WO2020140283A1 - 一种受激发射损耗显微镜的成像系统 - Google Patents

一种受激发射损耗显微镜的成像系统 Download PDF

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WO2020140283A1
WO2020140283A1 PCT/CN2019/070468 CN2019070468W WO2020140283A1 WO 2020140283 A1 WO2020140283 A1 WO 2020140283A1 CN 2019070468 W CN2019070468 W CN 2019070468W WO 2020140283 A1 WO2020140283 A1 WO 2020140283A1
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light
unit
excitation light
loss
gaussian
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French (fr)
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严伟
屈军乐
王璐玮
叶彤
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Shenzhen University
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Shenzhen University
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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
    • G01N21/62—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
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes

Definitions

  • the invention relates to the field of optical microscope imaging, in particular to an imaging system of a stimulated emission loss microscope.
  • the image spot formed by the object point after passing through the imaging system is called a point spread function (PSF).
  • the point spread function describes the ability of the imaging system to resolve object points, and is an important basis for judging the imaging resolution of the optical system. Limited by the diffraction limit, when two object points in the object plane are close to each other, they form two overlapping image spots through the optical system. The distance between the image spots corresponds to the distance between the object points.
  • the image spots are far apart, their point spread functions are separated from each other, and it is easy to be distinguished; when the image spots are close to each other, the two PSFs highly overlap, and Unable to distinguish the respective image spots; when the distance between the image spots is between the two, the PSF partially overlaps, and the image spots at this time are between the distinguishable and unresolvable, at a critical distance, and the image is smaller than this distance Spots will become indistinguishable after superposition.
  • the determination of the critical distance usually adopts the Rayleigh criterion, that is, the center of one image spot and the first dark ring of another image spot are re-cooperated as the distinguishing criterion. At this time, the distance between the center of the two image spots is equal to the radius of the Airy spot, Airy spot The size of the radius is related to the wavelength of the light wave and the numerical aperture of the objective lens.
  • German scientist Stefan W. Hell proposed stimulated emission loss microscopy (Stimulated Emission Depletion, STED), which improved the resolution of the optical microscope by an order of magnitude. This outstanding work won him the Nobel Prize in Chemistry in 2014. In principle, all optical super-resolution techniques revolve around how to obtain a smaller point spread function. Stefan The stimulated emission loss microscopy technology proposed by W.
  • Hell is a purely physical super-resolution technology.
  • a typical STED super-resolution system requires two beams of light: one is excitation light and the other is lossy light.
  • the core idea of STED super-resolution technology is to use stimulated emission to selectively deplete the excited state fluorescent molecules in the edge region of the spot, thereby reducing the luminous range of effective fluorescence and compressing the effective PSF scale to improve the system resolution.
  • the main purpose of the present invention is to provide an imaging system of stimulated emission loss microscope, which can solve the technical problem of using excessively high loss laser in the prior art to improve the imaging resolution, thereby affecting the sample life.
  • the present invention provides an imaging system of a stimulated emission loss microscope, characterized in that the system includes a loss light unit, an excitation light unit, an imaging unit and a counter unit;
  • the lossy light unit is used to generate a ring-shaped loss light with a preset pulse frequency and adjust and control the light intensity of the ring-shaped loss light, and the laser wavefront of the ring-loss light is ring-shaped;
  • the lossy light unit is connected to the excitation light unit, and the excitation light unit is used to generate a Gaussian-type excitation light of the pulse frequency under the trigger of the lossy light unit, and divide the Gaussian-type excitation light into A first Gaussian type excitation light and a second Gaussian type excitation light, the laser wavefront of the Gaussian type excitation light is Gaussian type, and the pulse peak interval between the Gaussian type excitation light and the ring loss light is a preset pulse Interval value
  • the ring loss light and the first Gaussian excitation light are merged and overlapped and input to the imaging unit, and the imaging unit is used to perform area scan imaging on the sample and collect the fluorescent signal reflected by the sample, the fluorescent signal And the second Gaussian-type excitation light respectively enter the counter unit, and the counter unit is used to perform fluorescence intensity imaging and fluorescence lifetime imaging based on the fluorescence signal and the second Gaussian-type excitation light.
  • the invention provides an imaging system of a stimulated emission loss microscope.
  • a loss light unit generates ring loss light and adjusts and controls the intensity of the ring loss light to keep the power value of the ring loss light at a low level.
  • the low-power ring loss light can increase the fluorescence lifetime in the sample, and as the laser irradiation time increases, the image resolution of the sample also gradually increases with the increase in fluorescence lifetime.
  • FIG. 1 is a schematic structural diagram of an imaging system of a stimulated emission loss microscope in an embodiment of the present invention
  • FIG. 2 is a schematic structural diagram of a lossy optical unit 1 in an embodiment of the present invention.
  • FIG. 3 is a schematic structural diagram of an excitation light unit 2 in an embodiment of the present invention.
  • FIG. 4 is a schematic structural diagram of an imaging unit 3 in an embodiment of the present invention.
  • FIG. 5 is a schematic structural diagram of a counter unit 4 in an embodiment of the present invention.
  • FIG. 6 is a schematic structural diagram of Gaussian-type excitation light spot, ring loss light spot, and spatial overlap of the two light spots in an embodiment of the present invention
  • FIG. 7 is a schematic diagram comparing the lifespan of fluorescent beads in a long-time laser scanning area and a first scanning area in a confocal imaging mode according to an embodiment of the present invention
  • FIG. 8 is a schematic diagram comparing the lifespan of fluorescent beads in the laser long-time scanning area and the first scanning area in the STED imaging mode in the embodiment of the present invention.
  • the present invention proposes an imaging system of a stimulated emission loss microscope.
  • the loss optical unit generates ring loss light and adjusts and controls the intensity of the ring loss light to keep the power value of the ring loss light at Lower level.
  • the low-power ring loss light can increase the fluorescence lifetime in the sample, and as the laser irradiation time increases, the image resolution of the sample also gradually increases with the increase in fluorescence lifetime.
  • FIG. 1 is a schematic structural diagram of an imaging system of a stimulated emission loss microscope according to an embodiment of the present invention, where an arrow indicates a laser transmission direction.
  • the system includes a loss light unit 1, an excitation light unit 2, an imaging unit 3, and a counter unit 4;
  • the lossy optical unit 1 is used to generate ring-loss light with a preset pulse frequency and adjust and control the light intensity of the ring-loss light.
  • the laser wavefront of the ring-loss light is ring-shaped;
  • the lossy light unit 1 is connected to the excitation light unit 2.
