CN103558193A - Two-photon microscope - Google Patents
Two-photon microscope Download PDFInfo
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- CN103558193A CN103558193A CN201310507178.0A CN201310507178A CN103558193A CN 103558193 A CN103558193 A CN 103558193A CN 201310507178 A CN201310507178 A CN 201310507178A CN 103558193 A CN103558193 A CN 103558193A
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Abstract
The embodiment of the invention relates to the technical field of optics and discloses a two-photon microscope. The microscope comprises a first light splitting element, a second light splitting element, a reflection element, a first lens, a second lens, a third lens, a dichroscope, an objective lens, an objective table and a photomultiplier, wherein an incident pulse light beam sequentially passes through the first light splitting element and the second light splitting element and then is emitted to the reflection element; after being reflected by the reflection element, the pulse light beam sequentially passes through the first lens, the second lens, the dichroscope and the objective lens to be emitted to the objective table; fluorescent light excited by the incident pulse light beam on a sample on the objective table is collected by the objective lens and then emitted to the dichroscope and then is reflected by the dichroscope to be emitted to the photomultiplier by the third lens. With the adoption of the embodiment of the invention, simultaneously-focused space and time point scanning imaging can be carried out on the sample and the imaging detection depth is increased.
Description
Technical field
The present invention relates to optical technical field, be specifically related to a kind of Two Photon Fluorescence.
Background technology
In biological vital tissue fluorescence imaging field, Two Photon Fluorescence has become a kind of requisite imaging tool.Compare single photon technology, its most important advantage is to carry out high-resolution imaging to tissue sample.Because Two Photon Fluorescence has subcellular resolution to scattering biological sample, make this kind of microscope become one of at present most popular micro-imaging technique, yet this kind of probe microscope degree of depth is limited at present.Increase along with imaging depth, because sample tissue is to the absorption of light and scattering process, incident laser power can present the decay of index, the signal at focus place is weakened, the Noise enhancement at non-focus place, thus signal to noise ratio (S/N ratio) (being the signal at focus place and the ratio of the noise outside the focus) variation of imaging caused.After a certain certain depth, the ground unrest outside focus finally can surpass focus signal, now resoluting signal and noise again, and this degree of depth is the limit investigation depth of this imaging device.Therefore, improving imaging signal to noise ratio (S/N ratio) is the key that increases investigation depth.
One of prior art is while utilizing wide field pulsed field to propagate in sample, the characteristic that its recurrence interval changes with the increase of the degree of depth, make pulse when arriving focal plane, there is the shortest recurrence interval, thereby in focal plane, produce stronger signal and suppressed focal plane ground unrest in addition simultaneously.This technology has realized the wide field two-photon micro-imaging function with optical section (optical section) ability, has improved the speed of two-photon micro-imaging.But, scattering process due to tissue, this wide field imaging (face scanning) technology is compared the Two Photon Fluorescence of general point scanning, and its spatial discrimination is along with the deterioration degree of imaging depth is more serious, so the increase imaging depth that this technology can not be real.
Two of prior art is by biprism grating, to realize incident light pulse to carry out time focusing in X-Z plane, and at perpendicular Y-Z Planar realization space clustering, thereby make light pulse form at place, imaging focal plane the line that the elapsed time focuses on, this kind of formation method coordinates scanning galvanometer can realize two-dimentional two-photon micro-imaging.Although this technology can improve imaging depth to a certain extent, but incident light pulse can only be focused on into a line, then the mode by line sweep obtains image, such design has inevitably reduced the lateral resolution of image, cause the fuzzy of image, so very limited to the raising of the actual imaging degree of depth.
Summary of the invention
The embodiment of the invention discloses a kind of Two Photon Fluorescence, can carry out the Mapping that room and time focuses on simultaneously to sample, increase the imaging detection degree of depth.
The embodiment of the present invention provides a kind of Two Photon Fluorescence, and described microscope comprises the first beam splitter, the second beam splitter, reflecting element, first lens, the second lens, the 3rd lens, dichroic mirror, object lens, objective table and photomultiplier, wherein:
Incident pulse light beam is inciding on described reflecting element successively after described the first beam splitter and the second beam splitter, and through described first lens, the second lens, dichroic mirror and object lens, incides described objective table successively after by described reflecting element reflection;
The fluorescence exciting on the sample of described incident pulse light beam on described objective table by described object lens, collected after dichroic mirror described in directive, and by after described dichroic mirror reflects through photomultiplier described in described the 3rd lens directive.
In above-mentioned Two Photon Fluorescence, described the first beam splitter and the second beam splitter are respectively grating, stagger relative with the second beam splitter of described the first beam splitter be arranged in parallel, so that described incident pulse light beam is producing light splitting successively after described the first beam splitter and the second beam splitter, form the parallel pulsed light beam of diffraction.
