CN106980174A - A kind of comprehensive fluorescence super-resolution microscopic imaging device - Google Patents
A kind of comprehensive fluorescence super-resolution microscopic imaging device Download PDFInfo
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- CN106980174A CN106980174A CN201710114306.3A CN201710114306A CN106980174A CN 106980174 A CN106980174 A CN 106980174A CN 201710114306 A CN201710114306 A CN 201710114306A CN 106980174 A CN106980174 A CN 106980174A
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0068—Optical details of the image generation arrangements using polarisation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0056—Optical details of the image generation based on optical coherence, e.g. phase-contrast arrangements, interference arrangements
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
Abstract
The present invention discloses a kind of comprehensive fluorescence super-resolution microscopic imaging device, light beam is divided into symmetrical four bundles light road by three polarizing beam splitter mirrors first, then after the modulation that beam propagation angle and position are carried out by 4f galvanometer systems, beam is closed by two pieces of beam splitters, transmitted again by 4f systems in the form of converging to the back focal plane of microcobjective, finally it is incident on sample in four beam directional light modes by microcobjective, and interfere；By the shutter in four bundles light road, vortex phase plate and scanning galvanometer position adjustments, latticed, horizontal stripe shape and longitudinal stripe shape interference structure optical illumination pattern can be formed, and structure photoperiod, phase and direction arbitrarily can quickly be adjusted.The present invention can be achieved to include the full function such as interior angle reflected fluorescent light micro-imaging and Fourier's shift frequency iteration micro-imaging of Structured Illumination micro-imaging, three-dimensional structure optical illumination micro-imaging, annular.
Description
Technical field
The present invention relates to micro-imaging technique field, more particularly to a kind of comprehensive fluorescence super-resolution micro-imaging dress
Put.
Background technology
Cell is the basic structure and functional unit of all life activity, and people can be helped by probing into intracellular vital movement
Understand the vital movement of mankind itself.Fluorescence super-resolution micro-imaging technique has lossless, high specificity, penetration depth depth etc.
Advantage, can observe life of such as genetic transcription, the albumen synthesis with transport, the numerous generations of mass exchange in each organelle
Activity, thus the research of fluorescence associated super-resolution micro-imaging technique and device also emerges in an endless stream, and have important science valency
Value and practical significance.
At present, the super-resolution microtechnic of main flow has stimulated radiation to exhaust microscope (Stimulated emission
Depletion microscopy, STED), random optical rebuilds microscopy (stochastic optical
Reconstruction microscopy, STORM), photoactivation position finding microscope (photoactivated localization
Microscopy, PALM), Structured Illumination microscope (structured-illumination microscopy, SIM) is glimmering
Equation of light micrometric(al) microscope (Fluorescence emission difference, FED) etc., each of which has oneself uniqueness
Operation principle, independent system, independent mutually, it is difficult often to possess that this, which results in the micro- product of existing super-resolution, if needed
Multiple functions are realized, it is necessary to access disparate modules on same micro-platform, system complexity, operation difficulty has been significantly greatly increased
And cost.
The features such as SIM microscopes are due to wide visual field, low illumination luminous power, smaller phototoxicity is super in living body biological cell
There is larger application prospect in resolution imaging field.The SIM microscopic structures of current main flow mainly by physical grating or
Spatial light modulator (SLM) produces ± 1 order diffraction light beam, and light beam is being loaded onto into sample by lens combination and microcobjective
Surface, forms periodicity interference fringe in the way of interference, and then carries out Structured Illumination to sample, by Mechanical Moving or
The patten regulations loaded on SLM, carry out phase shift and striped rotation, finally obtain the necessary number needed for super resolution image restores
According to.For physical grating and SLM, the former needs accurate displacement servo-drive system to translate and rotate it, and the latter is limited
In SLM material properties (majority is liquid crystal), the time that they obtain a two field picture is general in tens of ms magnitudes, reports at present most
High shooting speed is converted into super resolution image frame number about in 6 frames or so, lived for changing faster biology in 100 frames or so
Body motion process is still not enough.