  • the excitation light unit 2 is used to generate a Gaussian-type excitation light with a pulse frequency under the trigger of the lossy light unit 1, and divide the Gaussian-type excitation light into the first Gaussian-type excitation light and the first Two Gaussian excitation light, the laser wavefront of the Gaussian excitation light is Gaussian, and the pulse peak interval between the Gaussian excitation light and the ring loss light is the preset pulse interval value;
  • the ring loss light merges with the first Gaussian excitation light and enters the imaging unit 3.
  • the imaging unit 3 is used to perform area scan imaging on the sample and collect the fluorescent signal reflected by the sample.
  • the fluorescence signal and the second Gaussian excitation light are incident respectively
  • the counter unit 4 is used to perform fluorescence intensity imaging and fluorescence lifetime imaging based on the fluorescence signal and the second Gaussian-type excitation light.
  • the wavelength of the ring loss light is 760 nm, and the wavelengths of the first Gaussian excitation light, the second Gaussian excitation light, and the Gaussian excitation light are all 635 nm.
  • the pulse peak interval between the Gaussian excitation light and the ring loss light is a preset pulse interval value, and the pulse interval value is maintained in the range of about 200 ps, so as to ensure that the ring loss light more thoroughly generates the Gaussian excitation light
  • the excited state of electrons returns to the ground state in the form of stimulated radiation.
  • the ordinary STED microscope can only collect fluorescence intensity signals
  • the present invention uses a fluorescence lifetime microscope (Fluorescence Lifetime Imaging Microscopy (FLIM), which uses FLIM to describe the spatial distribution of fluorescence lifetime, can be applied to the imaging of fixed cells and live cells to reveal more biological information (such as changes in the microenvironment of the cell).
  • FLIM Fluorescence Lifetime Imaging Microscopy
  • the imaging system of the stimulated emission loss microscope of the present invention combines FLIM technology and STED technology to extract the information of fluorescence intensity and fluorescence lifetime from the fluorescence signal, and then realizes fluorescence lifetime imaging with super-resolution information, that is, STED-FLIM image.
  • the imaging system of the stimulated emission loss microscope in the present invention is a super-resolution imaging system that can collect the information of fluorescence intensity and fluorescence lifetime at the same time, and aims to study the influence of laser intensity and irradiation time on the fluorescence lifetime and imaging resolution, and belongs to STED super-resolution Microscopic imaging system can significantly improve the system spatial resolution.
  • the system further includes a second mirror 5, a third mirror 6, a first dichromatic mirror 7 and a second dichromatic mirror 8;
  • the first Gaussian excitation light is reflected by the second mirror 5, the third mirror 6 and the first dichroic mirror 7 in turn, and the ring loss light is transmitted through the second dichroic mirror 8 and the first dichroic mirror 7 in turn
  • the ring loss light transmitted by a dichroic mirror 7 merges and overlaps with the first Gaussian-type excitation light reflected by the first dichroic mirror 7 and enters the imaging unit 3;
  • the fluorescent signal passes through the first dichromatic mirror transmission 7 and the second dichromatic mirror 8 in order, and then enters the counter unit 4.
  • the second mirror 5 and the third mirror 6 are used to reflect incident light and change the propagation direction of the light beam.
  • the first dichroic mirror 7 is not only used to reflect the first Gaussian excitation light and transmit the ring loss light, but also to fine-tune the transmission direction of the first Gaussian excitation light, so that the first Gaussian excitation light and the ring loss light can be well Confluence overlaps.
  • the second dichroic mirror 8 is used to transmit ring-loss light while reflecting the fluorescent signal.
  • FIG. 2 is a schematic structural diagram of a lossy optical unit 1 according to an embodiment of the present invention, where an arrow indicates a light transmission direction.
  • the lossy optical unit 1 includes a femtosecond laser 11, a first adjustment unit 12, a pulse stretching unit 13, a second adjustment unit 14, and a spiral phase plate 15;
  • the femtosecond laser 1 is used to generate femtosecond pulse-type loss light with a pulse frequency.
  • the femtosecond pulse-type loss light enters the first adjustment unit 12, and the first adjustment unit 12 performs linear polarization adjustment and light intensity adjustment to output the first line.
  • Polarized loss light, the first linearly polarized loss light enters the pulse stretching unit 13, and after the pulse stretching unit 13 performs pulse stretching, a preset pulse width of the extended loss light is output.
  • the broadened loss light enters the second adjustment unit 14 and passes through the second The adjustment unit 14 performs linear polarization adjustment and light intensity adjustment to output second linear polarization loss light, and the second linear polarization loss light passes through the spiral phase plate 15 to output ring loss light;
  • the femtosecond laser 11 is connected to the excitation light unit 2.
  • the femtosecond laser 11 is also used to trigger the excitation light unit 2 to generate Gaussian type excitation light.
  • spiral phase plate 15 (VortexPhase Plate, VPP) is used to convert the wavefront of the lost light from Gaussian to circular.
  • the first adjustment unit 12 includes a first half-wave plate 121 and a first Glan prism 122;
  • the femtosecond pulsed loss light enters the first half-wave plate 121, is adjusted to the linearly polarized light of the preset first polarization direction by the first half-wave plate 121, enters the first Glan prism 122, and passes through the first Glan prism 122 Adjust the light intensity to output the first linear polarization loss light.
  • the first half-wave plate 121 is used to adjust the femtosecond pulsed loss light to linearly polarized light and adjust its polarization direction, and cooperates with the first Glan prism 122 to control the intensity of the laser light.
  • the pulse stretching unit 13 includes a glass rod 131, a first lens 132, a single-mode polarization maintaining fiber 133 and a second lens 134;
  • the first linear polarization loss light is incident on the glass rod 131, pulse-stretched through the glass rod 131, and then incident on the first lens 132, focused on the first lens 132, and then incident on the single-mode polarization-maintaining fiber 133, and through the single-mode polarization-maintaining fiber 133 After the pulse is broadened again, it is incident on the second lens 134, and after being focused by the second lens 134, the broadened loss light is output.
  • the glass rod 131 (Glass Rod, GR) is used to broaden the pulse of the first linear polarization loss light to about 1 picosecond
  • the single-mode polarization maintaining fiber 133 (Polarization Maintaining Fiber (PMF) is a 100-meter single-mode polarization-maintaining fiber, which is used to further widen the loss of light and make its pulse width reach about 200 picoseconds.
  • the second adjustment unit 14 includes a second half-wave plate 141 and a second Glan prism 142;
  • the broadened loss light enters the second half-wave plate 141, is linearly polarized by the second half-wave plate 141 adjusted to a preset second polarization direction, and enters the second Glan prism 142, and the light is adjusted by the second Glan prism 142 After strong, output the second linear polarization loss light.
