CN110187487B - Single-wavelength two-photon STED and two-wavelength single-photon STED coupled imaging device and method - Google Patents
Single-wavelength two-photon STED and two-wavelength single-photon STED coupled imaging device and method Download PDFInfo
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- CN110187487B CN110187487B CN201910510783.0A CN201910510783A CN110187487B CN 110187487 B CN110187487 B CN 110187487B CN 201910510783 A CN201910510783 A CN 201910510783A CN 110187487 B CN110187487 B CN 110187487B
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Abstract
本发明涉及一种单波长双光子STED与双波长单光子STED耦合成像装置，包括：飞秒激光器、皮秒激光器、第I电动快门、第I二分之一波片、第Ⅰ分光器、第Ⅱ分光器或第Ⅰ反射镜可切换装置、第Ⅱ二分之一波片、第Ⅰ四分之一波片、第Ⅱ电动快门、第Ⅱ反射镜、第III反射镜、第I延时反射镜组、第IV反射镜、第I二色镜、第V反射镜、第II延时反射镜组、第VI反射镜、玻璃棒、第III二分之一波片、偏振器、第VII反射镜、第I透镜、保偏光纤、第II透镜、第VIII反射镜、相位板、第IX反射镜、提升光路反射镜组和共聚焦扫描显微镜。本发明可以根据需要分时切换成单波长双光子STED成像系统与双波长单光子成像系统。
The invention relates to a single-wavelength two-photon STED and two-wavelength single-photon STED coupling imaging device, comprising: a femtosecond laser, a picosecond laser, the first electric shutter, the first half-wave plate, the first optical splitter, the first II beam splitter or I mirror switchable device, II half wave plate, I quarter wave plate, II motor shutter, II reflection mirror, III reflection mirror, I delay reflection Mirror Group, Mirror IV, Dichroic Mirror I, Mirror V, Delay Mirror Group II, Mirror VI, Glass Rod, Half Wave Plate III, Polarizer, Reflection VII Mirror, I lens, polarization maintaining fiber, II lens, VIII reflector, phase plate, IX reflector, mirror group for lifting light path, and confocal scanning microscope. The present invention can be switched into a single-wavelength two-photon STED imaging system and a two-wavelength single-photon imaging system in a time-division as needed.
Description
Technical Field
The invention relates to the technical field of microscopic imaging, in particular to a single-wavelength two-photon STED and dual-wavelength single-photon STED coupling imaging device and method.
Background
Due to the diffraction limit of the resolution of the conventional fluorescence microscope, some super-resolution imaging technologies that break through the diffraction limit of the resolution, such as stimulated radiation depletion microscopy (STED), and structured light illumination microscopy (SIM), light activated positioning microscopy (PALM), random optical reconstruction microscopy (STORM), have been developed for over a decade. Wherein, STED introduces another annular loss light with longer wavelength than the exciting light on the basis of the laser scanning confocal fluorescence microscope. A focused laser beam excites the fluorophore to a high energy state (excited state) while an annular loss spot of a different wavelength is focused superimposed on the previous spot. Therefore, the fluorescence of the excited state in the overlapped region of the two can be reduced to the lowest energy level (namely, the ground state), only a small region of the central region emits fluorescence signals, the effective size of the excited Point Spread Function (PSF) is smaller than that of the diffraction limit PSF, and the purpose of improving the resolution ratio is achieved. Thus, STED super-resolution imaging techniques typically require two light sources. Early STED techniques were only available for single photon imaging, and later researchers developed two-photon STED techniques. In 2011, Teodora Scheul et al proposed a single-wavelength two-photon STED technology, but the experiment only used a solution sample and did not involve a cell experiment. 2012, Paolo Bianchini et al published the results of cell experiments performed by single-wavelength two-photon STED. The single-wavelength two-photon STED technology simplifies a system that the original STED needs two beams of light (one beam of exciting light and one beam of STED light), reduces the use of a light filter and reduces the cost of the system. However, the currently developed STED super-resolution imaging platform performs either two-wavelength single photon STED, two-wavelength two-photon STED, or single-wavelength two-photon STED. There is no platform for coupling single photon and two-photon STED imaging, so that researchers can select corresponding single photon or two-photon STED imaging according to research needs.
Disclosure of Invention
In view of the above, the present invention provides a single-wavelength two-photon STED and two-wavelength single-photon STED coupled imaging apparatus and method, which can be switched to a single-wavelength two-photon STED imaging system and a two-wavelength single-photon imaging system at different times according to research needs.