Fourier's shift frequency iteration micro-imaging (Fourier ptychographic microscopy, FPM) falls within width
Field microtechnic, is illuminated by specific structure light or speckle to sample, using the shift frequency iteration different from SIM image restorations
Algorithm, reduces the pseudomorphism in image restoration, and recovering quality will be substantially better than SIM.
Ring-type utilizing total internal reflection fluorescence microscope (Ring-TIRF) is to produce suddenly to die in medium another side using light total reflection
The characteristic of ripple, excites fluorescence molecule to observe the very thin region of fluorescence calibration sample, the dynamic range of observation generally 200nm with
Under.Ring-type TIRF resides in reduced laser speckle influence using a ring-shaped light circle formation TIRF illumination imaging, its advantage, soon
The multi-angle imaging of speed reduces the aberration that the time of 3D imagings and single angle imaging are produced, and breaks there is provided image 3D is carried out
The possibility that layer is rebuild, makes system possess Z axis nano-precision chromatography function.
The content of the invention
It is an object of the invention to provide a kind of comprehensive fluorescence super-resolution microscopic imaging device, it can be obtained by beam splitting system
Four beam coherent beams, and incoming position of the light beam at microcobjective entrance pupil is arbitrarily adjusted by 4f- galvanometer systems, so as to realize
Including Structured Illumination micro-imaging (SIM), three-dimensional structure optical illumination micro-imaging (3D-SIM), the full interior angle reflected fluorescent light of annular
Micro-imaging (Ring-TIRF), the function such as Fourier's shift frequency iteration micro-imaging (FPM).
To achieve the above object, the invention provides following scheme：
A kind of comprehensive fluorescence super-resolution microscopic imaging device, including light source, in addition：
With the first polarization beam apparatus being arranged in light path, and respectively positioned at the first polarization beam apparatus reflection and thoroughly
The second polarization beam apparatus and the 3rd polarization beam apparatus penetrated in light path, the incident beam for light source to be sent are divided into four beam intensities
Equal light beam；
With the beam splitter being arranged on four bundles light beam optical path, for closing beam to four bundles light Shu Jinhang；
With a microcobjective, for the four beam directional lights closed after beam to be incident on into sample, and interfere, form knot
Structure optical illumination；
Also there is the optics being separately positioned on four bundles light beam optical path to open the door, for selectively opened/corresponding light of closing
Beam, forms the Both wide field illumination or TIRF light illumination modes of 2 beam light, 3 beam light and 4 beam optical interference patterns or 1 beam light.
It is preferred that：With the polarizer between the light source and the first polarization beam apparatus, for entering of sending light source
Irradiating light beam is changed into linearly polarized light, and described linearly polarized light is divided into intensity identical p light and s after by the first polarization beam apparatus
Light；
With the one 1/2 wave plate being separately positioned in the light path of the p light and s light, for by described p light and s light
The second polarization beam apparatus and the 3rd polarization beam apparatus are respectively enterd after rotation 45° angle, the equal light beam of four beam intensities is formed.
As preferred, transmitted in second polarization beam apparatus and the 2nd 1/2 ripple is set in the light path for the p-polarization light to be formed
Piece, s polarised lights are become for p-polarization light to be rotated by 90 °；
3rd 1/2 wave plate is set in the s polarised light light paths that the 3rd polarization beam apparatus is reflected to form, for s is inclined
The light that shakes, which is rotated by 90 °, becomes p-polarization light.
As preferred, with the 4f- galvanometer modules being located at respectively on four bundles light beam optical path, for each light beam of system aobvious
The Exit positions of speck mirror back focal plane, to change shape, the cycle of interference fringe.
As preferred, described 4f- galvanometers module including the first galvanometer galvanometer for x scanning directions, for y side
It is described to the second galvanometer galvanometer of scanning, first reflective/transmission-type 4f systems and second reflective/transmission-type 4f systems
The front focal plane of first reflective/transmission-type 4f systems should be overlapped with the first galvanometer galvanometer, second reflective/transmission-type 4f systems
The back focal plane of system should be overlapped with the second galvanometer galvanometer, the back focal plane of first reflective/transmission-type 4f systems and the second reflection
The front focal plane of formula/transmission-type 4f systems should be overlapped, and the first galvanometer galvanometer and the second galvanometer galvanometer are in conjugate position.