  • the second half-wave plate 141 is used to adjust the broadened loss light to linearly polarized light and adjust its polarization direction, and cooperates with the second Glan prism 142 to control the intensity of the laser light.
  • the femtosecond pulsed lossy light is generated by the femtosecond laser 1, and first passes through the first half-wave plate 121 and the first Glan prism 122 to adjust the linear polarization direction of the lost light and the laser intensity to ensure the entry into the optical path system
  • the lost light is linearly polarized light.
  • the glass rod 131 is used to broaden the femtosecond loss pulse light, and the pulse width after the broadening is about 1 picosecond.
  • the light beam After being focused by the first lens 132, the light beam is coupled into a 100-meter single-mode polarization-maintaining fiber, further widening the pulse width of the lost light to about 200 picoseconds.
  • the outgoing beam is expanded by the second lens 134 and enters the second half-wave plate 141 and the second Glan prism 142, and the polarization characteristic and intensity of the lost light are adjusted again. After the light beam passes through the spiral phase plate 15, the wavefront changes from Gaussian to circular.
  • FIG. 3 is a schematic structural diagram of an excitation light unit 2 according to an embodiment of the present invention, where an arrow indicates a light transmission direction.
  • the excitation light unit 2 includes a picosecond laser 21, a single-mode fiber 22, a third lens 23, and an adjustment control unit 24;
  • the picosecond laser 21 is connected to the lossy optical unit 1.
  • the picosecond laser 21 is used to generate a picosecond pulsed excitation light with a pulse frequency under the trigger of the lossy optical unit 1.
  • the picosecond pulsed excitation light enters the single-mode fiber 22 and passes through
  • the single-mode optical fiber 22 performs mode adjustment and then enters the third lens 23, after being enlarged by the third lens 23, enters the adjustment control unit 24, and performs linear polarization adjustment, pulse interval adjustment, and light intensity adjustment through the adjustment control unit 24 to output laser light
  • the wavefront is Gaussian-type excitation light.
  • the single mode fiber 22 (Single Mode Fiber, SMF) is mainly used for mode control of the excitation light emitted from the picosecond laser 21.
  • the adjustment control unit 24 includes a third half-wave plate 241, a corner reflector 242, and a third Glan prism 243;
  • the laser light amplified by the third lens 23 enters the third half-wave plate 241, and is adjusted to the linearly polarized light of the preset third polarization direction by the third half-wave plate 241, and then passes through the corner reflector 242.
  • the corner reflector 242 is used to The pulse interval between the Gaussian excitation light and the ring-loss light is adjusted.
  • the laser light whose pulse interval is adjusted by the angle reflector 242 is incident on the third Glan prism 243, and after the third Glan prism 243 adjusts the light intensity, it is output separately The first Gaussian type excitation light and the second Gaussian type excitation light.
  • the angle reflector 242 can change the optical path of the excitation light, and is used to control the pulse interval between the excitation light and the loss light in time.
  • changing the optical path of the excitation light is usually extended The optical path of the excitation light or shorten the optical path of the excitation light.
  • the third half-wave plate 241 is used to adjust the spread loss light to linearly polarized light and adjust its polarization direction.
  • the third Granite prism 243 divides the Gaussian excitation light into two paths, which are the first Gaussian excitation light and the second Gaussian excitation light, respectively.
  • the excitation light is generated by the picosecond laser 21 and is triggered by the depleted laser signal to emit laser light of the same repetition frequency. Coupling through a single-mode fiber 22, magnified by the third lens 23 and sequentially incident on the third half-wave plate 241, the corner reflector 242 and the third Glan prism 243 to adjust the polarization characteristics and intensity of the excitation light to ensure the excitation entering the optical path system
  • the light is linearly polarized light.
  • the excitation light passes through the corner reflector 242 to change the optical path of the optical path, thereby controlling the pulse interval between the excitation light and the loss light.
  • FIG. 4 is a schematic structural diagram of an imaging unit 3 according to an embodiment of the present invention, where an arrow indicates a light transmission direction.
  • the imaging unit 3 includes a fourth lens 31, a galvanometer scanning system 32, a fifth lens 33, a quarter glass slide 34, a high numerical aperture objective 35, and a stage 36 on which a sample is placed;
  • the ring-loss light and the first Gaussian excitation light merge and overlap, pass through the fourth lens 31, the galvanometer scanning system 32, and the fifth lens 33 in sequence, and then enter the quarter glass 34, and then pass the quarter glass 34 After adjusting the first Gaussian excitation light and the ring loss light after being merged and overlapped from linearly polarized light to circularly polarized light, it is incident on the stage 36 after passing through the high numerical aperture objective lens 35;
  • the high numerical aperture objective lens 35 is used to collect the fluorescent signal reflected by the sample.
  • the fluorescent signal passes through the quarter glass 34, the fifth lens 33, the galvanometer scanning system 32, and the fourth lens 31, and then enters the counter unit 5;
  • the galvanometer scanning system 32 is used to synchronously scan the first Gaussian-type excitation light and the ring-loss light after the convergence and overlap to realize the area scan imaging of the sample.
  • the high numerical aperture objective lens 35 has a magnification of 100 times and a numerical aperture of 1.4, and is used to focus the overlapped first Gaussian-type excitation light and ring loss light, and collect the fluorescent signal reflected back from the sample.
  • Quarter-Wave Plate 34 (Quarter-Wave Plate, QWP) is used to convert ring-loss light from linearly polarized light to circularly polarized light to ensure that the ring-loss light has better spot quality.
  • the ring-loss light is enlarged by the fourth lens 31 and the fifth lens 33 to the spot diameter equal to or slightly larger than the aperture of the high-power numerical aperture objective lens 35, and the incident parallel light is focused on the sample of the stage 36.
  • FIG. 5 is a schematic structural diagram of a counter unit 4 according to an embodiment of the present invention, where an arrow indicates a light transmission direction.
  • the counter unit 4 includes a sixth lens 41, a filter 42, a multimode optical fiber 43, a photomultiplier tube 44, a first mirror 45, a seventh lens 46, a detector 47, and a time-dependent single photon counter 48;
  • the fluorescent signal After the fluorescent signal is transmitted through the sixth lens 41 and the stray light and the multimode optical fiber 43 are filtered by the filter 42, it enters the photomultiplier tube 44 and the photomultiplier tube 44 amplifies the fluorescent signal and enters the time-related single photon counter 48;
  • the second Gaussian excitation light After the second Gaussian excitation light is reflected by the first mirror 45 and transmitted by the seventh lens 46, it enters the detector 47, is converted into an electrical signal by the detector 47, and is input into the time-dependent single photon counter 48;
  • the time-dependent single photon counter 48 is used for fluorescence intensity imaging and fluorescence lifetime imaging based on the second Gaussian excitation light and fluorescence signal.
