CN103698309A - STED (stimulated emission depletion) super-resolution microscope based on tunable laser - Google Patents
STED (stimulated emission depletion) super-resolution microscope based on tunable laser Download PDFInfo
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Abstract
The invention relates to the technical field of microscopes and provides an STED (stimulated emission depletion) super-resolution microscope based on tunable laser. The STED super-resolution microscope comprises an exciting light unit, a loss light unit, a first dichroic mirror and a scanning imaging unit, wherein the exciting light unit comprises a first tunable laser source; the loss light unit comprises a second tunable laser source, a half slide, a vortex phase plate and a quarter-wave plate; the scanning imaging unit comprises a second dichroic mirror, a scanning galvanometer, a scanning lens, a cylindrical mirror, an objective lens, a pinhole lens and a photomultiplier. According to the STED super-resolution microscope, the tunable laser sources are adopted as an exciting light source and a loss light source respectively; as the wavelengths of exciting light and loss light can be adjusted according to the needs, the proper wavelengths of the exciting light and the loss light can be found for a given fluorescent dye, and the application range of the STED super-resolution microscope is expanded.
Description
Technical field
The present invention relates to microscopy field, especially relate to a kind of STED super-resolution microscope based on tunable laser.
Background technology
Super-resolution optical microscopy has revolutionary impetus to the research in the fields such as biomedical and materialogy, and its progress has all produced far-reaching influence in a lot of scientific domains.Super-resolution optical microscopy is because implementation method is different, there is polytype: stimulated radiation loss (Stimulated Emission Depletion, STED) microscopy is a kind of optical ultra-discrimination microscopy being based upon on laser co-focusing microscopy basis, that first to propose be also the far-field optics microscopy of directly customer service optical diffraction limit, super-resolution microscopy with respect to other type, its image taking speed is relatively very fast, can carry out imaging to living cells, in biomedical research, can survey meticulousr structure, also for material science research provides new tool.
In STED micro imaging system, need two kinds of laser lightings, a kind of laser forms the hot spot that approximate Ai Li distributes at object focal point place, fluorescent material is excited, and makes fluorescent material send fluorescence, and this laser illuminator is exciting light; Another kind forms central light intensity at object focal point place be zero annular hot spot, making to be positioned at the fluorescent material in excited state on annulus loses, emitting fluorescence no longer, the fluorescent material that is only positioned at annular focal spot central authorities dark space could produce fluorescence, because the diameter of central dark space is much smaller than diffraction limit, therefore can obtain the image that surmounts the optical resolution limit.
The exciting light of setted wavelength can only be used for exciting absorption peak to be positioned near the fluorescent dye of this wavelength, the loss light of setted wavelength can only be used for de excitation fluoresce emission wavelength red end and the approaching fluorescent dye of this loss optical wavelength, and the absorption emission wavelength of excitation wavelength, loss optical wavelength and fluorescent dye will mate.Mostly current STED micro imaging system is to adopt exciting light or the loss light of fixed wave length, and so applicable fluorescent dye quantity is just very limited, and the biological tissue that can observe is also just very limited; Adopt double-colored or polychrome STED to two or more fluorescent dyes, to excite simultaneously, expanded the scope of application of system, even if but adopt double-colored or polychrome STED also just very limitedly to expand the scope of application of system, in actual biological experiment, fluorescent dye varies, and limited several excitation wavelengths or loss optical wavelength can not meet the demands.
Summary of the invention
The object of the invention is: a kind of STED super-resolution microscope based on tunable laser is provided, and excitation wavelength and loss optical wavelength all can regulate setting as required, have expanded the scope of application of STED super-resolution micro imaging system.