As preferred, first described reflective/transmission-type 4f systems and second reflective/transmission-type 4f systems be from
Axis paraboloidal mirror or achromatic lens.
In the present invention, described beam splitter includes：First beam splitter, for the second polarization beam apparatus beam splitting to be obtained into two beams
Light beam closes beam；Second beam splitter, beam is closed for the 3rd polarization beam apparatus beam splitting to be obtained into two light beams；4th polarization beam apparatus,
For closing beam to the first beam splitter and the light beam of the second beam splitter outgoing.
Further, vortex phase plate is set respectively on the emitting light path of first beam splitter and the second beam splitter,
For being rotated to light beam polarization direction, light beam is set to produce interference in sample surfaces with s polarization modes.
It is preferred that, the regulation light in the transmission of the second polarization beam apparatus and the 3rd polarization beam apparatus or/and reflected light path
Path difference mechanism, for adjusting the optical path difference between light beam to change the phase of interference fringe.
It is to be coated with the straight of metallic reflective coating by what piezoelectric position moving stage drove as preferred, described regulation optical path difference mechanism
Angle prism.
The fluorescence super-resolution microscopic imaging device of the present invention, can not only realize SIM, 3D-SIM, FPM and Ring-TIRF
Microtechnic, and the different super-resolution microtechnics imaging to biological sample can be realized by same Optical devices, to sample
More comprehensive super-resolution imaging research is carried out, to carry out more deep understanding to cyto-dynamics, while again can be very big
System module quantity and system complexity are simplified.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to institute in embodiment
The accompanying drawing needed to use is briefly described, it should be apparent that, drawings in the following description are only some implementations of the present invention
Example, for those of ordinary skill in the art, without having to pay creative labor, can also be according to these accompanying drawings
Obtain other accompanying drawings.
Fig. 1 is comprehensive fluorescence super-resolution microscopic imaging device structural representation of the invention；
Fig. 2 is 4f- galvanometers modular structure schematic diagram of the present invention；
Fig. 3 is another structural representation of 4f- galvanometers module of the present invention；
Fig. 4 is vortex phase plate optical direction spatial distribution map of the present invention；
Fig. 5 is polarisation distribution schematic diagram of the line polarisation after vortex phase plate；
Fig. 6 is incoming position schematic diagram of the focal beam spot in microcobjective entrance pupil face；Wherein, Fig. 6 (a) is under the pattern of wide field
Incoming position figure, Fig. 6 (b) is the incident beam location drawing under Ring-TIRF patterns, and Fig. 6 (c) is SIM/TIRF-SIM pattern tripping in
The irradiating light beam location drawing, Fig. 6 (d) is the incident beam location drawing under TIRF-SIM patterns, and Fig. 6 (e) is incident light under 3D-SIM patterns
Beam position figure, Fig. 6 (f) is the incident beam location drawing under FPM patterns；
Fig. 7 is the part-structure light effect schematic diagram produced by apparatus of the present invention, wherein, Fig. 7 (a) is interference fringe striped
Axial distribution map, Fig. 7 (b) is latticed interference fringe picture.