  • the filter 42 is used to transmit the fluorescence signal in the band to be collected and filter the stray light outside this band.
  • the multimode fiber 43 is used to transmit the collected fluorescence signal to the photomultiplier tube 44.
  • the fiber core of the optical fiber 43 can be used as a small hole to receive the fluorescent signal focused by the sixth lens 41 and exclude the influence of stray light.
  • the detector 47 is used to detect the second Gaussian-type excitation light pulse as a reference signal for measuring the fluorescence lifetime of the sample.
  • the time-correlated single photon counter 48 can divide the fluorescence signal collected by the photomultiplier tube 44 into two channels, one for fluorescence intensity Imaging, the other way is used for fluorescence lifetime imaging.
  • FIG. 6 is a schematic structural diagram of a Gaussian-type excitation light spot, a ring loss light spot, and a spatial overlap of the two spots in the embodiment of the present invention.
  • a represents a Gaussian-type excitation light spot
  • b represents a ring-loss light spot
  • c represents a spatial overlap diagram of the Gaussian-type excitation light spot and the ring-loss light spot.
  • FIG. 7 is a schematic diagram of the lifetime comparison of the fluorescent beads in the laser long-time scanning area and the first scanning area in the confocal imaging mode in the embodiment of the present invention.
  • the dotted area in the center is the imaging area irradiated by the laser for a long time
  • the rest is the imaging area for the first scan.
  • t represents the average life of the entire area
  • t1 represents the life of the peripheral area
  • t2 represents the life of the central area.
  • the fluorescence lifetime of the central area is higher than that of the peripheral area, indicating that the lifetime of the fluorescent beads changes to a long lifetime under long-term laser irradiation.
  • photobleaching will cause a decrease in fluorescence intensity after prolonged irradiation of the sample, the excitation light intensity is only in the order of microwatts, so the intensity change is not obvious.
  • FIG. 8 is a schematic diagram comparing the lifespan of the fluorescent beads in the laser long-time scanning area and the first scanning area in the STED imaging mode in the embodiment of the present invention.
  • the center dotted line area is the imaging area that has been irradiated by the laser for a long time, and the rest is the imaging area for the first scan.
  • T represents the average life of the entire area
  • T1 represents the life of the peripheral area
  • T2 represents the life of the central area.
  • long-term laser irradiation will make the life of the fluorescent beads in the scanning area change more obviously.
  • the fluorescent beads in the central area show a darker color, and their lifespan is much longer than that of the peripheral area. Due to the high laser energy loss, prolonged laser irradiation increases the photobleaching rate of the fluorophore, which in turn reduces the number of autofluorescent photons, so the fluorescence intensity of the central scanning area and the peripheral scanning area are different.
