WO2017079902A1 - Système de balayage aléatoire - Google Patents

Système de balayage aléatoire Download PDF

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Publication number
WO2017079902A1
WO2017079902A1 PCT/CN2015/094214 CN2015094214W WO2017079902A1 WO 2017079902 A1 WO2017079902 A1 WO 2017079902A1 CN 2015094214 W CN2015094214 W CN 2015094214W WO 2017079902 A1 WO2017079902 A1 WO 2017079902A1
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WO
WIPO (PCT)
Prior art keywords
incident
mirror
light
imaging
scanning
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PCT/CN2015/094214
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English (en)
Chinese (zh)
Inventor
屈军乐
严伟
邵永红
叶彤
田蜜
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深圳大学
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Priority to PCT/CN2015/094214 priority Critical patent/WO2017079902A1/fr
Publication of WO2017079902A1 publication Critical patent/WO2017079902A1/fr

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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

Definitions

  • the invention belongs to the field of microscopic imaging, and in particular relates to a random scanning system for STED super-resolution microscopic imaging.
  • the optical method achieves super-resolution imaging beyond the diffraction limit, so it is very suitable for the application of living cell super-resolution imaging, but STED super-resolution imaging has a disadvantage that the intensity of the loss of light is large, and there is a certain damage to living cells. .
  • the commonly used method is to reduce the intensity of the loss of light to reduce the damage of light to living cells. Although this reduces the light damage to the cells, it greatly reduces the resolution of the system and limits its wide range of applications.
  • the technical problem to be solved by the present invention is to provide a system for random scanning, which aims to ensure that the living cells are damaged by light damage based on the resolution of the STED super-resolution microscopic imaging system.
  • the present invention is achieved by a random scanning system including a dispersion pre-compensation unit, a random scanning unit, and an imaging unit;
  • the dispersion pre-compensation unit is configured to perform chromatic dispersion pre-compensation and optical path adjustment on the incident light, and then vertically incident to the random scanning unit to implement scanning of an arbitrary region;
  • the random scanning unit is further configured to collect a fluorescent signal generated after performing an arbitrary area scan, and process the fluorescent signal and then incident on the imaging unit for imaging.
  • the incident light includes STED loss light and excitation light.
  • the dispersion pre-compensation unit includes a first prism, a second mirror, a first mirror group, and a second mirror group;
  • the STED loss light is pre-compensated by the first prism and then incident on the first mirror group, and is optically adjusted by the first mirror group and then vertically incident to the random scanning unit;
  • the excitation light is pre-compensated by the second prism and then incident on the second mirror group, and is optically adjusted by the second mirror group and then vertically incident to the random scanning unit.
  • first prism and the second mirror are both placed at an angle of 45° in the horizontal direction.
  • the random scanning unit includes:
  • a first two-dimensional acousto-optic deflector a first data acquisition card connected to the first two-dimensional acousto-optic deflector
  • a mirror a first dichroic mirror, a second dichroic mirror, and an objective lens
  • the dispersion-precompensated STED loss light is incident perpendicularly to the first two-dimensional acousto-optic deflector, and the first two-dimensional acousto-optic deflector loses light to the incident STED under the control of the first data acquisition card Performing beam modulation and deflection, and the modulated and deflected STED loss light is redirected by the mirror reflection, projected by the first dichroic mirror, and incident on the objective lens;
  • the dispersion-pre-compensated excitation light is incident perpendicularly to the second two-dimensional acousto-optic deflector, and the second two-dimensional acousto-optic deflector beams the incident excitation light under the control of the second data acquisition card Modulating and deflecting, the modulated and deflected excitation light is incident on the objective lens after being projected by the second dichroic mirror;
  • the objective lens is used for focusing the incident excitation light and the STED loss light to be incident on the sample, thereby exciting the sample to generate a fluorescence signal exceeding the diffraction limit; and also for collecting the fluorescence signal generated by the sample and irradiating the second dichroic mirror;
  • the second dichroic mirror is further configured to transmit the fluorescent signal to the imaging unit after being reflected by the first dichroic mirror.