Symbol description：
Collimation lens 1, the polarizer 2, plane mirror 3, the first polarizing beam splitter cube (PBS) 4 (a), the second polarization point
Beam device 4 (b), the 3rd polarization beam apparatus 4 (c), the 4th polarization beam apparatus 4 (d), the first beam splitter (BS) 5 (a) and the second beam splitter
(BS) 5 (b), the first 4f- galvanometer modules 6 (a), the 2nd 4f- galvanometer modules 6 (b), the 3rd 4f- galvanometer modules 6 (c), the 4th 4f-
Galvanometer module 6 (d), the first electronic shutter7 (a), the second electronic shutter7 (b), the 3rd electronic shutter7 (c), the 4th
Electronic shutter7 (d), the first one-dimensional piezoelectric position moving stage 8 (a) and the second one-dimensional piezoelectric position moving stage 8 (b), first to be coated with metal anti-
The right-angle prism 9 (a) of film is penetrated, second is coated with the right-angle prism 9 (b) of metallic reflective coating, and the 3rd is coated with the right angle of metallic reflective coating
Prism 9 (c), the 4th is coated with the right-angle prism 9 (d) of metallic reflective coating, the wave plate 10 (a) of the first achromatism 1/2nd, and second disappears
The wave plate 10 (b) of aberration 1/2nd, the wave plate 10 (c) of the 3rd achromatism 1/2nd, the wave plate 10 of the 4th achromatism 1/2nd
(d), the first D type speculums 11 (a), the 2nd D type speculums 11 (b), the 3rd D type speculums 11 (c), the 4th D types speculum 11
(d), the first vortex phase plate 12 (a) and the second vortex phase plate 12 (b), the first f- θ lens 13 (a), the 2nd f- θ lens 13
(b), the 3rd f- θ lens 13 (c), the 4th f- θ lens 13 (d), the first pipe mirror (Tube lens) 14 (a), the second pipe mirror (Tube
Lens) 14 (b), the 3rd pipe mirror (Tube lens) 15, microcobjective 16, dichroscope 17, imaging len 18, CCD camera 19,
First galvanometer galvanometer 20 (a) and the second galvanometer galvanometer 20 (b), first reflective/transmission-type 4f systems 21 and second reflect
Formula/transmission-type 4f systems 22.
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 carried out clear, complete
Site preparation is described, it is clear that described embodiment is only a part of embodiment of the invention, rather than whole embodiments.It is based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under the premise of creative work is not made
Embodiment, belongs to the scope of protection of the invention.
The comprehensive fluorescence super-resolution microscopic imaging device of the present invention, can obtain four beam coherent beams by beam splitting system,
And incoming position of the light beam at microcobjective entrance pupil is arbitrarily adjusted by 4f- galvanometer systems, shone so as to realize including structure light
Bright micro-imaging (SIM), three-dimensional structure optical illumination micro-imaging (3D-SIM), the full interior angle reflected fluorescent light micro-imaging of annular
(Ring-TIRF), the function such as Fourier's shift frequency iteration micro-imaging (FPM).
In order to facilitate the understanding of the purposes, features and advantages of the present invention, it is below in conjunction with the accompanying drawings and specific real
Applying mode, the present invention is further detailed explanation.
As shown in figure 1, the comprehensive fluorescence super-resolution microscopic imaging device of the present invention includes：
Incident beam is reflected into the first polarization after being collimated via collimation lens 1 by the polarizer 2 and plane mirror 3
Beam splitter 4 (a) is divided into intensity identical p light and s light, then again by the 1/1st wave plate 10 (a) and the 1/2nd
Enter after wave plate 10 (b) rotation 45° angle in the second polarization beam apparatus 4 (b) and the 3rd polarization beam apparatus 4 (c), form 4 beam altogether
The equal light beam of intensity；Then the first 4f- galvanometer modules 6 (a), the 2nd 4f- galvanometer modules 6 (b), the 3rd 4f- galvanometers module 6 are passed through
(c) carried out with the 4th 4f- galvanometer modules 6 (d) after the modulation of beam propagation angle and position, by the first beam splitter 5 (a) and
Second beam splitter 5 (b) closes beam, then passes through the 3rd f- θ lens 13 (c), the 4th f- θ lens 13 (d), the first pipe mirror (Tube
Lens two groups of 4f systems and the 4th polarization beam apparatus 4 (d) that) 14 (a) and the second pipe mirror (Tube lens) 14 (b) are combined into
Close after beam, transmitted by the 3rd pipe mirror (Tube lens) 15 in the form of converging to the back focal plane of microcobjective 16, finally by micro-
It is incident on sample by object lens in four beam directional light modes, and is interfered, and forms Structured Illumination.
The s polarised lights reflected to form by the 3rd polarization beam apparatus 4 (c), should be rotated by 90 ° by the 3rd 1/2 wave plate 10 (c)
Become p-polarization light, transmit the p-polarization light formed by the second polarization beam apparatus 4 (b), should be revolved by the 4th 1/2 wave plate 10 (d)
Turn 90 ° and become s polarised lights.