  • the lossy light unit 1 in the imaging system generates ring-loss light and adjusts the intensity of the ring-loss light to keep the power value of the ring-loss light at a low level.
  • the fluorescence lifetime of the sample gradually increases with the increase of the laser irradiation time.
  • the increased fluorescence lifetime reduces the saturation intensity of the fluorescent sample, and the image resolution of the sample also increases with the fluorescence lifetime Gradually increase.

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Abstract

一种受激发射损耗显微镜的成像系统，包括损耗光单元(1)、激发光单元(2)、成像单元(3)与计数器单元(4)，损耗光单元(1)产生环形损耗光并控制环形损耗光的光强和偏振，激发光单元(2)被损耗光单元(1)触发后发出高斯型激发光，高斯型激发光经过第三格兰棱镜(243)分为第一高斯型激发光与第二高斯型激发光，环形损耗光与第一高斯型激发光重叠后输入成像单元(3)，成像单元(3)对样品进行扫描成像并收集荧光信号。第二高斯型激发光作为参考信号，与荧光信号一并进入计数器单元(4)，用于荧光强度成像和荧光寿命成像。当施加较低的环形损耗光能量时，样品的荧光寿命随着激光照射时间而增加，增加的荧光寿命降低了荧光样品的饱和强度，进而可以实现成像分辨率的进一步提高。

Description

一种受激发射损耗显微镜的成像系统 技术领域
本发明涉及光学显微镜成像领域，尤其涉及一种受激发射损耗显微镜的成像系统。
 背景技术
在光学系统中，物点经成像系统后形成的像斑称为点扩展函数(Point Spread Function, PSF)。点扩展函数描述了成像系统对物点的解析能力，是判断光学系统成像分辨率高低的重要依据。受衍射极限的限制，当物平面中两个物点距离较近时，它们经过光学系统形成两个重叠的像斑。像斑之间的距离与物点间的距离相对应，当像斑相距较远时，其点扩展函数相互分离，则容易被分辨；当像斑相距很近时，两个PSF高度重叠，进而无法区分各自的像斑；而当像斑间的距离介于两者之间时，PSF部分重叠，此时的像斑处于可分辨和不可分辨之间，处于一个临界距离，小于该距离时像斑叠加后将变得无法区分。该临界距离的确定通常采用瑞利判据，即以一个像斑的中心与另一个像斑的第一暗环重合作为区分标准，此时两像斑中心距离等于艾里斑半径，艾里斑半径的大小与光波波长和物镜的数值孔径有关。
19世纪末，德国物理学家Ernst K. Abbe根据几何光学中的正弦条件，确定了光学显微镜的分辨率极限在可见光波长的一半左右。这意味着通过传统的光学显微镜可以分辨出单个细胞，以及细胞内的细胞器成分，却无法分辨尺寸更小的物体，比如更小尺寸的病毒、单个的蛋白质或者其他小分子物质。1994年，德国科学家Stefan W.Hell提出了受激发射损耗显微技术(Stimulated Emission Depletion, STED)，使光学显微镜的分辨率提高了一个数量级，这一杰出的工作使他在2014年获得了诺贝尔化学奖。原则上说，所有的光学超分辨技术都是围绕着如何得到一个尺寸更小的点扩展函数而展开的。Stefan W.Hell提出的受激发射损耗显微技术是一个纯物理的超分辨技术。一个典型的STED超分辨系统中需要两束光：一束为激发光，另一束为损耗光。STED超分辨技术的核心思想是利用受激发射选择性损耗激发光斑中边沿区域的激发态荧光分子，从而减少有效荧光的发光范围，压缩有效PSF尺度来提高系统分辨率。
普通的STED显微镜只能采集荧光强度信号，而忽略了荧光分子的其他信息。在STED显微镜技术中，为了提高成像分辨率，需要对样品施加极高的激光功率（>100 mW），而过高的损耗激光功率会损伤样品，并使荧光分子发生光漂白效应，因此限制了其在活细胞成像等方面的应用，是STED显微镜最大的缺点之一。。
 技术问题
本发明的主要目的在于提供一种受激发射损耗显微镜的成像系统，可以解决现有技术中为了提高成像分辨率而使用过高的损耗激光，从而影响样品寿命的技术问题。
 技术解决方案
为实现上述目的，本发明提供一种受激发射损耗显微镜的成像系统，其特征在于，所述系统包括损耗光单元、激发光单元、成像单元与计数器单元；
所述损耗光单元用于产生预设脉冲频率的环形损耗光并调节控制所述环形损耗光的光强，所述环形损耗光的激光波前为环形；
所述损耗光单元与所述激发光单元连接，所述激发光单元用于在所述损耗光单元的触发下产生所述脉冲频率的高斯型激发光，并将所述高斯型激发光分为第一高斯型激发光与第二高斯型激发光，所述高斯型激发光的激光波前为高斯型，所述高斯型激发光与所述环形损耗光之间的脉冲峰值间隔为预设脉冲间隔值；
所述环形损耗光与所述第一高斯型激发光汇合重叠后输入所述成像单元，所述成像单元用于对样品进行面阵扫描成像并收集所述样品反射的荧光信号，所述荧光信号和所述第二高斯型激发光分别射入所述计数器单元，所述计数器单元用于基于所述荧光信号和所述第二高斯型激发光进行荧光强度成像和荧光寿命成像。
 有益效果
本发明提供一种受激发射损耗显微镜的成像系统，该成像系统中损耗光单元产生环形损耗光并调节控制环形损耗光的光强，使环形损耗光的功率值保持在较低的水平。低功率的环形损耗光能增加样品中的荧光寿命，且随着激光照射时间的增加，样品的图像分辨率也随着荧光寿命的增加而逐渐提高。
 附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域技术人员来讲，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为本发明实施例中一种受激发射损耗显微镜的成像系统的结构示意图；
图2为本发明实施例中损耗光单元1的结构示意图；
图3为本发明实施例中激发光单元2的结构示意图；
图4为本发明实施例中成像单元3的结构示意图；
图5为本发明实施例中计数器单元4的结构示意图；
图6为本发明实施例中高斯型激发光光斑、环形损耗光光斑及二者光斑空间重叠的结构示意图；
图7为本发明实施例中共聚焦成像模式下荧光珠在激光长时间扫描区域和首次扫描区域的寿命对比示意图；
图8为本发明实施例中STED成像模式下荧光珠在激光长时间扫描区域和首次扫描区域的寿命对比示意图。
