CN103698309B - STED super-resolution microscope based on tunable laser - Google Patents
STED super-resolution microscope based on tunable laser Download PDFInfo
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- CN103698309B CN103698309B CN201310732247.8A CN201310732247A CN103698309B CN 103698309 B CN103698309 B CN 103698309B CN 201310732247 A CN201310732247 A CN 201310732247A CN 103698309 B CN103698309 B CN 103698309B
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Abstract
The present invention relates to microscopy field, it is provided that the STED super-resolution microscope based on tunable laser include exciting light unit, loss light unit, the first dichroscope and scanning and imaging unit；Exciting light unit includes the first tunable laser source；Loss light unit includes the second tunable laser source, half slide, vortex phase sheet and quarter-wave plate；Scanning and imaging unit includes the second dichroscope, scanning galvanometer, scanning lens, cylinder mirror, object lens, apeture lens and photomultiplier tube.The present invention adopts tunable laser source as the exciting light sources in STED super-resolution microscope and loss radiant, all can be adjusted as required due to excitation wavelength and loss optical wavelength arranging, for given fluorescent dye, applicable excitation wavelength and loss optical wavelength can be found, expand the microscopical scope of application of STED super-resolution.
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
The research in the fields such as biomedical and materialogy is had revolutionary impetus by super-resolution optical microscopy, and its progress all creates far-reaching influence in a lot of scientific domains.Super-resolution optical microscopy is owing to realizing method difference, occur in that polytype: stimulated radiation loss (StimulatedEmissionDepletion, STED) microscopy is built upon a kind of optical ultra-discrimination microscopy on Laser confocal scanning light microscopy basis, it is first propose also to be the far-field optics microscopy of the most directly customer service optical diffraction limit, relative to other type of super-resolution microscopy, its image taking speed is relatively fast, living cells can be carried out imaging, biomedical research can detect finer structure, also new tool is provided for material science research.
Needing two kinds of laser lightings in STED micro imaging system, a kind of laser forms the hot spot of approximate Ai Li distribution at object focal point place, fluorescent material is excited so that fluorescent material sends fluorescence, and this laser illuminator is exciting light；Another kind forms the annular hot spot that central authorities' light intensity is zero at object focal point place, the fluorescent material making to be positioned on annulus and being in excited state loses, no longer emitting fluorescence, the fluorescent material being only positioned at annular focal spot central authorities dark space could produce fluorescence, owing to the diameter of central authorities dark space is much smaller than diffraction limit, therefore the image surmounting optical resolution limit can be obtained.
The exciting light of setted wavelength can only be used for exciting absorption peak to be positioned at the fluorescent dye near this wavelength, the loss light of setted wavelength can only fluoresce for de excitation and launch the fluorescent dye that wavelength red end is close with this loss optical wavelength, and the absorption transmitting wavelength of excitation wavelength, loss optical wavelength and fluorescent dye to mate.Mostly current STED micro imaging system is to adopt exciting light or the loss light of fixed wave length, and the fluorescent dye quantity being so suitable for is just very limited, it is possible to the biological tissue of observation is also just very limited；Adopt double-colored or polychrome STED two or more fluorescent dyes to be excited simultaneously, expand the scope of application of system, even if adopting double-colored or polychrome STED also simply 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 requirement.
Summary of the invention
It is an object of the invention to: provide a kind of STED super-resolution microscope based on tunable laser, excitation wavelength and loss optical wavelength all can be adjusted arranging as required, expand the scope of application of STED super-resolution micro imaging system.