By the first electronic shutter7 (a) in four bundles light road, the second electronic shutter7 (b), the 3rd electronic
Shutter7 (c) and the 4th electronic shutter7 (d) alternative beat the corresponding light beam of opening/closing, 2 beam light of formation, 3 beam light with
And 4 beam optical interference patterns, or 1 beam light Both wide field illumination or TIRF light illumination modes.
Light beam polarization direction is entered by the first vortex phase plate 12 (a) as shown in Figure 4 and the second vortex phase plate 12 (b)
Row rotation, produces interference, to form optimal fringe contrast with s polarization modes in sample surfaces all the time to ensure light beam.
As shown in figure 5, the polarization direction of incident light should be with vortex phase plate fast axle relative position relation：Incident light polarization
Direction should be vertical with 0 ° of quick shaft direction of vortex phase plate, thereby may be ensured that the polarization direction of emergent light is inclined in s all the time
Shake.
Pass through the first 4f- galvanometer modules 6 (a), the 2nd 4f- galvanometer modules 6 (b), the 3rd 4f- galvanometer modules 6 (c) and the 4th
Galvanometer in 4f- galvanometer modules 6 (d) carries out x, the modulation in y directions to light beam so that the Exit positions per Shu Guang can be at micro-
The optional position of object lens back focal plane, so as to change the shape of interference fringe, cycle.
Pass through movable, the regulation light beam of the first one-dimensional piezoelectric position moving stage 8 (a) and the second one-dimensional piezoelectric position moving stage 8 (b)
Between optical path difference, so as to change the phase of interference fringe.
First vortex phase plate 12 (a) and the second vortex phase plate 12 (b) should be at the f- θ lens 13 (a) of lens the first, the
Near the back focal plane of two f- θ lens 13 (b), the 3rd f- θ lens 13 (c) and the 4th f- θ lens 13 (d), with ensure four bundles light with
Convergence form passes through, so that light beam polarization direction is rotated to tangential polarization direction by same fast axle.
As shown in Fig. 2 the first 4f- galvanometer modules 6 (a), the 2nd 4f- galvanometer modules 6 (b), the 3rd 4f- galvanometer modules 6 (c)
Should be anti-with reference to first by the first galvanometer galvanometer 20 (a) and the second galvanometer galvanometer 20 (b) with the 4th 4f- galvanometer modules 6 (d)
The formula of penetrating/transmission-type 4f systems 21 and second are reflective/and transmission-type 4f systems 22 constitute, wherein the first galvanometer galvanometer 20 (a) is negative
X scanning directions are blamed, the second galvanometer galvanometer 20 (b) is responsible for y scanning directions；
The front focal plane of first reflective/transmission-type 4f systems 21 should be overlapped with the first galvanometer galvanometer 20 (a), and second is anti-
The back focal plane of the formula of penetrating/transmission-type 4f systems 22 should be overlapped with the second galvanometer galvanometer 20 (b), first reflective/transmission-type 4f
The back focal plane of system 21 is reflective with second/and the front focal planes of transmission-type 4f systems 22 should overlap, it is ensured that the first galvanometer galvanometer 20
(a) it is in conjugate position with the second galvanometer galvanometer 20 (b).
In the present embodiment, if the first 4f- galvanometer modules 6 (a), the 2nd 4f- galvanometer modules 6 (b), the 3rd 4f- galvanometers module 6
(c) it is and in the 4th 4f- galvanometer modules 6 (d) reflective structure, then first reflective/transmission-type 4f systems 21 and second reflect
Formula/transmission-type 4f systems 22 should select off axis paraboloidal mirror, if as shown in figure 3, the first 4f- galvanometer modules 6 (a), the 2nd 4f- shake
It is transmission-type structure, then the first reflection in mirror module 6 (b), the 3rd 4f- galvanometer modules 6 (c) and the 4th 4f- galvanometer modules 6 (d)
Formula/transmission-type 4f systems 21 and second are reflective/and transmission-type 4f systems 22 should select achromatic lens.