 本发明的实施方式
为使得本发明的发明目的、特征、优点能够更加的明显和易懂，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而非全部实施例。基于本发明中的实施例，本领域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，都属于本发明保护的范围。
由于现有技术中存在为了提高成像分辨率而使用过高的损耗激光，从而影响样品寿命的技术问题。
为了解决上述技术问题，本发明提出一种受激发射损耗显微镜的成像系统，该成像系统中损耗光单元产生环形损耗光并调节控制环形损耗光的光强，使环形损耗光的功率值保持在较低的水平。低功率的环形损耗光能增加样品中的荧光寿命，且随着激光照射时间的增加，样品的图像分辨率也随着荧光寿命的增加而逐渐提高。
请参阅图1，为本发明实施例中一种受激发射损耗显微镜的成像系统的结构示意图，其中，箭头表示激光的传输方向。
该系统包括损耗光单元1、激发光单元2、成像单元3与计数器单元4；
损耗光单元1用于产生预设脉冲频率的环形损耗光并调节控制环形损耗光的光强，环形损耗光的激光波前为环形；
损耗光单元1与激发光单元2连接，激发光单元2用于在损耗光单元1的触发下产生脉冲频率的高斯型激发光，并将高斯型激发光分为第一高斯型激发光与第二高斯型激发光，高斯型激发光的激光波前为高斯型，高斯型激发光与环形损耗光之间的脉冲峰值间隔为预设脉冲间隔值；
环形损耗光与第一高斯型激发光汇合重叠后输入成像单元3，成像单元3用于对样品进行面阵扫描成像并收集样品反射的荧光信号，荧光信号和第二高斯型激发光分别射入计数器单元4，计数器单元4用于基于荧光信号和第二高斯型激发光进行荧光强度成像和荧光寿命成像。
优选的，环形损耗光的波长为760纳米，第一高斯型激发光、第二高斯型激发光及高斯型激发光的波长均为635纳米。
优选的，高斯型激发光与环形损耗光之间的脉冲峰值间隔为预设脉冲间隔值，该脉冲间隔值保持在200ps左右的范围，这样才能保证环形损耗光更彻底地将高斯型激发光产生的激发态电子以受激辐射的形式返回到基态。
需要说明的是，普通的STED显微镜只能采集荧光强度信号，本发明采用荧光寿命显微镜(Fluorescence Lifetime Imaging Microscopy, FLIM)，用FLIM来描述荧光寿命空间分布，可以应用于固定细胞和活细胞的成像中，来揭示更多的生物信息（如细胞内微环境的变化）。本发明的受激发射损耗显微镜的成像系统是结合FLIM技术与STED技术从荧光信号中提取荧光强度和荧光寿命的信息，进而实现具有超分辨信息的荧光寿命成像，即STED-FLIM图像。本发明中的受激发射损耗显微镜的成像系统是可同时收集荧光强度和荧光寿命信息的超分辨成像系统，旨在研究激光强度和照射时间对荧光寿命和成像分辨率的影响，属于STED超分辨显微成像系统，能显著提高系统空间分辨率。
进一步的，该系统还包括第二反射镜5、第三反射镜6、第一双色镜7和第二双色镜8；
第一高斯型激发光依次经过第二反射镜5反射、第三反射镜6反射及第一双色镜7反射，环形损耗光依次经过第二双色镜8透射和第一双色镜7透射，经过第一双色镜7透射的环形损耗光与经过第一双色镜7反射的所第一高斯型激发光汇合重叠后射入成像单元3；
荧光信号依次经过第一双色镜透射7和第二双色镜8反射后，射入计数器单元4。
需要说明的是，第二反射镜5和第三反射镜6用于反射入射光，改变光束的传播方向。第一双色镜7不仅用于反射第一高斯型激发光，透射环形损耗光，还用于微调第一高斯型激发光的传输方向，使第一高斯型激发光和环形损耗光能够很好的汇合重叠。第二双色镜8用于透射环形损耗光，同时反射荧光信号。
进一步的，请参阅图2，为本发明实施例中损耗光单元1的结构示意图，其中，箭头表示光的传输方向。损耗光单元1包括飞秒激光器11、第一调节单元12、脉冲展宽单元13、第二调节单元14和螺旋相位板15；
飞秒激光器1用于产生脉冲频率的飞秒脉冲型损耗光，飞秒脉冲型损耗光射入第一调节单元12，经第一调节单元12进行线偏振调节和光强调节后输出第一线偏振损耗光，第一线偏振损耗光射入脉冲展宽单元13，经脉冲展宽单元13进行脉冲展宽后输出预设脉冲宽度的展宽损耗光，展宽损耗光射入第二调节单元14，经第二调节单元14进行线偏振调节和光强调节后输出第二线偏振损耗光，第二线偏振损耗光经过螺旋相位板15后输出环形损耗光；
飞秒激光器11与激发光单元2连接，飞秒激光器11还用于触发激发光单元2产生高斯型激发光。
需要说明的是，螺旋相位板15（VortexPhase Plate，VPP）用于将损耗光的波前由高斯型转换成环形。
进一步的，第一调节单元12包括第一半波片121和第一格兰棱镜122；
飞秒脉冲型损耗光射入第一半波片121，经第一半波片121调节为预设第一偏振方向的线偏振光后射入第一格兰棱镜122，经第一格兰棱镜122调节光强后输出第一线偏振损耗光。
需要说明的是，第一半波片121用于将飞秒脉冲型损耗光调节为线偏振光并调整其偏振方向，搭配第一格兰棱镜122，控制激光的强度。
进一步的，脉冲展宽单元13包括玻璃棒131、第一透镜132、单模保偏光纤133和第二透镜134；
第一线偏振损耗光射入玻璃棒131，经玻璃棒131进行脉冲展宽后射入第一透镜132，经第一透镜132聚焦后射入单模保偏光纤133，经单模保偏光纤133进行脉冲再次展宽后射入第二透镜134，经第二透镜134聚焦后输出展宽损耗光。
需要说明的是，玻璃棒131（Glass Rod，GR）用于将第一线偏振损耗光的脉冲展宽至约1皮秒，单模保偏光纤133（Polarization Maintaining Fiber ，PMF）为100米单模保偏光纤，用于进一步展宽损耗光，使其脉冲宽度达到200皮秒左右。
进一步的，第二调节单元14包括第二半波片141和第二格兰棱镜142；
展宽损耗光射入第二半波片141，经第二半波片141调节为预设第二偏振方向的线偏振光后射入第二格兰棱镜142，经第二格兰棱镜142调节光强后输出第二线偏振损耗光。
需要说明的是，第二半波片141用于将展宽损耗光调节为线偏振光并调整其偏振方向，搭配第二格兰棱镜142，控制激光的强度。
优选的，飞秒脉冲型损耗光由飞秒激光器1产生，首先通过第一半波片121和第一格兰棱镜122，用来调节损耗光的线偏振方向和激光强度，确保进入光路系统的损耗光为线偏振光。玻璃棒131用于将飞秒级的损耗脉冲光进行展宽，展宽后的脉冲宽度约为1皮秒。光束经第一透镜132聚焦后耦合进入100米单模保偏光纤，将损耗光的脉冲宽度进一步展宽至约200皮秒。出射光束经第二透镜134扩束后进入第二半波片141和第二格兰棱镜142，再一次对损耗光的偏振特性和强度进行调节。光束经过螺旋相位板15之后，波前由高斯型转变为环形。
进一步的，请参阅图3，为本发明实施例中激发光单元2的结构示意图，其中，箭头表示光的传输方向。
激发光单元2包括皮秒激光器21、单模光纤22、第三透镜23和调节控制单元24；
皮秒激光器21与损耗光单元1连接，皮秒激光器21用于在损耗光单元1的触发下产生脉冲频率的皮秒脉冲型激发光，皮秒脉冲型激发光射入单模光纤22，经单模光纤22进行模式调控后射入第三透镜23，经第三透镜23进行放大后射入调节控制单元24，经调节控制单元24进行线偏振调节、脉冲间隔调节和光强调节后输出激光波前为高斯型的高斯型激发光。