The technical scheme is that
A kind of STED super-resolution microscope based on tunable laser, it is characterised in that include exciting light unit, loss light unit, the first dichroscope and scanning and imaging unit；
Described exciting light unit includes the first tunable laser source；Described loss light unit includes the second tunable laser source, half slide, vortex phase sheet and quarter-wave plate, and the second tunable laser source, half slide, vortex phase sheet and quarter-wave plate set gradually along the luminous light path of the second tunable laser source；Described scanning and imaging unit includes the second dichroscope, scanning galvanometer, scanning lens, cylinder mirror, object lens, apeture lens and photomultiplier tube, scanning galvanometer, scanning lens, cylinder mirror, object lens set gradually along the second dichroiscopic reflected light path, and apeture lens and photomultiplier tube set gradually along the second dichroiscopic transmitted light path；
Described first dichroscope is used for connecting exciting light unit and loss light unit, and the exciting light of exciting light unit outgoing is carried out transmission by the first dichroscope, and the loss light of loss light unit outgoing is reflected；Exciting light after the first dichroscope transmission and the loss light co-incident after reflection are to the second dichroscope；Incident loss light and exciting light are reflected by the second dichroscope；
The excitation wavelength that the first tunable laser source in described exciting light unit sends can be configured 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, form the first hot spot at object lens focal plane place, described first hot spot is for exciting the fluorescent material in sample thus producing fluorescence；
The loss optical wavelength that the second tunable laser source in described loss light unit sends can be configured as required, half slide is for changing the polarization direction of incident loss light, vortex phase sheet for introducing the vortex phase distribution of 0-2 π in loss light light beam, quarter-wave plate is for being transferred loss light to rotatory polarization by line polarisation, the loss light of loss light unit outgoing, through the first dichroscope, the second dichroscope, scanning galvanometer reflection and scanning lens, cylinder mirror, object lens transmission, forms the second hot spot at object lens focal plane place；Described first hot spot and the second hot spot are overlapping；
The fluorescence that fluorescent material in described sample sends reflects through cylinder mirror, scanning lens transmission and scanning galvanometer after object lens are collected, and enters to inject the second dichroscope, and is collected by photomultiplier tube through apeture lens after the second dichroscope transmission.
Below technique scheme is explained further:
The exciting light of exciting light unit outgoing is had high-transmission rate by described first dichroscope, and the loss light of loss light unit outgoing is had high reflectance；Incident loss light and exciting light are had high reflectance by the second dichroscope, and the fluorescence that excitation is produced has high-transmission rate.
Described first hot spot is Airy disk shaped laser spot；
Described second hot spot is donut-like hot spot, and the middle position light intensity of hot spot is close to zero and peripheral cyclic region light intensity is higher.
Described Airy disk shaped laser spot and donut-like hot spot are overlapping, make to be positioned at the fluorescence molecule being in fluorescence emission stage wherein in the Airy disk outer peripheral areas light de excitation that is depleted and send out, no longer produce fluorescence.
Being provided with pin hole between described photomultiplier tube and apeture lens, described pin hole is positioned at the focal point of apeture lens；The fluorescence that fluorescent material in described sample sends focuses on pin hole place through apeture lens, and the fluorescence through pin hole is collected by photomultiplier tube.
The invention have the advantage that
The present invention adopts tunable laser source as the exciting light sources in STED super-resolution microscope and loss radiant, all can be adjusted as required due to excitation wavelength and loss optical wavelength arranging, for given fluorescent dye, applicable excitation wavelength and loss optical wavelength can be found, 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: based on the STED super-resolution microscope 100 of tunable laser, exciting light unit the 110, first tunable laser source 111, loss light unit the 120, second tunable laser source 121, half slide 122, vortex phase sheet 123, quarter-wave plate the 124, first dichroscope 130, scanning and imaging unit the 140, second dichroscope 141, scanning galvanometer 142, scanning lens 143, cylinder mirror 144, object lens 145, apeture lens 146, pin hole 147, photomultiplier tube 148, sample 200.
Detailed description of the invention
Refer to Fig. 1.The light path indicating single arrow in Fig. 1 is laser propagation light path；The light path indicating double-head arrow is that fluorescence propagates light path.
Embodiment: the STED super-resolution microscope 100 based on tunable laser includes exciting light unit 110, loss light unit the 120, first dichroscope 130 and scanning and imaging unit 140.
Exciting light unit 110 includes the first tunable laser source 111, and the excitation wavelength that this first tunable laser source 111 sends can be configured as required.
Loss light unit 120 includes the second tunable laser source 121, half slide 122, vortex phase sheet 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 second tunable laser source 121 sends is configured also dependent on needs.Wherein, half slide 122 is for changing the polarization direction of incident loss light, and vortex phase sheet 123 for introducing the vortex phase distribution of 0-2 π in loss light light beam, and quarter-wave plate 124 is for being transferred loss light to rotatory polarization by line polarisation.
First dichroscope 130 is used for connecting exciting light unit 110 and loss light unit 120, and the exciting light of exciting light unit 110 outgoing is carried out transmission, and the loss light of loss light unit 120 outgoing is reflected.In a preferred embodiment, the exciting light of exciting light unit 110 outgoing is had high-transmission rate by the first dichroscope 130, and the loss light of loss light unit 120 outgoing is had high reflectance.