  • a distance between the first prism and the first two-dimensional acousto-optic deflector is 35 cm; a distance between the second prism and the second two-dimensional acousto-optic deflector is 35CM .
  • the imaging unit includes: a beam splitter, a photomultiplier tube, an image sensor, and an imaging module;
  • the beam splitter is configured to divide the incident fluorescent signal into a first fluorescent signal and a second fluorescent signal according to a ratio
  • the first fluorescent signal is amplified by the photomultiplier tube, and then transmitted to the imaging module for processing and imaging;
  • the second fluorescent signal is optically adjusted by the image sensor, and then transmitted to the imaging module for real-time display.
  • the beam splitter splits the incident fluorescent signal into a first fluorescent signal and a second fluorescent signal in a ratio of 9:1.
  • the invention has the beneficial effects that the invention uses the scanning technology based on the dual sound deflector, avoids the mechanical offset of the galvanometer scanning, has higher scanning precision and faster scanning speed, and at the same time
  • the scanning technology of the acousto-optic deflector can also realize the selection of the fast region of interest and scan imaging, which greatly reduces the optical damage while ensuring the resolution is unchanged, and realizes substantially no photo damage in the non-interest area in the field of view. .
  • FIG. 1 is a schematic structural diagram of a random scanning system according to an embodiment of the present invention.
  • FIG. 2 is a schematic structural diagram of a random scanning system according to an embodiment of the present invention.
  • Figure 3 is a diffraction pattern of the AOD at the center frequency.
  • Figure 4 is a schematic illustration of the overlap of two beams of light after passing through the AOD.
  • Figure 5 is a schematic diagram of a 4 x 4 dot pattern formed by excitation light passing through AOD.
  • FIG. 6 is a schematic diagram showing the formation of a 4 ⁇ 4 dot matrix superposition after the excitation light and the STED loss light respectively pass through the respective AODs.
  • FIG. 7 is a schematic diagram of a method for finding two sets of AOD frequencies by using fixed lattice coordinates to realize coincidence of two light-matrix arrays.
  • Figure 8 is a schematic diagram of STD super-resolution arbitrary addressing scanning using two sets of two-dimensional AOD techniques.
  • Figure 9 is a schematic diagram of the letter "A" STED super-resolution scan imaging using the AOD technique.
  • the commonly used STED super-resolution system uses point scanning, and its imaging speed is slow.
  • the scanning system used is galvanometer scanning. This scanning method requires scanning and imaging of the entire field of view, so the live STED super-resolution is displayed. During microimaging experiments, cells in the entire field of view are subject to photodamage.
  • the present application invents a dual two-dimensional acousto-optic deflector (Acousto-optic) in a STED super-resolution imaging system.
  • Defector, AOD any selection scanning technique that has the advantage of fast and arbitrary region-of-region scanning imaging, which will greatly reduce optical damage and non-interest areas in the field of view while maintaining the same resolution. There is basically no light damage.
  • STED super-resolution uses the stimulated radiation effect to reduce the effective fluorescent light-emitting area.
  • two beams are needed, one for excitation and the other for depletion.
  • the excitation light illuminates the fluorescent sample
  • the fluorescent molecules in the range of the diffraction spot are excited, and the electrons therein will transition to the excited state, and then the circular depletion light is superimposed on the excitation light, and the light is exhausted.
  • the electrons in the overlapping partial excited state return to the ground state in the form of stimulated radiation, and the other excited state electrons located at the center of the laser spot continue to be fluorescently returned to the ground state in the form of spontaneous radiation because they are not affected by the depletion light.
  • the photons received by the detector after filtering are generated by the autofluorescence of the fluorescent sample located at the center of the excitation spot.
  • the light-emitting area of such effective fluorescence is reduced, thereby increasing the spatial resolution of the system.
  • the embodiment of the present invention provides a system for random scanning as shown in FIG. 1, including a dispersion pre-compensation unit 1, a random scanning unit 2, and an imaging unit 3;
  • the dispersion pre-compensation unit 1 is configured to perform chromatic dispersion pre-compensation and optical path adjustment on the incident light, and then vertically incident to the random scanning unit 2 for realizing scanning of an arbitrary area;
  • the random scanning unit 2 is further configured to collect a fluorescent signal generated after scanning an arbitrary region, and process the fluorescent signal and then incident on the imaging unit 3 for imaging.