In order to achieve the purpose, the invention adopts the following technical scheme:
a single wavelength two-photon STED and dual wavelength single photon STED coupled imaging device, comprising: the device comprises a femtosecond laser, a picosecond laser, an I electric shutter, an I half wave plate, an I beam splitter, an II beam splitter or an I reflector switchable device, an II half wave plate, an I quarter wave plate, an II electric shutter, an II reflector, an III reflector, an I time-delay reflector group, an IV reflector, an I dichroic mirror, a V reflector, an II time-delay reflector group, a VI reflector, a glass rod, an III half wave plate, a polarizer, a VII reflector, an I lens, a polarization-maintaining optical fiber, an II lens, a VIII reflector, a phase plate, an IX reflector, a lifting optical path reflector group and a confocal scanning microscope; the light emitted by the femtosecond laser sequentially passes through an I electric shutter, an I half wave plate and an I beam splitter and then is divided into two beams, wherein one beam is used as a two-photon excitation light source, and the other beam is used as an STED light source of two photons and a single photon; the two-photon excitation light source is guided into the confocal scanning microscope for two-photon scanning imaging after sequentially passing through a second beam splitter, a second half wave plate, a first quarter wave plate, a first IX reflector and a lifting light path reflector group in the switchable device; the STED light source of the two-photon and single-photon enters the glass rod to perform pulse broadening after sequentially passing through the V-th reflector, the II-th time delay reflector group and the VI-th reflector, and then is guided into the polarization-maintaining optical fiber to perform further pulse broadening after passing through the III-half wave plate, the polarizer, the VII-th reflector and the I-th lens; the emergent light after the polarization maintaining fiber is expanded passes through a second lens to adjust a light beam into parallel light, then passes through a VIII reflector, and then passes through a phase plate to be modulated into doughnut-shaped light spots; the ring-shaped light spot sequentially passes through the I dichroic mirror, the II beam splitter or the I reflecting mirror in the switchable device, the II half wave plate, the I quarter wave plate, the IX reflecting mirror and the lifting light path reflecting mirror group, and then is guided into the confocal scanning microscope 29 to be used as a loss light beam.
Further, the switchable dichroic film inside the confocal scanning microscope is selected to be a dichroic film suitable for two-photon imaging.
Furthermore, the picosecond laser and the femtosecond laser are triggered to be used as a single photon excitation light source, light beams emitted by the picosecond laser sequentially pass through a second electric shutter, a second reflecting mirror, a third reflecting mirror, a first time delay reflecting mirror group, a fourth reflecting mirror, a first dichroic mirror and a first reflecting mirror in the switchable device and then coincide with the two-photon excitation light beams, and are guided into the single photon confocal scanning microscope to perform scanning imaging after passing through a second half wave plate, a first quarter wave plate, a first IX reflecting mirror and a lifting light path reflecting mirror group
Furthermore, the switchable dichromatic plate in the confocal scanning microscope is selected to be a dichromatic plate suitable for single photon imaging.
A switching method of a single-wavelength two-photon STED and dual-wavelength single-photon STED coupling imaging device comprises the following steps:
step S1, when switching to the single wavelength two-photon STED imaging mode, the I electric shutter is opened, the II electric shutter is closed, the switching device is switched to the II optical splitter, and the switchable dichromatic sheet in the confocal scanning microscope is switched to a dichromatic sheet suitable for two-photon imaging;
and step S2, when the double-wavelength single-photon imaging mode is switched, the I electric shutter is opened, the II electric shutter is opened, the switchable device is switched to the I reflecting mirror, and the switchable dichromatic film in the confocal scanning microscope is switched to a dichromatic film suitable for single-photon imaging.
Compared with the prior art, the invention has the following beneficial effects:
the invention overcomes the defects that the conventional STED super-resolution imaging platform can execute the STED of a double-wavelength single photon, the STED of a double-wavelength double photon or the STED of a single-wavelength double photon, and researchers can switch the STED into a single-wavelength double-photon STED imaging system and a double-wavelength single photon imaging system in a time-sharing manner according to the research needs.
Drawings
FIG. 1 is a schematic diagram of an embodiment of the present invention
In the figure: 1-1-femtosecond laser, 2-picosecond laser, 3-I electric shutter, 4-I half wave plate, 5-I beam splitter, 6-II beam splitter or I reflector switching device, 7-II half wave plate, 8-I quarter wave plate, 9-II electric shutter, 10-II reflector, 11-III reflector, 12-I time delay reflector set, 13-IV reflector, 14-I dichroic mirror, 15-V reflector, 16-II time delay reflector set, 17-VI reflector, 18-glass rod, 19-III half wave plate, 20-polarizer, 21-VII reflector, 22-the I lens, 23-the polarization maintaining fiber, 24-the II lens, 25-the VIII reflector, 26-the phase plate, 27-the IX reflector, 28-the lifting optical path reflector group, 29-the confocal scanning microscope, 30-the switchable dichroism slice in the confocal scanning microscope.
Detailed Description
The invention is further explained below with reference to the drawings and the embodiments.