Technical scheme of the present invention is:
A STED super-resolution microscope based on tunable laser, is characterized in that, comprises exciting light unit, loss light unit, the first dichroscope and scanning imagery unit;
Described exciting light unit comprises the first tunable laser source; Described loss light unit comprises the second tunable laser source, half slide, vortex phase plate and quarter-wave plate, and the second tunable laser source, half slide, vortex phase plate and quarter-wave plate set gradually along the luminous light path of the second tunable laser source; Described scanning imagery unit comprises the second dichroscope, scanning galvanometer, scanning lens, cylinder mirror, object lens, apeture lens and photomultiplier, scanning galvanometer, scanning lens, cylinder mirror, object lens set gradually along the second dichroiscopic reflected light path, and apeture lens and photomultiplier set gradually along the second dichroiscopic transmitted light path;
Described the first dichroscope is used for connecting exciting light unit and loss light unit, and the first dichroscope carries out transmission to the exciting light of exciting light unit outgoing, and the loss light of loss light unit outgoing is reflected; Loss light after exciting light after the first dichroscope transmission and reflection is incident to the second dichroscope jointly; The second dichroscope reflects loss light and the exciting light of incident;
The excitation wavelength that the first tunable laser source in described exciting light unit sends can arrange as required, the exciting light of exciting light unit outgoing is through the first dichroscope transmission, the second dichroscope reflection, scanning galvanometer reflection and scanning lens, cylinder mirror, object lens transmission, at object lens focal plane, place forms the first hot spot, thereby described the first hot spot is for exciting the fluorescent material of sample to produce fluorescence;
The loss optical wavelength that the second tunable laser source in described loss light unit sends can arrange as required, half slide is used for changing incident loss polarisation of light direction, vortex phase plate is for introducing the vortex PHASE DISTRIBUTION of 0-2 π at loss light light beam, quarter-wave plate is for transferring loss light to rotatory polarization by line polarisation, the loss light of loss light unit outgoing is through the first dichroscope, the second dichroscope, scanning galvanometer reflection and scanning lens, cylinder mirror, object lens transmission, and at object lens focal plane, place forms the second hot spot; Described the first hot spot and the second hot spot are overlapping;
The fluorescence that fluorescent material in described sample sends is collected by cylinder mirror, scanning lens transmission and scanning galvanometer reflection through object lens, enters to inject the second dichroscope, and by photomultiplier, is collected by apeture lens by the second dichroscope transmission.
Below technique scheme is further explained:
Described the first dichroscope has high-transmission rate to the exciting light of exciting light unit outgoing, and the loss light of loss light unit outgoing is had to high reflectance; The second dichroscope has high reflectance to loss light and the exciting light of incident, and the fluorescence that excitation is produced has high-transmission rate.
Described the first hot spot is Airy disk shaped laser spot;
Described the second hot spot is donut-like hot spot, and the middle position light intensity of hot spot is close to zero and around annular section light intensity is higher.
Described Airy disk shaped laser spot and donut-like hot spot are overlapping, make to be positioned in Airy disk outer peripheral areas the light de excitation that is depleted of the fluorescence molecule in fluorescent emission state and send out, and no longer produce fluorescence.
Between described photomultiplier and apeture lens, be provided with pin hole, described pin hole is positioned at the focus place of apeture lens; The fluorescence that fluorescent material in described sample sends focuses on pin hole place through apeture lens, and the fluorescence that sees through pin hole is collected by photomultiplier.
Advantage of the present invention is:
The present invention adopts tunable laser source as the exciting light sources in STED super-resolution microscope and loss radiant, because excitation wavelength and loss optical wavelength all can regulate setting as required, for given fluorescent dye, can find applicable excitation wavelength and loss optical wavelength, expand the microscopical scope of application of STED super-resolution.
Accompanying drawing explanation
The STED super-resolution microscopic structure schematic diagram based on tunable laser that Fig. 1 provides for the embodiment of the present invention.
Wherein: the STED super-resolution microscope 100 based on tunable laser, exciting light unit 110, the first tunable laser source 111, loss light unit 120, the second tunable laser source 121, half slide 122, vortex phase plate 123, quarter-wave plate 124, the first dichroscope 130, scanning imagery unit 140, the second dichroscope 141, scanning galvanometer 142, scanning lens 143, cylinder mirror 144, object lens 145, apeture lens 146, pin hole 147, photomultiplier 148, sample 200.
Embodiment
Please refer to Fig. 1.The light path that indicates single arrow in Fig. 1 is laser propagation light path; The light path that indicates double-head arrow is that fluorescence is propagated light path.