  • the dispersion pre-compensation unit 1 includes a first prism 1 and a second prism 2.
  • the first mirror group and the second mirror group, the first mirror group includes a mirror M2 and a mirror M1
  • the second mirror group includes a mirror M5 and a mirror M4.
  • the random scanning unit 2 includes:
  • Second two-dimensional acousto-optic deflector 2A-AOD 2 and said second two-dimensional acousto-optic deflector 2D-AOD 2 phase connected second data acquisition card DAQ2;
  • a mirror M3 a first dichroic mirror DM1, a second dichroic mirror DM2, and an objective lens;
  • the imaging unit 3 includes a beam splitter BS, a photomultiplier tube PMT, an image sensor CCD, and an imaging module; the imaging module is a computer in this embodiment.
  • AOD-based random scanning system for STED super-resolution microscopy including:
  • First Mirror (Prism 1), mainly to pre-compensate the dispersion of STED loss light, the purpose is to offset the dispersion generated by the rear AOD, note that the prism here should be placed in the horizontal direction by about 45°;
  • Second Mirror (Prism 2), mainly to pre-compensate the dispersion of the excitation light, the purpose is to offset the dispersion generated by the rear AOD, note that the prism here should be placed in the horizontal direction by about 45°;
  • a second two-dimensional acousto-optic deflector (2D-AOD 2) for performing random arbitrary selection scanning of green excitation light
  • the first mirror M1 and the second mirror M2 are mainly used to adjust the STED loss light after passing through the mirror to ensure that it enters the 2D-AOD vertically. 1;
  • the fourth mirror M4 and the fifth mirror M5 are mainly used for adjusting the excitation light after passing through the prism to ensure that it enters the 2D-AOD 2 vertically;
  • DM1 a first dichroic mirror for transmitting 780 nm STED loss light, reflecting fluorescence of about 680 nm;
  • a second dichroic mirror (DM2) is used to reflect excitation light at 633 nm and to transmit fluorescence at about 680 nm.
  • a beam splitter (BS) is used to split the signal collected by the objective into two parts (10%: 90%), 10% of the fluorescent signal enters the CCD for adjusting the optical path, and 90% of the fluorescent signal enters the PMT for super-resolution imaging.
  • the data acquisition card (DAQ1 and DAQ2) is used to generate control signals to realize real-time control of the two pairs of acousto-optic deflectors.
  • a clock signal is shared between the two DAQs, so that two DAQs can be realized.
  • Two sets of AODs are synchronized to produce different scanning frequencies.
  • a photomultiplier tube amplifies the fluorescent signal and transmits the amplified signal to a computer for imaging.
  • the image sensor CCD is used to collect the fluorescent signal when adjusting the optical path, and transmits the signal to the computer for real-time display.
  • Computer is used to control the data acquisition card and the photomultiplier tube, and simultaneously process the signal of the photomultiplier tube to form a complete super-resolution image.
  • the detailed process of the present invention is as follows: Firstly, it is assumed that both the excitation light and the STED loss light have been adjusted to meet the STED imaging requirements when the random scanning system is reached, and the adjusted excitation light is first subjected to dispersion pre-compensation through the mirror 2, and then through the mirror ( M4 and M5) adjust the height of the beam, and then enter the two-dimensional acousto-optic deflector.
  • the dispersion generated in the acousto-optic deflector is exactly offset by the dispersion compensated by the prism, and the excitation light emitted by the acousto-optic deflector should be It is a circular spot, which is then reflected by the dichroic mirror DM2 and then focused by the objective lens.
  • the STED loss light which has also been adjusted, is pre-compensated by the prism, and then the height of the loss light is adjusted by the mirrors (M1 and M2), and the horizontally incident two-dimensional acousto-optic modulator passes through the acousto-optic modulator.
  • the pre-compensated dispersion of the prism should cancel out the dispersion generated by the acousto-optic modulator.