Referring to fig. 1, the present invention provides a single-wavelength two-photon STED and dual-wavelength single-photon STED coupled imaging device, including: a femtosecond laser 1, a picosecond laser 2, an I electric shutter 3, an I half wave plate 4, an I beam splitter 5, an II beam splitter or I reflector switchable device 6, an II half wave plate 7, an I quarter wave plate 8, an II electric shutter 9, an II reflector 10, an III reflector 11, an I time delay reflector group 12, an IV reflector 13, an I dichroic mirror 14, a V reflector 15, an II time delay reflector group 16, a VI reflector 17, a glass rod 18, an III half wave plate 19, a polarizer 20, a VII reflector 21, an I lens 22, a polarization maintaining optical fiber 23, an II lens 24, a VIII reflector 25, a phase plate 26, an IX reflector 27, a lifting optical path reflector group 28, a confocal scanning microscope 29 and a confocal scanning microscope internal switchable dichroic plate 30; the light emitted by the femtosecond laser 1 is divided into two beams after passing through an I electric shutter 3, an I half wave plate 4 and an I beam splitter 5 in sequence, wherein one beam is used as a two-photon excitation light source, and the other beam is used as an STED light source of two photons and single photons. The light path as the two-photon excitation light source passes through the second beam splitter, the second half-wave plate 7, the first quarter-wave plate 8, the IX reflecting mirror 27 and the lifting light path reflecting mirror group 28 in the switchable device 6 in sequence, and then is guided into the confocal scanning microscope 29 for two-photon scanning imaging, and the switchable dichroic plate 30 in the confocal scanning microscope is selected as a dichroic plate suitable for two-photon imaging. The light path of the STED light source which is used as two-photon and single-photon sequentially passes through the V-shaped reflector 15, the II-shaped time delay reflector group 16 and the VI-shaped reflector 17, and then enters the glass rod 18 to perform pulse broadening. And then introduced into a polarization maintaining fiber 23 through a third half wave plate 19, a polarizer 20, a VII-th mirror 21, and an I-th lens 22 to further expand the pulse. The light beam emitted from the polarization maintaining fiber 23 is adjusted into parallel light by the II lens 24, passes through the VIII reflecting mirror 25, and then passes through the phase plate 26 to generate an annular light beam. Then the light passes through the I dichroic mirror 14, the II beam splitter or the I reflector in the switchable device 6, the II half wave plate 7, the I quarter wave plate 8, the IX reflector 27 and the lifting optical path reflector group 28 in sequence, and then is guided into the confocal scanning microscope 29 as a loss light beam.
In an embodiment of the invention, a picosecond laser 2 serving as a single photon excitation light source and a femtosecond laser 1 are synchronously output, a light beam emitted by the picosecond laser 2 sequentially passes through a second electric shutter 9, a second reflecting mirror 10, a third reflecting mirror 11, a first delay reflecting mirror group 12, a fourth reflecting mirror 13, a first dichroic mirror 14 and a first reflecting mirror in a switchable device 6 and then is superposed with a two-photon excitation light beam, and then passes through a second half-wave plate 7, a first quarter-wave plate 8, a first IX reflecting mirror 27 and a lifting light path reflecting mirror group 28, and then is guided into a confocal scanning microscope 29 for single photon scanning imaging, and a switchable dichroic plate 30 in the confocal scanning microscope is selected as a dichroic plate suitable for single photon imaging.