Embodiment: the STED super-resolution microscope 100 based on tunable laser comprises exciting light unit 110, loss light unit 120, the first dichroscope 130 and scanning imagery unit 140.
Exciting light unit 110 comprises the first tunable laser source 111, and the excitation wavelength that this first tunable laser source 111 sends can arrange as required.
Loss light unit 120 comprises the second tunable laser source 121, half slide 122, vortex phase plate 123 and quarter-wave plate 124, and they set gradually along the luminous light path of the second tunable laser source 121 respectively.The loss optical wavelength that the second tunable laser source 121 sends also can arrange as required.Wherein, half slide 122 is for changing incident loss polarisation of light direction, and vortex phase plate 123 is for introducing the vortex PHASE DISTRIBUTION of 0-2 π at loss light light beam, and quarter-wave plate 124 is for transferring loss light to rotatory polarization by line polarisation.
The first dichroscope 130, for connecting exciting light unit 110 and loss light unit 120, carries out transmission to the exciting light of exciting light unit 110 outgoing, and the loss light of loss light unit 120 outgoing is reflected.In a preferred embodiment, the exciting light of the first 130 pairs of dichroscopes exciting light unit, 110 outgoing has high-transmission rate, and the loss light of loss light unit 120 outgoing is had to high reflectance.
Scanning imagery unit 140 comprises the second dichroscope 141, scanning galvanometer 142, scanning lens 143, cylinder mirror 144, object lens 145, apeture lens 146 and photomultiplier 148, scanning galvanometer 142, scanning lens 143, cylinder mirror 144, object lens 145 set gradually along the reflected light path of the second dichroscope 141, and apeture lens 146 and photomultiplier 148 set gradually along the transmitted light path of the second dichroscope 141.Loss light and exciting light that loss light after exciting light after the first dichroscope 130 transmissions and reflection is incident to 141 pairs of incidents of the second dichroscope 141, the second dichroscopes jointly reflect.In a preferred embodiment, loss light and the exciting light of 141 pairs of incidents of the second dichroscope have high reflectance.
The exciting light of exciting light unit 110 outgoing is through the first dichroscope 130 transmissions, the second dichroscope 141 reflections, scanning galvanometer 142 reflection and scanning lenses 143, cylinder mirror 144, object lens 145 transmissions, at object lens, 145 focal plane places form the first hot spot, thereby this first hot spot is for exciting the fluorescent material of sample 200 to produce fluorescence, fluorescence is collected by cylinder mirror 144 through object lens 145, scanning lens 143 transmissions and scanning galvanometer 142 reflections, enter to inject the second dichroscope 141, and by photomultiplier 148, collected by apeture lens 146 by the second dichroscope 141 transmissions, photomultiplier 148 is surveyed fluorescence and converts fluorescence to electric signal, in a preferred embodiment, the fluorescence that the second 141 pairs of dichroscopes excitation produces has high-transmission rate.The loss light of loss light unit 120 outgoing is through the first dichroscope 130, the second dichroscope 141, scanning galvanometer 142 reflections and scanning lens 143, cylinder mirror 144, object lens 145 transmissions, and at object lens, 145 focal plane places form the second hot spot.The first hot spot and the second hot spot are overlapping.
In a preferred embodiment, the first hot spot is Airy disk shaped laser spot; The second hot spot is donut-like hot spot, and the middle position light intensity of hot spot is close to zero and around annular section light intensity is higher.Airy disk shaped laser spot and donut-like hot spot overlap, making to be positioned in Airy disk outer peripheral areas the light de excitation that is depleted of the fluorescence molecule in fluorescent emission state sends out, no longer produce fluorescence, thereby reduced effective emitting area of fluorescence, realize optical ultra-discrimination imaging.