需要说明的是，单模光纤22(Single Mode Fiber，SMF)主要用于对皮秒激光器21中出射的激发光进行模式调控。
进一步的，调节控制单元24包括第三半波片241、角反射器242和第三格兰棱镜243；
经第三透镜23放大后的激光射入第三半波片241，经第三半波片241调节为预设第三偏振方向的线偏振光后经过角反射器242，角反射器242用于调节高斯型激发光与环形损耗光之间的脉冲间隔，经角反射器242进行脉冲间隔调节后的激光射入第三格兰棱镜243，经第三格兰棱镜243调节光强后，分别输出第一高斯型激发光和第二高斯型激发光。
需要说明的是，角反射器242(Retro Reflector，RR)可改变激发光的光路，用于在时间上控制激发光和损耗光之间的脉冲间隔，一般的，改变激发光的光路通常为延长激发光的光路或者缩短激发光的光路，同时，第三半波片241用于将展宽损耗光调节为线偏振光并调整其偏振方向，搭配第三格兰棱镜243，控制激光的强度，同时，第三格兰棱镜243将高斯型激发光分成两路，分别为第一高斯型激发光和第二高斯型激发光。
激发光由皮秒激光器21产生，被损耗激光信号触发后发出相同重复频率的激光。通过单模光纤22耦合，由第三透镜23放大并依次入射第三半波片241、角反射器242和第三格兰棱镜243，调节激发光的偏振特性和强度，确保进入光路系统的激发光为线偏振光。激发光通过角反射器242来改变光路的光程，从而控制激发光和损耗光之间的脉冲间隔。
进一步的，请参阅图4，为本发明实施例中成像单元3的结构示意图，其中，箭头表示光的传输方向。
成像单元3包括第四透镜31、振镜扫描系统32、第五透镜33、四分之一玻片34、高数值孔径物镜35和载物台36，载物台36上放置样品；
环形损耗光与第一高斯型激发光汇合重叠后依次经过第四透镜31、振镜扫描系统32和第五透镜33后，射入四分之一玻片34，经四分之一玻片34将汇合重叠后的第一高斯型激发光和环形损耗光由线偏振光调节为圆偏振光后，经过高数值孔径物镜35后入射至载物台36；
高数值孔径物镜35用于收集样品反射的荧光信号，荧光信号依次经过四分之一玻片34、第五透镜33、振镜扫描系统32、第四透镜31后射入计数器单元5；
振镜扫描系统32用于对汇合重叠后的第一高斯型激发光和环形损耗光进行同步扫描，实现对样品的面阵扫描成像。
需要说明的是，高数值孔径物镜35的放大倍率为100倍，数值孔径为1.4，用于聚焦重叠的第一高斯型激发光和环形损耗光，同时收集样品反射回来的荧光信号。四分之一玻片34（Quarter-Wave Plate，QWP）用于将环形损耗光由线偏振光转化成圆偏振光，以确保环形损耗光具有更好的光斑质量。其中，环形损耗光经第四透镜31和第五透镜33将光斑直径放大至等于或略大于高倍数值孔径物镜35的孔径，并将入射的平行光聚焦到载物台36的样品上。
进一步的，请参阅图5，为本发明实施例中计数器单元4的结构示意图，其中，箭头表示光的传输方向。
计数器单元4包括第六透镜41、滤镜42、多模光纤43、光电倍增管44、第一反射镜45、第七透镜46、探测器47和时间相关单光子计数器48；
荧光信号依次经过第六透镜41透射、滤镜42滤除杂散光及多模光纤43后，射入光电倍增管44，光电倍增管44将荧光信号放大后输入时间相关单光子计数器48；
第二高斯型激发光经过第一反射镜45反射和第七透镜46透射后，射入探测器47，经探测器47转化成电信号后输入时间相关单光子计数器48；
时间相关单光子计数器48用于基于第二高斯型激发光和荧光信号进行荧光强度成像和荧光寿命成像。
需要说明的是，滤镜42用于透过所要收集波段的荧光信号，并过滤此波段以外的杂散光，多模光纤43，用于将收集到的荧光信号传输给光电倍增管44，多模光纤43的光纤纤芯可作为小孔，接收第六透镜41聚焦的荧光信号，并排除杂散光的影响。探测器47用于探测第二高斯型激发光脉冲，作为测量样品荧光寿命的参考信号，时间相关单光子计数器48能将光电倍增管44收集到的荧光信号分为两路，一路用于荧光强度成像，另一路用于荧光寿命成像。
为更好的理解本发明，请参阅图6，为本发明实施例中高斯型激发光光斑、环形损耗光光斑及二者光斑空间重叠的结构示意图。其中图6中a表示高斯型激发光光斑，b表示环形损耗光光斑，c表示高斯型激发光光斑和环形损耗光光斑的空间重叠示意图。
进一步的，请参照图7，为本发明实施例中共聚焦成像模式下荧光珠在激光长时间扫描区域和首次扫描区域的寿命对比示意图。在共聚焦成像模式下形成的Confocal-FLIM图像中，中心虚线区域为经激光长时间照射的成像区域，而其余部分为首次扫描成像区域。t表示整个区域的平均寿命，t1表示外围区域的寿命，t2表示中心区域的寿命。在寿命图像中，中心区域的荧光寿命高于外围区域的荧光寿命，说明了荧光珠的寿命在长时间的激光照射下向长寿命变化。虽然对样品进行长时间照射后光漂白会导致荧光强度的降低，但是激发光强度只有微瓦量级，因此强度的变化并不明显。
进一步的，请参照图8，为本发明实施例中STED成像模式下荧光珠在激光长时间扫描区域和首次扫描区域的寿命对比示意图。在STED成像模式下形成的STED-FLIM图像中，中心虚线区域为经激光长时间照射的成像区域，而其余部分为首次扫描成像区域。T表示整个区域的平均寿命，T1表示外围区域的寿命，T2表示中心区域的寿命。在STED成像模式下，长时间的激光照射会使扫描区域内荧光珠的寿命发生更加明显的变化，中心区域荧光珠呈现出的颜色更深，其寿命远高于外围区域的荧光寿命。由于损耗激光能量较高，长时间激光照射增加了荧光团的光漂白率，进而降低了自发荧光的光子数，因此中心扫描区域与外围扫描区域的荧光强度不同。
STED超分辨成像技术需要用到损耗光与激发光，其分辨率随着损耗光能量的增加而提高，由公式表示为：R=λ/[2NA(1+I STED/I S) 1/2]，其中I S=hc/λτσ，λ为光波波长，NA为物镜的数值孔径，I STED为损耗激光的能量，I S为样品的饱和能量，由所用样品的荧光寿命(τ)和损耗光的吸收截面(σ)共同决定。因此，在固定的损耗激光能量下，荧光寿命的增加会导致饱和功率的降低，这也意味着可以在较低激光能量下实现分辨率的提升。在Confocal图像中，由于衍射极限的限制，许多荧光珠相距很近而无法被区分。当施加较低功率的损耗激光后，图像分辨率相比共聚焦图像略有提高。在STED成像模式下增加激光的照射时间，图像分辨率随着荧光寿命的增加而逐渐提高。因此在本系统中，能利用增加荧光寿命的方法在低功率下实现STED成像分辨率的进一步提升。
在本发明实施例中，该成像系统中损耗光单元1产生环形损耗光并调节控制环形损耗光的光强，使环形损耗光的功率值保持在较低的水平。当环形损耗光能量较低时，样品的荧光寿命随着激光照射时间的增加而逐渐增加，增加的荧光寿命降低了荧光样品的饱和强度，进而样品的图像分辨率也随着荧光寿命的增加而逐渐提高。
 序列表自由内容
在上述实施例中，对各个实施例的描述都各有侧重，某个实施例中没有详述的部分，可以参见其它实施例的相关描述。
以上为对本发明所提供的一种受激发射损耗显微镜的成像系统的描述，对于本领域的技术人员，依据本发明实施例的思想，在具体实施方式及应用范围上均会有改变之处，综上，本说明书内容不应理解为对本发明的限制。