In above-mentioned Two Photon Fluorescence, described the first beam splitter and the second beam splitter are respectively Amici prism, a limit opposing parallel of one side of described the first beam splitter and the second beam splitter arranges, so that described incident pulse light beam is producing light splitting successively after described the first beam splitter and the second beam splitter, form the parallel pulsed light beam of diffraction.
In above-mentioned Two Photon Fluorescence, described reflecting element is galvanometer.
In above-mentioned Two Photon Fluorescence, the mobile object lens of described object lens for moving along input path.
In above-mentioned Two Photon Fluorescence, described reflecting element is fixing catoptron.
In above-mentioned Two Photon Fluorescence, described objective table is D translation platform.
In above-mentioned Two Photon Fluorescence, described first lens is scanning lens.
In above-mentioned Two Photon Fluorescence, described the second object lens are tube lens.
In above-mentioned Two Photon Fluorescence, described the 3rd object lens are collecting lens.
In the embodiment of the present invention, incident pulse light beam is inciding on described reflecting element successively after described the first beam splitter and the second beam splitter, form diffraction directional light, and pass through successively described first lens, the second lens, dichroic mirror and object lens after by described reflecting element reflection, be finally focused into and be mapped to described objective table; The fluorescence exciting on the sample of described incident pulse light beam on described objective table by described object lens, collected after dichroic mirror described in directive, and by after described dichroic mirror reflects through photomultiplier described in described the 3rd lens directive.Minute light action due to the first beam splitter and the second beam splitter, incident light pulse will produce light splitting, the light of different wave length occupies the diverse location of light beam, form the pulse of diffraction directional light, make before arriving object focal point, incident pulse width is by broadening, only in the just overlapping focusing of object focal point place all wavelengths, according to the Fourier transform relation of spectral width and pulse length, incident pulse possesses short pulse at focus place, thereby the time of realizing incident light pulse focuses on.In addition, due to the space-focusing effect of object lens to incident pulse light, then can carry out the Mapping that room and time focuses on simultaneously to sample, increase the imaging detection degree of depth.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme of the embodiment of the present invention, to the accompanying drawing of required use in embodiment be briefly described below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skills, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Fig. 1 is the structural representation of a kind of Two Photon Fluorescence of providing of the embodiment of the present invention;
Light path schematic diagram when Fig. 2 is the incident beam that provides of the embodiment of the present invention through two Amici prisms in Two Photon Fluorescence;
Fig. 3 is the situation of change schematic diagram that incident pulse light signal that the embodiment of the present invention provides is subject to recurrence interval modulation and pulse-length modulation in whole Two Photon Fluorescence system;
Fig. 4 is the structural representation of the another kind of Two Photon Fluorescence that provides of the embodiment of the present invention.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is a part of embodiment of the present invention, rather than whole embodiment.Embodiment based in the present invention, those of ordinary skills, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.
The embodiment of the invention discloses a kind of Two Photon Fluorescence, can carry out the Mapping that room and time focuses on simultaneously to sample, increase the imaging detection degree of depth.Following examples are elaborated respectively.
Refer to Fig. 1, Fig. 1 shows the structural representation of the Two Photon Fluorescence that the embodiment of the present invention provides.This Two Photon Fluorescence comprises the first beam splitter 1, the second beam splitter 2, reflecting element 3, first lens 4, the second lens 5, the 3rd lens 9, dichroic mirror 6, object lens 7, objective table 8 and photomultiplier 10, wherein:
In embodiment, the first beam splitter 1 and the second beam splitter 2 are grating, and certainly, in other embodiments, the first beam splitter 1 and the second beam splitter 2 can also be other beam splitters, for example, and Amici prism.When the first beam splitter 1 and the second beam splitter 2 are grating, as shown in Figure 1, the first beam splitter 1 can relatively stagger and be arranged in parallel with the second beam splitter 2,2. 1. the parallel pulsed light beam of incident incide the rear formation pulsed diffracted beam of the first beam splitter 1,2. light beam further incides after the second beam splitter 2, due to the reversibility of light path, 3. 2. light beam will become the parallel pulsed light beam of diffraction after the second beam splitter 2 reflections, and light beam is the light λ of middle different wave length 3.
1～λ
noccupy different positions; When the first beam splitter 1 and the second beam splitter 2 are Amici prism, as shown in Figure 2,2. 1. the parallel pulsed light beam of incident see through the rear formation pulsed diffracted beam of the first beam splitter 1,3. 2. light beam further see through the parallel pulsed light beam of the rear formation diffraction of the second beam splitter 2, and light beam is the light λ of middle different wave length 3.
1～λ
noccupy different positions.