The fluorescence signal that sample is produced by Structured Illumination should be reflexed on imaging len 18 by dichroscope 17, and by
Imaging len 18 is imaged to CCD camera 19；
Four bundles light road should be as symmetrical as possible, and to ensure the coherence of four bundles light, light path difference slightly should be by second
The right-angle prism 9 (b) and the 3rd that are coated with metallic reflective coating are coated with the anterior-posterior translation of the right-angle prism 9 (c) of metallic reflective coating to mend
Repay.
Comprehensive fluorescence super-resolution microscopic imaging device in the present embodiment includes following several mode of operations：
Wide field pattern is：
The second electronic shutter7 (b), the 3rd electronic shutter7 (c) and the 4th electronic shutter7 (d) are closed, is opened
First electronic shutter7 (a), the galvanometer of the first 4f- galvanometer modules 6 (a) is in zero-bit, and light beam is in microcobjective entrance pupil
Heart position is incident, shown in such as Fig. 6 (a)；
Ring-TIRF patterns are：
The second electronic shutter7 (b), the 3rd electronic shutter7 (c) and the 4th electronic shutter7 (d) are closed, is opened
Asin (ω t) is loaded on first electronic shutter7 (a), the first galvanometer galvanometer 20 (a) of the first 4f- galvanometer modules 6 (a) to drive
Acos (ω t) drive signal is loaded on dynamic signal, the second galvanometer galvanometer 20 (b) so that incident beam is in such as Fig. 6 (b) institutes
It is shown into the TIRF regions in pupil face, formula, A is voltage, ω is frequency, by changing A values, thus it is possible to vary TIRF angles；
SIM/TIRF-SIM patterns are：
Electronic 3rd electronic shutter7 (c) and the 4th electronic shutter7 (d) are closed, the first electronic shutter7 is opened
(a) with the second electronic shutter7 (b), first, the first 4f- galvanometer modules 6 (a) and the first of the 2nd 4f- galvanometer modules 6 (b)
DC driven signal A is loaded on galvanometer galvanometer 20 (a), the second galvanometer galvanometer 20 (b) is in zero-bit so that incident light is in
0 ° of position as shown in Fig. 6 (c), so as to form horizontal direction interference fringe, is walked respectively using the first one-dimensional piezoelectric position moving stage 8 (a)
Enter the distance of 0, λ/6,2 λ/6, the phase shift of 0,2 π/3,4 π/3 is entered to striped, and using under CCD camera sync pulse jamming to respective phase
Photo；
Secondly, on the first galvanometer galvanometer 20 (a) of the first 4f- galvanometer modules 6 (a) and the 2nd 4f- galvanometer modules 6 (b)
DC driven signal A is loaded, in the loading of the second galvanometer galvanometer 20 (b)DC driven signal so that incident light is in
60 ° of positions as shown in Fig. 6 (c), so as to form 60 ° of direction interference fringes, are walked respectively using the first one-dimensional piezoelectric position moving stage 8 (a)
Enter the distance of 0, λ/6,2 λ/6, the phase shift of 0,2 π/3,4 π/3 is entered to striped, and using under CCD camera sync pulse jamming to respective phase
Photo；
Again, on the first galvanometer galvanometer 20 (a) of the first 4f- galvanometer modules 6 (a) and the 2nd 4f- galvanometer modules 6 (b)
DC driven signal-A is loaded, in the loading of the second galvanometer galvanometer 20 (b)DC driven signal so that incident light is in
120 ° of positions as shown in Fig. 6 (c), so as to form 120 ° of direction interference fringes, using the first one-dimensional piezoelectric position moving stage 8 (a) respectively
Stepping 0, the distance of λ/6,2 λ/6 enters the phase shift of 0,2 π/3,4 π/3 to striped, and using under CCD camera sync pulse jamming to respective phase
Photo；
Finally, 9 resulting data, corresponding super-resolution image is obtained by SIM algorithm invertings.
TIRF-SIM patterns are：
Basically identical with SIM in TIRF-SIM operations, difference is that DC voltage value A is bigger than in the case of SIM, to ensure
Incident beam is in the TIRF regions on entrance pupil face, i.e., 0 ° of position as shown in Fig. 6 (d), so that system intervention light is suddenly to die
Wave interference, 9 resulting data should obtain corresponding super-resolution image by TIRF-SIM algorithm invertings.