Claims (10)

  1. 一种受激发射损耗显微镜的成像系统，其特征在于，所述系统包括损耗光单元、激发光单元、成像单元与计数器单元；
    所述损耗光单元用于产生预设脉冲频率的环形损耗光并调节控制所述环形损耗光的光强，所述环形损耗光的激光波前为环形；
    所述损耗光单元与所述激发光单元连接，所述激发光单元用于在所述损耗光单元的触发下产生所述脉冲频率的高斯型激发光，并将所述高斯型激发光分为第一高斯型激发光与第二高斯型激发光，所述高斯型激发光的激光波前为高斯型，所述高斯型激发光与所述环形损耗光之间的脉冲峰值间隔为预设脉冲间隔值；
    所述环形损耗光与所述第一高斯型激发光汇合重叠后输入所述成像单元，所述成像单元用于对样品进行面阵扫描成像并收集所述样品反射的荧光信号，所述荧光信号和所述第二高斯型激发光分别射入所述计数器单元，所述计数器单元用于基于所述荧光信号和所述第二高斯型激发光进行荧光强度成像和荧光寿命成像。
  2. 根据权利要求1所述的成像系统，其特征在于，所述损耗光单元包括飞秒激光器、第一调节单元、脉冲展宽单元、第二调节单元和螺旋相位板；
    所述飞秒激光器用于产生所述脉冲频率的飞秒脉冲型损耗光，所述飞秒脉冲型损耗光射入所述第一调节单元，经所述第一调节单元进行线偏振调节和光强调节后输出第一线偏振损耗光，所述第一线偏振损耗光射入所述脉冲展宽单元，经所述脉冲展宽单元进行脉冲展宽后输出预设脉冲宽度的展宽损耗光，所述展宽损耗光射入所述第二调节单元，经所述第二调节单元进行线偏振调节和光强调节后输出第二线偏振损耗光，所述第二线偏振损耗光经过所述螺旋相位板后输出所述环形损耗光；
    所述飞秒激光器与所述激发光单元连接，所述飞秒激光器还用于触发所述激发光单元产生所述高斯型激发光。
  3. 根据权利要求2所述的成像系统，其特征在于，所述第一调节单元包括第一半波片和第一格兰棱镜；
    所述飞秒脉冲型损耗光射入所述第一半波片，经所述第一半波片调节为预设第一偏振方向的线偏振光后射入所述第一格兰棱镜，经所述第一格兰棱镜调节光强后输出所述第一线偏振损耗光。
  4. 根据权利要求2所述的成像系统，其特征在于，所述脉冲展宽单元包括玻璃棒、第一透镜、单模保偏光纤和第二透镜；
    所述第一线偏振损耗光射入所述玻璃棒，经所述玻璃棒进行脉冲展宽后射入所述第一透镜，经所述第一透镜聚焦后射入所述单模保偏光纤，经所述单模保偏光纤进行脉冲再次展宽后射入所述第二透镜，经所述第二透镜聚焦后输出所述展宽损耗光。
  5. 根据权利要求2所述的成像系统，其特征在于，所述第二调节单元包括第二半波片和第二格兰棱镜；
    所述展宽损耗光射入所述第二半波片，经所述第二半波片调节为预设第二偏振方向的线偏振光后射入所述第二格兰棱镜，经所述第二格兰棱镜调节光强后输出所述第二线偏振损耗光。
  6. 根据权利要求1所述的成像系统，其特征在于，所述激发光单元包括皮秒激光器、单模光纤、第三透镜和调节控制单元；
    所述皮秒激光器与所述损耗光单元连接，所述皮秒激光器用于在所述损耗光单元的触发下产生所述脉冲频率的皮秒脉冲型激发光，所述皮秒脉冲型激发光射入所述单模光纤，经所述单模光纤进行模式调控后射入所述第三透镜，经所述第三透镜进行放大后射入所述调节控制单元，经所述调节控制单元进行线偏振调节、脉冲间隔调节和光强调节后输出激光波前为高斯型的高斯型激发光。
  7. 根据权利要求6所述的成像系统，其特征在于，所述调节控制单元包括第三半波片、角反射器和第三格兰棱镜；
    经所述第三透镜放大后的激光射入所述第三半波片，经所述第三半波片调节为预设第三偏振方向的线偏振光后经过所述角反射器，所述角反射器用于调节所述高斯型激发光与所述环形损耗光之间的脉冲间隔，经所述角反射器进行脉冲间隔调节后的激光射入所述第三格兰棱镜，经所述第三格兰棱镜调节光强后，分别输出所述第一高斯型激发光和所述第二高斯型激发光。
  8. 根据权利要求1所述的成像系统，其特征在于，所述成像单元包括第四透镜、振镜扫描系统、第五透镜、四分之一玻片、高数值孔径物镜和载物台，所述载物台上放置所述样品；
    所述环形损耗光与所述第一高斯型激发光汇合重叠后依次经过所述第四透镜、所述振镜扫描系统和所述第五透镜后，射入所述四分之一玻片，经所述四分之一玻片将汇合重叠后的所述第一高斯型激发光和所述环形损耗光由线偏振光调节为圆偏振光后，经过所述高数值孔径物镜后入射至所述载物台；
    所述高数值孔径物镜用于收集所述样品反射的荧光信号，所述荧光信号依次经过所述四分之一玻片、所述第五透镜、所述振镜扫描系统、所述第四透镜后射入所述计数器单元；
    所述振镜扫描系统用于对汇合重叠后的所述第一高斯型激发光和所述环形损耗光进行同步扫描，实现对所述样品的面阵扫描成像。
  9. 根据权利要求1所述的成像系统，其特征在于，所述计数器单元包括第六透镜、滤镜、多模光纤、光电倍增管、第一反射镜、第七透镜、探测器和时间相关单光子计数器；
    所述荧光信号依次经过所述第六透镜透射、所述滤镜滤除杂散光及所述多模光纤后，射入所述光电倍增管，所述光电倍增管将所述荧光信号放大后输入所述时间相关单光子计数器；
    所述第二高斯型激发光经过所述第一反射镜反射和所述第七透镜透射后，射入所述探测器，经所述探测器转化成电信号后输入所述时间相关单光子计数器；
    所述时间相关单光子计数器用于基于所述第二高斯型激发光和所述荧光信号进行荧光强度成像和荧光寿命成像。
  10. 根据权利要求1-9任意一项所述的成像系统，其特征在于，所述系统还包括第二反射镜、第三反射镜、第一双色镜和第二双色镜；
    所述第一高斯型激发光依次经过所述第二反射镜反射、所述第三反射镜反射及所述第一双色镜反射，所述环形损耗光依次经过所述第二双色镜透射和所述第一双色镜透射，经过所述第一双色镜透射的所述环形损耗光与经过所述第一双色镜反射的所述第一高斯型激发光汇合重叠后射入所述成像单元；
    所述荧光信号依次经过所述第一双色镜透射和所述第二双色镜反射后，射入所述计数器单元。
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CN105467572A (zh) * 2016-01-18 2016-04-06 北京大学 单波长实现多光子脉冲sted-spim显微系统
DE202017100426U1 (de) * 2017-01-26 2017-02-07 Abberior Instruments Gmbh Vorrichtung zum Aufrüsten eines einen Kameraanschluss aufweisenden Lichtmikroskops zu einem STED- Mikroskop
CN106841149A (zh) * 2017-03-17 2017-06-13 王富 受激辐射损耗显微方法及显微装置
CN108120702A (zh) * 2017-11-30 2018-06-05 浙江大学 一种基于并行探测的超分辨荧光寿命成像方法和装置

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