In another preferred embodiment, between photomultiplier 148 and apeture lens 146, be provided with pin hole 147, pin hole 147 is positioned at the focus place of apeture lens 146; The fluorescence that fluorescent material in sample 200 sends focuses on pin hole 147 places through apeture lens 146, and the fluorescence that sees through pin hole 147 is collected by photomultiplier 148.
the present embodiment course of work is as follows:
As shown in Figure 1, the first tunable laser source 111 sends exciting light light beam, after the first dichroscope 130 transmissions, the second dichroscope 141 reflections, scanning galvanometer 142 reflections and scanning lens 143, cylinder mirror 144, object lens 145 transmissions, at object lens, 145 focal plane places form the hot spot that an Airy disk distributes, thereby for exciting the fluorescent material of sample 200 to produce fluorescence; The loss light light beam that the second tunable laser source 121 sends is after half slide 122, vortex phase plate 123,1/4th slides 124, through the first dichroscope 130, the second dichroscope 141, scanning galvanometer 142 reflections and scanning lens 143, cylinder mirror 144, object lens 145 transmissions, at the focal plane place of object lens 145, form the hot spot of a loaf of bread cast, the middle position light intensity of hot spot is close to zero and around annular section light intensity is higher.The donut-like hot spot that the Airy disk shaped laser spot that exciting light forms at object lens 145 focal plane places and loss light form at object lens 145 focal plane places overlaps, making to be positioned in Airy disk outer peripheral areas the light de excitation that is depleted of the fluorescence molecule in fluorescent emission state sends out, no longer produce fluorescence, thereby reduced effective emitting area of fluorescence, realized optical ultra-discrimination imaging.The fluorescence that fluorescent material in sample 200 sends after object lens 145 are collected through cylinder mirror 144 and scanning lens 143, be scanned afterwards galvanometer 142 reflections, enter to inject the second dichroscope 141, by the second dichroscope 141 transmissions, through apeture lens 146, focus on pin hole 147 places, the fluorescence that sees through pin hole 147 is collected by photomultiplier 148.
The present invention adopts tunable laser source as the exciting light sources in STED super-resolution microscope and loss radiant, because excitation wavelength and loss optical wavelength all can regulate setting as required, for given fluorescent dye, can find applicable excitation wavelength and loss optical wavelength, expand the microscopical scope of application of STED super-resolution.
Certainly the STED super-resolution microscope based on tunable laser of the present invention also can have multiple conversion and remodeling, is not limited to the concrete structure of above-mentioned embodiment.In a word, protection scope of the present invention should comprise those apparent conversion or alternative and remodeling to those skilled in the art.
Claims (5)
1. the STED super-resolution microscope based on tunable laser, is characterized in that, comprises exciting light unit, loss light unit, the first dichroscope and scanning imagery unit;
Described exciting light unit comprises the first tunable laser source; Described loss light unit comprises the second tunable laser source, half slide, vortex phase plate and quarter-wave plate, and the second tunable laser source, half slide, vortex phase plate and quarter-wave plate set gradually along the luminous light path of the second tunable laser source; Described scanning imagery unit comprises the second dichroscope, scanning galvanometer, scanning lens, cylinder mirror, object lens, apeture lens and photomultiplier, scanning galvanometer, scanning lens, cylinder mirror, object lens set gradually along the second dichroiscopic reflected light path, and apeture lens and photomultiplier set gradually along the second dichroiscopic transmitted light path;
Described the first dichroscope is used for connecting exciting light unit and loss light unit, and the first dichroscope carries out transmission to the exciting light of exciting light unit outgoing, and the loss light of loss light unit outgoing is reflected; Loss light after exciting light after the first dichroscope transmission and reflection is incident to the second dichroscope jointly; The second dichroscope reflects loss light and the exciting light of incident;
The excitation wavelength that the first tunable laser source in described exciting light unit sends can arrange as required, the exciting light of exciting light unit outgoing is through the first dichroscope transmission, the second dichroscope reflection, scanning galvanometer reflection and scanning lens, cylinder mirror, object lens transmission, at object lens focal plane, place forms the first hot spot, thereby described the first hot spot is for exciting the fluorescent material of sample to produce fluorescence;
The loss optical wavelength that the second tunable laser source in described loss light unit sends can arrange as required, half slide is used for changing incident loss polarisation of light direction, vortex phase plate is for introducing the vortex PHASE DISTRIBUTION of 0-2 π at loss light light beam, quarter-wave plate is for transferring loss light to rotatory polarization by line polarisation, the loss light of loss light unit outgoing is through the first dichroscope, the second dichroscope, scanning galvanometer reflection and scanning lens, cylinder mirror, object lens transmission, and at object lens focal plane, place forms the second hot spot; Described the first hot spot and the second hot spot are overlapping;
The fluorescence that fluorescent material in described sample sends is collected by cylinder mirror, scanning lens transmission and scanning galvanometer reflection through object lens, enters to inject the second dichroscope, and by photomultiplier, is collected by apeture lens by the second dichroscope transmission.
