焦图像及初始超分辨图像，根据图像处理规则对共聚焦图像及初始超分辨图像进行分析处理后得到分辨率进一步提升 的目标超分辨图像。通过上述方法，采用低功率的损耗激光减小了对生物样品的破坏，降低了荧光染料的光漂白效应，延长了超分辨成像的有效时间，结合图像增强处理得到包含细微结构特征的高分辨率图像，在实际应用过程中取得了良好的技术效果。
本申请实施例还提供一种基于低功率受激发射损耗的超分辨成像***，该基于低功率受激发射损耗的超分辨成像***可用于实现前述基于低功率受激发射损耗的超分辨成像方法的任一实施例。具体地，请参阅图5-图6，图5为本申请实施例提供的基于低功率受激发射损耗的超分辨成像***的示意图，图6为本申请实施例提供的成像处理终端的示意性框图，所述成像***包括信号采集装置10及成像处理终端20。
所述信号采集装置10，用于采集得到所述激发光脉冲信号作为参考信号，并采集所述样品被照射后产生的荧光信号。
具体的，所述信号采集装置包括激发激光器101、损耗激光器102、分光镜103、第一双色镜104、第二双色镜105、所述角反射器106、螺旋相位板107、扫描振镜108、物镜109、所述载物台110、前置放大器111、第一探测器112、第二探测器113、时间相关单光子计数器(TCSPC)114。
其中，所述激发激光器101，用于发射高斯型脉冲的激发激光；所述损耗激光器102，用于发射高斯型脉冲的损耗激光；所述分光镜，用于对所述激发激光进行分光，以使一部分所述激发激光沿所述第二光路传播、另一部分所述激发激光射入所述第二探测器；所述角反射器106，用于对沿所述第一光路传播的所述损耗激光进行反射以使其传播至所述螺旋相位板；所述螺旋相位板107，用于将入射的所述高斯型脉冲的损耗激光转换为环形损耗激光以使其传播至所述第二双色镜；所述第一双色镜104，用于对沿所述第二光路传播的激发激光进行反射以使其传播至所述第二双色镜，并对所述样品被照射后产生的荧光信号进行透射；所述第二双色镜105，用于对所述环形损耗激光进行反射、对沿所述第二光路传播的激发激光进行透射以使两束激光重合后传播至所述扫描振镜，并对所述荧光信号进行透射；所述扫描振镜108，用于对入射的激发激光及环形损耗激光进行同步扫描，以实现对样品的面阵成像；所述物镜109，用于对入射的激光进行聚焦后照射所述样品；所述载物台110，用于放置和固定样品，并对所述样品进行三维移动；所述第一探测器112，用于探测和收集荧光染料被照射后发出的荧光光子信号；所述第二探测器113，用于对入射的激发激光进行探测以 得到所述激发光脉冲信号；所述前置放大器111，用于对来自所述第一探测器的荧光光子信号进行放大和滤波；所述时间相关单光子计数器(TCSPC)114，用于信号存储和荧光寿命成像以得到所述荧光信号。
更具体的，激发激光器101与损耗激光器102之间进行电连接，激发激光器101由损耗激光器102同步触发，使两束激光脉冲保持稳定的脉冲间隔。
所述成像处理终端20，用于对所述信号采集装置所采集的所述激发光脉冲信号及所述荧光信号进行处理以得到所述目标超分辨图像。
所述成像处理终端20即是用于获取信号采集装置所采集的激发光脉冲信及荧光信号后进行成像处理得到目标超分辨图像的终端设备，例如工作站、台式电脑、笔记本电脑、平板电脑或手机等。
成像处理终端20可执行以下步骤：根据所述激发光脉冲信号及预置的分割规则从所述荧光信号中分离出共聚焦图像及初始超分辨图像；根据预置的图像处理规则对所述共聚焦图像及所述初始超分辨图像进行分析处理，以得到高分辨率的目标超分辨图像。
在一实施例中，如图6所示，所述成像处理终端20包括荧光信号分割单元210及图像处理单元220。
荧光信号分割单元210，用于根据所述激发光脉冲信号及预置的分割规则从所述荧光信号中分离出共聚焦图像及初始超分辨图像；图像处理单元220，用于根据预置的图像处理规则对所述共聚焦图像及所述初始超分辨图像进行分析处理，以得到更高分辨率的目标超分辨图像。
以上所述，仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到各种等效的修改或替换，这些修改或替换都应涵盖在本申请的保护范围之内。因此，本申请的保护范围应以权利要求的保护范围为准。

Claims (10)