Light beam is 3. by inciding on objective table 8 through first lens 4, the second lens 5, dichroic mirror 6 and object lens 7 successively after reflecting element 3 reflections.Particularly, reflecting element 3 is galvanometer, as preferred embodiment a kind of, reflecting element 3 is scanning galvanometer, light beam is 3. by changing light beam into 4. after reflecting element 3 reflections, 5. 4. light beam become light beam after first lens 4, 6. 5. light beam become light beam after the second lens 5, be to form parallel pulsed light beam 6. after 4. light beam expands with the second lens 5 through first lens 4, first lens 4 can be scanning lens, the second lens 5 can be tube lens, 7. 6. light beam focus on the objective table 8 at the place, focal plane that is positioned at object lens 7 through changing light beam after dichroic mirror 6 and object lens 7 into, due to minute light action of the first beam splitter 1 and the second beam splitter 2,3. light beam will be a branch of diffraction parallel beam, the light λ of different wave length
1～λ
ndifferent positions will be occupied, according to optical imaging concept, the pulsed light inciding on the first beam splitter 1 will arrive the focal plane of object lens 7 simultaneously, form the focus light with incident pulse light same pulse width, yet any point before light pulse arrives the focal plane of object lens 7, due to different diffraction light λ
1～λ
nthe path of experience is different, will form a more macrocyclic pulsed light, in like manner, in light pulse, through any point after the focal plane of object lens 7, can form equally one compared with the pulsed light in long pulse cycle, Fig. 3 shows the 1. situation of change of recurrence interval modulation and pulse-length modulation in whole system of incident beam, wherein, 1. 1 1. 1 time cycle of showing and space width of incident light pulse is shown, 7. represent light beam time cycle and space width 7., other pattern represent be respectively subject to the first beam splitter 1, the second beam splitter 2, reflecting element 3,, the variation of first lens 4, the second lens 5 and the lower incident light pulses of object lens 7 effect, by such optical focus modulator approach, can effectively reduce the fluorescent radiation probability of non-focus place sample tissue, strengthen the signal to noise ratio (S/N ratio) of fluorescent radiation signal.
The fluorescence that 7. light beam excites on the sample on objective table 8 by object lens 7, collected after directive dichroic mirror 6, and by after dichroic mirror 6 reflections through the 3rd lens 9 directive photomultipliers 10, wherein, the 3rd lens 9 can be collecting lens.In the present embodiment, whole Two Photon Fluorescence can carry out grid scanning by reflecting element 3 and form two dimensional image, and due to the mobile object lens of object lens 7 for moving along input path, can further by object lens 7, form 3-D scanning, be that the direction that object lens 7 can be arranged in turn along reflecting element 3, first lens 4, the second lens 5, dichroic mirror 6 and object lens 7 vertically moves, and coordinate reflecting element 3(galvanometer) grid scanning, thereby form the 3-D scanning image of sample.
Refer to Fig. 4, Fig. 4 shows the structural representation of the another kind of Two Photon Fluorescence that the embodiment of the present invention provides.This Two Photon Fluorescence comprises the first beam splitter 1 as shown in Figure 1, the second beam splitter 2, first lens 4, the second lens 5, the 3rd lens 9, dichroic mirror 6, object lens 7, objective table 8 and photomultiplier 10, therefore these parts do not repeat them here, difference is, radiated element 32 in Fig. 4 is catoptron, further, reflecting element 32 is static catoptron, moreover, objective table 82 is D translation platform, therefore, embodiment shown in Fig. 4 is the different of three-dimensional imaging mode from the difference of the embodiment shown in Fig. 1, what the embodiment in Fig. 4 adopted is to carry out three-dimensional imaging by the movement of objective table 82.
The focal spot modulation scanning imaging technology that the embodiment of the present invention provides a kind of room and time to combine, can be used for the imaging of thick sample tissue, the core of the Two Photon Fluorescence that the embodiment of the present invention provides is, not only spatially there is focusing, also there is in time a during focusing, can be by introducing spatial frequency distribution (light of different wave length occupy light beam without position), make only just overlapping at object focal point place all wavelengths, and according to the Fourier transform relation of spectral width and pulse length, guarantee only at focus place, just to possess short pulse, thereby focus on when realizing time and space, and, by Mapping, obtain image, compare wide field scanning and line sweep, the image blur effect having caused due to tissue scatter while having reduced same optical section surface imaging, when guaranteeing image high-resolution, the real imaging detection degree of depth that improves.
The Two Photon Fluorescence above embodiment of the present invention being provided is described in detail, applied specific case herein principle of the present invention and embodiment are set forth, the explanation of above embodiment is just for helping to understand method of the present invention and core concept thereof; , for one of ordinary skill in the art, according to thought of the present invention, all will change in specific embodiments and applications, in sum, this description should not be construed as limitation of the present invention meanwhile.