  • the light emitted from the acousto-optic deflector should be a circular spot after being focused by the objective lens. Since the data acquisition cards DAQ1 and DAQ2 controlling the two sets of AOD signals share one clock. Therefore, the excitation light and the STED loss light are scanned synchronously. However, in the pulse interval, the excitation light reaches the sample about 180ps faster than the STED loss light, so that a better super-resolution image can be obtained.
  • the AOD scanning system needs to be fine-tuned first, because the diffraction angle of the AOD will change slightly due to the different wavelengths. Therefore, after the system is adjusted, the spectrometer is placed at the exit of the light source of the excitation light and the STED loss light, and is monitored at any time. The change in wavelength minimizes the effect of wavelength on the scanning system.
  • the imaging control data acquisition card allows the AOD to be fixed at the center frequency when the two beams of light pass through the AOD (AOD frequency range is 6000 Hz-9000 Hz, center frequency is 7500 Hz).
  • the AOD at this time is equivalent to a fixed grating, and the diffracted spot appears at the AOD light exit after the two beams pass through the respective AODs.
  • the (-1, -1) point is selected, and then the fluorescent light is selected.
  • the CCD is used to collect the spot on the road for real-time imaging.
  • the fine-tuning mirror and the two-color mirror are used to ensure that the (-1, -1) points of the two beams are completely coincident. Then look for the one-to-one correspondence between each pixel coordinate and the AOD scanning frequency.
  • the excitation light and the STED loss light of the embodiment of the present invention have been adjusted by default, and the work of the present invention is to ensure the high overlap of the excitation light and the STED loss light at any time, and construct the optical path according to the method of FIG. .
  • the excitation light path is established, the excitation light is kept horizontal, the position of the prism is adjusted, and the excitation light is incident at about 45°, and the prism itself is placed at an angle of about 45° with the horizontal plane, and the excitation light passes through the mirror dispersion. After pre-compensation, it is combined by a double mirror to adjust the height and direction of the excitation light.
  • Diffraction point (as shown in Figure 3), where the (-1,-1)-order diffraction point is the desired spot, and the shape of the diffraction spot is observed. If it is an ellipse, it can be adjusted by tilting the angle of the prism, AOD, or AOD. The distance to the mirror is changed to change the shape of the spot until the spot is round, as shown in Fig. 3, which is the modulated diffraction pattern. The same operation is also performed for the STED loss light.
  • the object with better reflectivity is used as the sample (the slide in this embodiment) for reflection imaging.
  • the excitation light is turned on, and a circular spot can be seen on the CCD.
  • the CCD allows the spot to be located in the center of the CCD, then turns off the excitation light to turn on the STED loss light.
  • a circular spot can be seen on the CCD.
  • the mirror M3 By adjusting the mirror M3, the ring spot is located at the center of the CCD, and finally the excitation light is turned on. Observe the degree of coincidence of the two beams. Make sure that the two beams are completely coincident by fine-tuning the mirror M3 and the dichroic mirror DM2, as shown in Figure 4.
  • each group of AODs is composed of two separate AODs (each AOD can perform one-dimensional line scanning), so that surface scanning can be performed through a set of AODs, in order to achieve random scanning.
  • the function must correspond to the scanning frequency of the two-dimensional AOD and the two-dimensional coordinates on the scanning surface.
  • the AOD scanning system is used for dot matrix scanning, and then the coordinates of the centroids of each point are found by using MATLAB, and then the lattice scanning frequency of AOD is increased, and the corresponding formula of lattice coordinates and frequency is found f k (x , y).
  • x, y are resolved as coordinate points
  • F 1x and F 1y are frequencies in the x direction and y direction, respectively
  • a 1x and A 1y are coefficients before frequency
  • B 1x and B 1y are constant, and known coordinate points
  • f 1 (x, y, F 1 ) can be determined, and the coordinates can be based on any position. Let the software automatically find the corresponding frequency through the formula f 1 (x, y, F 1 ) to realize the arbitrary selection scan imaging of AOD.
  • the same method is performed on the operation of STED loss of light.
  • the wavelength of the excitation light is 633 nm and the wavelength of the STED loss light is 780 nm, the diffraction angle of AOD to different wavelengths will be somewhat different. Therefore, when overlapping, there will be some shift in the position of the lattice center of the two beams. As shown in Figure 6.