  1. 一种基于低功率受激发射损耗的超分辨成像方法，应用于成像***，其特征在于，所述方法包括：
    将被荧光染料染色的样品置于载物台上并调整第一光路中角反射器的位置；
    发射激发激光及损耗激光，所述激发激光及所述损耗激光均为高斯型脉冲激光，其中，所述损耗激光的波长大于所述激发激光的波长；
    将沿所述第一光路传播的所述损耗激光转换为环形损耗激光并与沿第二光路传播的所述激发激光重合后，聚焦照射所述样品；
    同时采集所述激发激光的激发光脉冲信号以及所述样品被照射后产生的荧光信号，所述荧光信号中包含荧光光子的时间信息和空间信息；
    根据所述激发光脉冲信号及预置的分割规则从所述荧光信号中分离出共聚焦图像及初始超分辨图像；
    根据预置的图像处理规则对所述共聚焦图像及所述初始超分辨图像进行分析处理，以得到更高分辨率的目标超分辨图像。
  2. 根据权利要求1所述的基于低功率受激发射损耗的超分辨成像方法，其特征在于，所述根据所述激发光脉冲信号及预置的分割规则从所述荧光信号中分离出共聚焦图像及初始超分辨图像，包括：
    将采集到的所述激发光脉冲信号的时间点作为荧光寿命探测的起始时间，获取所述荧光光子在时间通道上的强度变化，以得到所述荧光信号的荧光衰减曲线；
    根据所述分割规则获取所述荧光衰减曲线中强度突变的时间通道位置作为分割点；
    根据所述荧光信号中位于所述分割点之前的荧光光子的空间信息组成所述共聚焦图像；
    根据所述荧光信号中位于所述分割点之后的荧光光子的空间信息组成所述初始超分辨图像。
  3. 根据权利要求1所述的基于低功率受激发射损耗的超分辨成像方法，其特征在于，所述根据预置的图像处理规则对所述共聚焦图像及所述初始超分辨图像进行分析处理，以得到高分辨率的目标超分辨图像，包括：
    将所述共聚焦图像的强度值减去所述初始超分辨图像的强度值得到环状图像；
    将所述环状图像与所述图像处理规则中的增强系数相乘以得到增强环状图像；
    将所述初始超分辨图像的强度值减去所述增强环状图像的强度值得到所述目标超分辨图像。
  4. 根据权利要求3所述的基于低功率受激发射损耗的超分辨成像方法，其特征在于，所述增强系数的取值大于1。
  5. 根据权利要求2所述的基于低功率受激发射损耗的超分辨成像方法，其特征在于，所述分割规则包括斜率值计算公式及斜率变化值计算公式，所述根据所述分割规则获取所述荧光衰减曲线中强度突变的时间通道位置作为分割点，包括：
    根据所述斜率值计算公式计算所述荧光衰减曲线中每一点的斜率值以得到对应的斜率曲线；
    根据所述斜率变化值计算公式计算所述斜率曲线中每一点的斜率变化值；
    获取数值最大的一个所述斜率变化值在所述荧光衰减曲线中对应的时间通道位置作为所述分割点。
  6. 根据权利要求1-5任一项所述的基于低功率受激发射损耗的超分辨成像方法，其特征在于，所述损耗激光的功率大于所述激发激光的功率。
  7. 根据权利要求6所述的基于低功率受激发射损耗的超分辨成像方法，其特征在于，所述激发激光的脉宽与所述损耗激光的脉宽均为百皮秒量级。
  8. 一种基于低功率受激发射损耗的超分辨成像***，所述成像***用于实现如权利要求1-7任一项所述的基于低功率受激发射损耗的超分辨成像方法，其特征在于，所述成像***包括信号采集装置及成像处理终端；
    所述信号采集装置，用于采集得到所述激发光脉冲信号作为参考信号，并采集所述样品被照射后产生的荧光信号；
    所述成像处理终端，用于对所述信号采集装置所采集的所述激发光脉冲信号及所述荧光信号进行处理以得到所述目标超分辨图像。
  9. 根据权利要求8所述的基于低功率受激发射损耗的超分辨成像***，其特征在于，所述成像处理终端包括：
    荧光信号分割单元，用于根据所述激发光脉冲信号及预置的分割规则从所 述荧光信号中分离出共聚焦图像及初始超分辨图像；
    图像处理单元，用于根据预置的图像处理规则对所述共聚焦图像及所述初始超分辨图像进行分析处理，以得到更高分辨率的目标超分辨图像。
  10. 根据权利要求8所述的基于低功率受激发射损耗的超分辨成像***，其特征在于，所述信号采集装置包括激发激光器、损耗激光器、分光镜、第一双色镜、第二双色镜、所述角反射器、螺旋相位板、扫描振镜、物镜、所述载物台、前置放大器、第一探测器、第二探测器、时间相关单光子计数器；
    所述激发激光器，用于发射高斯型脉冲的激发激光；
    所述损耗激光器，用于发射高斯型脉冲的损耗激光；
    所述分光镜，用于对所述激发激光进行分光，以使一部分所述激发激光沿所述第二光路传播、另一部分所述激发激光射入所述第二探测器；
    所述角反射器，用于对沿所述第一光路传播的所述损耗激光进行反射以使其传播至所述螺旋相位板；
    所述螺旋相位板，用于将入射的所述高斯型脉冲的损耗激光转换为环形损耗激光以使其传播至所述第二双色镜；
    所述第一双色镜，用于对沿所述第二光路传播的激发激光进行反射以使其传播至所述第二双色镜，并对所述样品被照射后产生的荧光信号进行透射；
    所述第二双色镜，用于对所述环形损耗激光进行反射、对沿所述第二光路传播的激发激光进行透射以使两束激光重合后传播至所述扫描振镜，并对所述荧光信号进行透射；
    所述扫描振镜，用于对入射的激发激光及环形损耗激光进行同步扫描，以实现对样品的面阵成像；
    所述物镜，用于对入射的激光进行聚焦后照射所述样品；
    所述载物台，用于放置和固定样品，并对所述样品进行三维移动；
    所述第一探测器，用于探测和收集荧光染料被照射后发出的荧光光子信号；
    所述第二探测器，用于对入射的激发激光进行探测以得到所述激发光脉冲信号；
    所述前置放大器，用于对来自所述第一探测器的荧光光子信号进行放大和滤波；
    所述时间相关单光子计数器，用于信号存储和荧光寿命成像以得到所述荧光信号，其中，所述荧光信号中包含荧光光子的时间信息和空间信息。
PCT/CN2020/096779 2020-06-04 2020-06-18 基于低功率受激发射损耗的超分辨成像方法及成像*** WO2021243754A1 (zh)

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