2. the STED super-resolution microscope based on tunable laser according to claim 1, is characterized in that, described the first dichroscope has high-transmission rate to the exciting light of exciting light unit outgoing, and the loss light of loss light unit outgoing is had to high reflectance; The second dichroscope has high reflectance to loss light and the exciting light of incident, and the fluorescence that excitation is produced has high-transmission rate.
3. the STED super-resolution microscope based on tunable laser according to claim 1, is characterized in that, described the first hot spot is Airy disk shaped laser spot;
Described the second hot spot is donut-like hot spot, and the middle position light intensity of hot spot is close to zero and around annular section light intensity is higher.
4. the STED super-resolution microscope based on tunable laser according to claim 3, it is characterized in that, described Airy disk shaped laser spot and donut-like hot spot are overlapping, make to be positioned in Airy disk outer peripheral areas the light de excitation that is depleted of the fluorescence molecule in fluorescent emission state and send out, and no longer produce fluorescence.
5. the STED super-resolution microscope based on tunable laser according to claim 1, is characterized in that, between described photomultiplier and apeture lens, be provided with pin hole, described pin hole is positioned at the focus place of apeture lens; The fluorescence that fluorescent material in described sample sends focuses on pin hole place through apeture lens, and the fluorescence that sees through pin hole is collected by photomultiplier.
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| CN104089935A (en) * | 2014-07-11 | 2014-10-08 | 中国科学院化学研究所 | Super-resolution fluorescence lifetime correlation spectroscopy system |
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| CN106442445B (en) * | 2016-09-18 | 2019-04-30 | 中国计量大学 | One kind being based on single pass polychrome super-resolution microscopic system and method |
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| CN110082897A (en) * | 2018-01-25 | 2019-08-02 | 中国科学院上海光学精密机械研究所 | A kind of high-speed imaging device and method |
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| CN108320758A (en) * | 2018-02-02 | 2018-07-24 | 中国科学院上海光学精密机械研究所 | A kind of reversible phase transition material high density memory Set |
| WO2020048021A1 (en) * | 2018-09-07 | 2020-03-12 | 中国科学院苏州生物医学工程技术研究所 | Composite microscope employing two-photon excitation and stimulated emission depletion techniques |
| US11550134B2 (en) | 2018-09-07 | 2023-01-10 | Suzhou Institute Of Biomedical Engineering And Technology, Chinese Academy Of Sciences | Composite microscope employing two-photon excitation and stimulated emission depletion techniques |
| WO2020056917A1 (en) * | 2018-09-21 | 2020-03-26 | 中国科学院苏州生物医学工程技术研究所 | Stimulated emission depletion super-resolution microscope realizing quick combination of light beams |
| US11366300B2 (en) | 2018-09-21 | 2022-06-21 | Suzhou Institute Of Biomedical Engineering And Technology, Chinese Academy Of Sciences | Stimulated emission depletion super-resolution microscope using quick combination of light beams |
| CN109188668A (en) * | 2018-09-21 | 2019-01-11 | 苏州国科医疗科技发展有限公司 | A kind of stimulated emission depletion super-resolution microscope realized light beam and quickly close beam |
| CN109683342A (en) * | 2018-12-25 | 2019-04-26 | 浙江大学 | Multimode fibre super-resolution imaging device and its facula correction method based on wavefront shaping |
| CN109683342B (en) * | 2018-12-25 | 2020-11-03 | 浙江大学 | Multi-mode fiber super-resolution imaging device based on wavefront shaping and light spot correction method thereof |
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