Scanning and imaging unit 140 includes the second dichroscope 141, scanning galvanometer 142, scanning lens 143, cylinder mirror 144, object lens 145, apeture lens 146 and photomultiplier tube 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 tube 148 set gradually along the transmitted light path of the second dichroscope 141.Exciting light after the first dichroscope 130 transmission and the loss light co-incident after reflection are to the second dichroscope 141, and incident loss light and exciting light are reflected by the second dichroscope 141.In a preferred embodiment, incident loss light and exciting light are had high reflectance by the second dichroscope 141.
The exciting light of exciting light unit 110 outgoing is through the first dichroscope 130 transmission, second dichroscope 141 reflects, scanning galvanometer 142 reflection and scanning lens 143, cylinder mirror 144, object lens 145 transmission, the first hot spot is formed at object lens 145 focal plane place, this first hot spot is for exciting the fluorescent material in sample 200 thus producing fluorescence, fluorescence through object lens 145 collect after through cylinder mirror 144, scanning lens 143 transmission and scanning galvanometer 142 reflect, enter to inject the second dichroscope 141, and collected by photomultiplier tube 148 through apeture lens 146 after the second dichroscope 141 transmission, photomultiplier tube 148 detects fluorescence and converts fluorescence to the signal of telecommunication, in a preferred embodiment, the fluorescence that excitation is produced by the second dichroscope 141 has high-transmission rate.The loss light of loss light unit 120 outgoing through first dichroscope the 130, second dichroscope 141, scanning galvanometer 142 reflects and scanning lens 143, cylinder mirror 144, object lens 145 transmission, forms the second hot spot at object lens 145 focal plane place.First hot spot and the second hot spot are overlapping.
In a preferred embodiment, the first hot spot is Airy disk shaped laser spot；Second hot spot is donut-like hot spot, and the middle position light intensity of hot spot is close to zero and peripheral cyclic region light intensity is higher.Airy disk shaped laser spot and donut-like hot spot overlap, and making to be positioned at the fluorescence molecule being in fluorescence emission stage wherein in the Airy disk outer peripheral areas light de excitation that is depleted and send out, no longer producing fluorescence, thus reducing effective emitting area of fluorescence, it is achieved optical ultra-discrimination imaging.
In another preferred embodiment, being provided with pin hole 147 between photomultiplier tube 148 and apeture lens 146, pin hole 147 is positioned at the focal point of apeture lens 146；The fluorescence that fluorescent material in sample 200 sends focuses on pin hole 147 place through apeture lens 146, and the fluorescence through pin hole 147 is collected by photomultiplier tube 148.
The present embodiment work process is as follows:
As shown in Figure 1, first tunable laser source 111 sends exciting light light beam, through the first dichroscope 130 transmission, the second dichroscope 141 reflects, scanning galvanometer 142 reflects and after scanning lens 143, cylinder mirror 144, object lens 145 transmission, the hot spot of an Airy disk distribution is formed, for exciting the fluorescent material in sample 200 thus producing fluorescence at object lens 145 focal plane place；The loss light light beam that second tunable laser source 121 sends is after half slide 122, vortex phase sheet 123,1/4th slide 124, through first dichroscope the 130, second dichroscope 141, scanning galvanometer 142 reflects and scanning lens 143, cylinder mirror 144, object lens 145 transmission, form the hot spot of a loaf of bread cast at the focal plane place of object lens 145, the middle position light intensity of hot spot is close to zero and peripheral cyclic region light intensity is higher.Airy disk shaped laser spot that exciting light is formed at object lens 145 focal plane place and the donut-like hot spot that loss light is formed at object lens 145 focal plane place overlap, make to be positioned at the fluorescence molecule being in fluorescence emission stage wherein in the Airy disk outer peripheral areas light de excitation that is depleted to send out, no longer produce fluorescence, thus reducing effective emitting area of fluorescence, it is achieved optical ultra-discrimination imaging.The fluorescence that fluorescent material in sample 200 sends through object lens 145 collect after through cylinder mirror 144 and scanning lens 143, scanned galvanometer 142 reflects afterwards, enter to inject the second dichroscope 141, by the second dichroscope 141 transmission, focusing on pin hole 147 place through apeture lens 146, the fluorescence through pin hole 147 is collected by photomultiplier tube 148.