Claims (10)
1. a Two Photon Fluorescence, is characterized in that, described microscope comprises the first beam splitter, the second beam splitter, reflecting element, first lens, the second lens, the 3rd lens, dichroic mirror, object lens, objective table and photomultiplier, wherein:
Incident pulse light beam is inciding on described reflecting element successively after described the first beam splitter and the second beam splitter, and through described first lens, the second lens, dichroic mirror and object lens, incides described objective table successively after by described reflecting element reflection;
The fluorescence exciting on the sample of described incident pulse light beam on described objective table by described object lens, collected after dichroic mirror described in directive, and by after described dichroic mirror reflects through photomultiplier described in described the 3rd lens directive.
2. Two Photon Fluorescence according to claim 1, it is characterized in that, described the first beam splitter and the second beam splitter are respectively grating, stagger relative with the second beam splitter of described the first beam splitter be arranged in parallel, so that described incident pulse light beam is producing light splitting successively after described the first beam splitter and the second beam splitter, form the parallel pulsed light beam of diffraction.
3. Two Photon Fluorescence according to claim 1, it is characterized in that, described the first beam splitter and the second beam splitter are respectively Amici prism, a limit opposing parallel of one side of described the first beam splitter and the second beam splitter arranges, so that described incident pulse light beam is producing light splitting successively after described the first beam splitter and the second beam splitter, form the parallel pulsed light beam of diffraction.
4. according to the Two Photon Fluorescence described in any one in claim 1～3, it is characterized in that, described reflecting element is galvanometer.
5. Two Photon Fluorescence according to claim 4, is characterized in that, the mobile object lens of described object lens for moving along input path.
6. according to the Two Photon Fluorescence described in any one in claim 1～3, it is characterized in that, described reflecting element is fixing catoptron.
7. Two Photon Fluorescence according to claim 6, is characterized in that, described objective table is D translation platform.
8. according to the Two Photon Fluorescence described in any one in claim 1～3, it is characterized in that, described first lens is scanning lens.
9. Two Photon Fluorescence according to claim 8, is characterized in that, described the second object lens are tube lens.
10. Two Photon Fluorescence according to claim 9, is characterized in that, described the 3rd object lens are collecting lens.
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| CN104155274A (en) * | 2014-08-07 | 2014-11-19 | 华中科技大学 | Double beam plate lighting microscan imaging method and microscope |
| CN104198458A (en) * | 2014-09-26 | 2014-12-10 | 哈尔滨工业大学 | Femtosecond laser two-photon fluorescent biological microimaging system and imaging method thereof |
| CN104279984A (en) * | 2014-11-05 | 2015-01-14 | 哈尔滨工业大学 | Two-photon-method-based device and method for measuring smooth free-form surface sample |
| CN106290277A (en) * | 2016-08-08 | 2017-01-04 | 中国科学院苏州生物医学工程技术研究所 | A kind of measure the device and method in conversion nano fluorescent particle life-span on single dispersing |
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| CN109870441A (en) * | 2019-03-27 | 2019-06-11 | 浙江大学 | Three-dimensional super-resolution light slice fluorescent microscopic imaging method and device based on shift frequency |
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| CN104155274A (en) * | 2014-08-07 | 2014-11-19 | 华中科技大学 | Double beam plate lighting microscan imaging method and microscope |
| CN104198458A (en) * | 2014-09-26 | 2014-12-10 | 哈尔滨工业大学 | Femtosecond laser two-photon fluorescent biological microimaging system and imaging method thereof |
| CN104198458B (en) * | 2014-09-26 | 2017-02-22 | 哈尔滨工业大学 | Femtosecond laser two-photon fluorescent biological microimaging system and imaging method thereof |
| CN104279984A (en) * | 2014-11-05 | 2015-01-14 | 哈尔滨工业大学 | Two-photon-method-based device and method for measuring smooth free-form surface sample |
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| CN108982431A (en) * | 2017-06-01 | 2018-12-11 | 深圳先进技术研究院 | Online fluorescence detection device |
| CN107677653A (en) * | 2017-08-24 | 2018-02-09 | 南开大学 | Three-dimensional image forming apparatus and imaging method based on two-photon fluorescence |
| CN109307930A (en) * | 2018-11-05 | 2019-02-05 | 中国科学院苏州生物医学工程技术研究所 | The Two Photon Fluorescence of two-dimensional high speed scanning imagery is carried out using the acousto-optic deflection device combination of two kinds of different velocities of sound |
| CN109307930B (en) * | 2018-11-05 | 2023-09-12 | 中国科学院苏州生物医学工程技术研究所 | Two-photon microscope for two-dimensional high-speed scanning imaging by adopting two acousto-optic deflectors with different sound speeds |
| CN109724954A (en) * | 2018-12-07 | 2019-05-07 | 北京超维景生物科技有限公司 | Phosphor collection device, miniature Two Photon Fluorescence and two photon imaging method |
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