In an embodiment of the invention, the device can be switched into a single-wavelength two-photon STED imaging system and a dual-wavelength single-photon imaging system in a time-sharing manner. When the scanning microscope is switched to the single-wavelength two-photon STED imaging mode, the I electric shutter 3 is opened, the II electric shutter 9 is closed, the switchable device 6 is switched to the II optical splitter, and the switchable dichroism slice 30 in the confocal scanning microscope is switched to a dichroism slice suitable for two-photon imaging. When the double-wavelength single-photon imaging mode is switched, the I electric shutter 3 is opened, the II electric shutter 9 is opened, the switchable device 6 is switched into the I reflecting mirror, and the switchable dichromatic film 30 in the confocal scanning microscope is switched into a dichromatic film suitable for single-photon imaging.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (5)
1. The utility model provides a single wavelength two-photon STED and dual wavelength single photon STED coupling image device which characterized in that includes: the device comprises a femtosecond laser, a picosecond laser, an I electric shutter, an I half wave plate, an I beam splitter, an II beam splitter or an I reflector switchable device, an II half wave plate, an I quarter wave plate, an II electric shutter, an II reflector, an III reflector, an I time-delay reflector group, an IV reflector, an I dichroic mirror, a V reflector, an II time-delay reflector group, a VI reflector, a glass rod, an III half wave plate, a polarizer, a VII reflector, an I lens, a polarization-maintaining optical fiber, an II lens, a VIII reflector, a phase plate, an IX reflector, a lifting optical path reflector group and a confocal scanning microscope; the light emitted by the femtosecond laser sequentially passes through an I electric shutter, an I half wave plate and an I beam splitter and then is divided into two beams, wherein one beam is used as a two-photon excitation light source, and the other beam is used as an STED light source of two photons and a single photon; the two-photon excitation light source is guided into the confocal scanning microscope for two-photon scanning imaging after sequentially passing through a second beam splitter, a second half wave plate, a first quarter wave plate, a first IX reflector and a lifting light path reflector group in the switchable device; the STED light source of the two-photon and single-photon enters the glass rod to perform pulse broadening after sequentially passing through the V-th reflector, the II-th time delay reflector group and the VI-th reflector, and then is guided into the polarization-maintaining optical fiber to perform further pulse broadening after passing through the III-half wave plate, the polarizer, the VII-th reflector and the I-th lens; the emergent light after the polarization maintaining fiber is expanded passes through a second lens to adjust a light beam into parallel light, then passes through a VIII reflector, and then passes through a phase plate to be modulated into doughnut-shaped light spots; the ring-shaped light spot sequentially passes through the I dichroic mirror, the II beam splitter or the I reflecting mirror in the switchable device, the II half wave plate, the I quarter wave plate, the IX reflecting mirror and the lifting light path reflecting mirror group, and then is guided into the confocal scanning microscope 29 to be used as a loss light beam.
2. The single-wavelength two-photon STED and two-wavelength single-photon STED coupled imaging device of claim 1, wherein: the switchable dichromatic plate in the confocal scanning microscope is selected as a dichromatic plate suitable for two-photon imaging.
3. The single-wavelength two-photon STED and two-wavelength single-photon STED coupled imaging device of claim 1, wherein: the picosecond laser device is triggered to serve as a single photon excitation light source, light beams emitted by the picosecond laser device sequentially pass through the second electric shutter, the second reflecting mirror, the third reflecting mirror, the first time-delay reflecting mirror group, the fourth reflecting mirror, the first dichroic mirror and the first reflecting mirror in the switchable device to be superposed with the two-photon excitation light beams, and are guided into the confocal scanning microscope to perform single photon scanning imaging after passing through the second half wave plate, the first quarter wave plate, the IX reflecting mirror and the lifting light path reflecting mirror group.
4. The single-wavelength two-photon STED and two-wavelength single-photon STED coupled imaging device of claim 3, wherein: the switchable dichromatic film in the confocal scanning microscope is selected as a dichromatic film suitable for single photon imaging.
5. The switching method of the single-wavelength two-photon STED and two-wavelength single-photon STED coupled imaging device according to claim 1, comprising the following steps:
step S1, when switching to the single wavelength two-photon STED imaging mode, the I electric shutter is opened, the II electric shutter is closed, the switching device is switched to the II optical splitter, and the switchable dichromatic sheet in the confocal scanning microscope is switched to a dichromatic sheet suitable for two-photon imaging;
and step S2, when the double-wavelength single-photon imaging mode is switched, the I electric shutter is opened, the II electric shutter is opened, the switchable device is switched to the I reflecting mirror, and the switchable dichromatic film in the confocal scanning microscope is switched to a dichromatic film suitable for single-photon imaging.
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| CN105467572A (en) * | 2016-01-18 | 2016-04-06 | 北京大学 | Multiphoton subpulse STED-SPIM microscopic system realized by single wavelength |
| CN107941353A (en) * | 2018-03-06 | 2018-04-20 | 中国计量大学 | A kind of mensuration based on the associated coherence vortex topological charge of two-photon |
| CN108981935A (en) * | 2017-12-28 | 2018-12-11 | 三维通信股份有限公司 | A kind of implementation method tangled based on the two-photon higher dimensional space converted under Spontaneous Parametric |
| CA2973361A1 (en) * | 2017-07-14 | 2019-01-14 | Peter A. Vokhmin | Multichannel line scan sted microscopy, method and device |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105467572A (en) * | 2016-01-18 | 2016-04-06 | 北京大学 | Multiphoton subpulse STED-SPIM microscopic system realized by single wavelength |
| CA2973361A1 (en) * | 2017-07-14 | 2019-01-14 | Peter A. Vokhmin | Multichannel line scan sted microscopy, method and device |
| CN108981935A (en) * | 2017-12-28 | 2018-12-11 | 三维通信股份有限公司 | A kind of implementation method tangled based on the two-photon higher dimensional space converted under Spontaneous Parametric |
| CN107941353A (en) * | 2018-03-06 | 2018-04-20 | 中国计量大学 | A kind of mensuration based on the associated coherence vortex topological charge of two-photon |
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