The present invention adopts tunable laser source as the exciting light sources in STED super-resolution microscope and loss radiant, all can be adjusted as required due to excitation wavelength and loss optical wavelength arranging, for given fluorescent dye, applicable excitation wavelength and loss optical wavelength can be found, expand the microscopical scope of application of STED super-resolution.
The STED super-resolution microscope based on tunable laser of certain present invention also can have multiple conversion and remodeling, it is not limited to the concrete structure of above-mentioned embodiment.In a word, protection scope of the present invention should include those apparent conversion or replacement and remodeling to those skilled in the art.
Claims (5)
1. the STED super-resolution microscope based on tunable laser, it is characterised in that include exciting light unit, loss light unit, the first dichroscope and scanning and imaging unit；
Described exciting light unit includes the first tunable laser source；Described loss light unit includes the second tunable laser source, half slide, vortex phase sheet and quarter-wave plate, and the second tunable laser source, half slide, vortex phase sheet and quarter-wave plate set gradually along the luminous light path of the second tunable laser source；Described scanning and imaging unit includes the second dichroscope, scanning galvanometer, scanning lens, cylinder mirror, object lens, apeture lens and photomultiplier tube, scanning galvanometer, scanning lens, cylinder mirror, object lens set gradually along the second dichroiscopic reflected light path, and apeture lens and photomultiplier tube set gradually along the second dichroiscopic transmitted light path；
Described first dichroscope is used for connecting exciting light unit and loss light unit, and the exciting light of exciting light unit outgoing is carried out transmission by the first dichroscope, and the loss light of loss light unit outgoing is reflected；Exciting light after the first dichroscope transmission and the loss light co-incident after reflection are to the second dichroscope；Incident loss light and exciting light are reflected by the second dichroscope；
The excitation wavelength that the first tunable laser source in described exciting light unit sends can be adjusted arranging as required, so can select suitable excitation wavelength according to given fluorescent dye, the exciting light of described 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, form the first hot spot at object lens focal plane place, described first hot spot is for exciting the fluorescent material in sample thus producing fluorescence；
The loss optical wavelength that the second tunable laser source in described loss light unit sends can be adjusted arranging as required, so can select suitable loss optical wavelength according to given fluorescent dye, half slide is for changing the polarization direction of incident loss light, vortex phase sheet for introducing the vortex phase distribution of 0-2 π in loss light light beam, quarter-wave plate is for being transferred loss light to rotatory polarization by line polarisation, the loss light of loss light unit outgoing is through the first dichroscope, second dichroscope, scanning galvanometer reflection and scanning lens, cylinder mirror, object lens transmission, the second hot spot is formed at object lens focal plane place；Described first hot spot and the second hot spot are overlapping；
The fluorescence that fluorescent material in described sample sends reflects through cylinder mirror, scanning lens transmission and scanning galvanometer after object lens are collected, and enters to inject the second dichroscope, and is collected by photomultiplier tube through apeture lens after the second dichroscope transmission.
2. the STED super-resolution microscope based on tunable laser according to claim 1, it is characterised in that the exciting light of exciting light unit outgoing is had high-transmission rate by described first dichroscope, and the loss light of loss light unit outgoing is had high reflectance；Incident loss light and exciting light are had high reflectance by the second dichroscope, 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, it is characterised in that described first hot spot is Airy disk shaped laser spot；
Described second hot spot is donut-like hot spot, and the middle position light intensity of hot spot is close to zero and peripheral cyclic region 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 at the fluorescence molecule being in fluorescence emission stage wherein in the Airy disk outer peripheral areas light de excitation that is depleted and send out, no longer produce fluorescence.
5. the STED super-resolution microscope based on tunable laser according to claim 1, it is characterised in that being provided with pin hole between described photomultiplier tube and apeture lens, described pin hole is positioned at the focal point of apeture lens；The fluorescence that fluorescent material in described sample sends focuses on pin hole place through apeture lens, and the fluorescence through pin hole is collected by photomultiplier tube.
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| CN107490568B (en) * | 2017-08-09 | 2020-08-18 | 四川大学 | Super-resolution microscopic imaging device and method based on stimulated emission loss characteristics |
| CN110082897B (en) * | 2018-01-25 | 2020-11-10 | 中国科学院上海光学精密机械研究所 | High-speed imaging device and method |
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