  • two sets of AODs (2D-AOD1 and 2D-AOD2) are used to respectively control the excitation light and the STED loss light, in order to make the lattices generated by the two beams coincide.
  • the excitation light generate a set of lattices as shown in Fig. 5.
  • the formula f 2 (x, y, F 2 ) can also be found.
  • the parameters of the formula at this time are different from the formula f1(x, y, F 1 ) of the excitation light, and therefore, the expression of f 2 (x, y, F 2 ) is assumed here as follows.
  • F 2x and F 2y are the frequencies in the x direction and the y direction, respectively, A 2x and A 2y are the coefficients before the frequency, and B 2x and B 2y are constants, in which the centroid coordinates and frequencies are known. Therefore, each parameter can be calculated by MATLAB software to determine the equation f 2 (x, y, F 2 ), and finally the two sets of equations f 1 (x, y, F 1 ) and f 2 (x, y, F 2 ) The parameters are respectively input to the corresponding scanning control software Scan imaging, run the software, perform 4 ⁇ 4 dot matrix scanning, and obtain the dot pattern as shown in FIG. 7 to explain the excitation light and the STED loss light in the field of view.
  • the points are coincident. Therefore, the STED super-resolution random addressing scan as shown in Fig. 8 can be performed.
  • the basic idea is as follows: First, determine the point of interest (x i , y j ) in the field of view, and then according to the equation f 1 (x, y , F 1) and f 2 (x, y, F 2 ), find the frequencies F 1 (x i , y j ) and F 2 (x i , y j ) corresponding to each point, and then execute the scanning software to realize the point STED super-resolution imaging of (x i , y j ).
  • the schematic diagram of the letter "A" STED super-resolution imaging is implemented by using the designed software in this embodiment. Therefore, two sets of two-dimensional AOD systems can be utilized to realize arbitrary selection imaging of the STED super-resolution system.

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  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
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  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
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Abstract

La présente invention concerne un système de balayage aléatoire comprenant une unité de précompensation de dispersion (1), une unité de balayage aléatoire (2) ; et une unité d'imagerie (3). L'unité de précompensation de dispersion (1) est configurée pour effectuer une précompensation de dispersion et un ajustement de trajet optique sur une lumière incidente pour permettre à la lumière incidente d'atteindre l'unité de balayage aléatoire (2) perpendiculairement, de manière à mettre en œuvre le balayage d'une région quelconque. L'unité de balayage aléatoire (2) est en outre configurée pour collecter des signaux fluorescents générés après le balayage d'une région quelconque, et traiter les signaux fluorescents et ensuite permettre à ceux-ci d'atteindre l'unité d'imagerie (3) pour imagerie. L'utilisation d'une technologie de balayage à base de double déflecteur acousto-optique évite un décalage mécanique de balayage de galvanomètre, et permet d'obtenir une précision de balayage élevée et une vitesse de balayage rapide. En outre, la technologie de balayage à base de déflecteur acousto-optique peut en outre mettre en œuvre une sélection, un balayage et une imagerie rapides d'une région d'intérêt, réduire considérablement la photodégradation tout en garantissant une résolution inchangée, et ne causer pratiquement aucune photodégradation dans une région de non-intérêt dans un champ de vision.
PCT/CN2015/094214 2015-11-10 2015-11-10 Système de balayage aléatoire WO2017079902A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
US20020149769A1 (en) * 2000-12-15 2002-10-17 Roorda Robert Dixon Beam-steering of multi-chromatic light using acousto-optical deflectors and dispersion-compensatory optics
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Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020149769A1 (en) * 2000-12-15 2002-10-17 Roorda Robert Dixon Beam-steering of multi-chromatic light using acousto-optical deflectors and dispersion-compensatory optics
CN203164118U (zh) * 2012-11-14 2013-08-28 深圳大学 一种荧光寿命显微成像系统
CN105352926A (zh) * 2015-11-10 2016-02-24 深圳大学 一种随机扫描的系统
CN205209960U (zh) * 2015-11-10 2016-05-04 深圳大学 一种随机扫描的系统

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