3D-SIM patterns are：
Close the 4th electronic shutter7 (d), open the first electronic shutter7 (a), the second electronic shutter7 (b) and
3rd electronic shutter7 (c), first, the first galvanometer of the first 4f- galvanometer modules 6 (a) and the 2nd 4f- galvanometer modules 6 (b)
DC driven signal A is loaded on galvanometer 20 (a), the second galvanometer galvanometer 20 (b) is in zero-bit, the 3rd 4f- galvanometer modules 6 (c)
Galvanometer be in zero-bit so that incident light be in as shown in Fig. 6 (e) 0 ° of position, so as to form the Three-beam Interfere of horizontal direction
Striped, axially distribution is as shown in Fig. 7 (a) for striped, using the first one-dimensional piezoelectric position moving stage 8 (a) stepping 0 respectively, λ/10,2 λ/10,
The distance of 3 λ/10,4 λ/10, the phase shift of 0,2 π/5,4 π/5,6 π/5,8 π/5 is entered to striped, and using CCD camera sync pulse jamming to accordingly
Photo under phase；
Secondly, on the first galvanometer galvanometer 20 (a) of the first 4f- galvanometer modules 6 (a) and the 2nd 4f- galvanometer modules 6 (b)
DC driven signal A is loaded, in the loading of the second galvanometer galvanometer 20 (b)DC driven signal so that incident light is in
60 ° of positions as shown in Fig. 6 (d), so as to form the Three-beam Interfere striped in 60 ° of directions, utilize the first one-dimensional piezoelectric position moving stage 8
(a) stepping 0 respectively, the distance of λ/10,2 λ/10,3 λ/10,4 λ/10 enters the phase shift of 0,2 π/5,4 π/5,6 π/5,8 π/5 to striped, and
Utilize the photo under CCD camera sync pulse jamming to respective phase；
Again, on the first galvanometer galvanometer 20 (a) of the first 4f- galvanometer modules 6 (a) and the 2nd 4f- galvanometer modules 6 (b)
DC driven signal-A is loaded, in the loading of the second galvanometer galvanometer 20 (b)DC driven signal so that incident light is in
120 ° of positions as shown in Fig. 6 (d), so as to form the Three-beam Interfere striped in 120 ° of directions, utilize the first one-dimensional piezoelectric position moving stage 8
(a) stepping 0 respectively, the distance of λ/10,2 λ/10,3 λ/10,4 λ/10 enters the phase shift of 0,2 π/5,4 π/5,6 π/5,8 π/5 to striped, and
Utilize the photo under CCD camera sync pulse jamming to respective phase；
Finally, 15 resulting data, corresponding super-resolution image is obtained by 3D-SIM algorithm invertings.
FPM patterns are：
First, the first electronic shutter7 (a) and the second electronic shutter7 (b), the first 4f- galvanometer modules 6 (a) are opened
With loading DC driven signal A, the second galvanometer galvanometer on the first galvanometer galvanometer 20 (a) of the 2nd 4f- galvanometer modules 6 (b)
20 (b) is in zero-bit so that incident light is in 0 ° of position as shown in Fig. 6 (e), so as to form horizontal direction interference fringe；3rd
DC driven signal A is loaded on second galvanometer galvanometer 20 (b) of 4f- galvanometer modules 6 (c) and the 4th 4f- galvanometer modules 6 (d),
First galvanometer galvanometer 20 (a) is in zero-bit so that incident light is in 90 ° of positions as shown in Fig. 6 (e), so as to form Vertical Square
To interference fringe, and then form the latticed interference fringe as shown in Fig. 7 (b)；
Secondly, using the stepping 0 respectively of the first one-dimensional piezoelectric position moving stage 8 (a), the distance of the λ of λ/20,2 λ/20 ... 9/20, to level
Striped is carried out after the phase shift of the π of 0, π/10,2 π/10 ... 9/10, using the distance of the second one-dimensional piezoelectric position moving stage 8 (b) stepping λ/20, to hanging down
Vertical bar line carries out the phase shift of π/10, repeats the mobile first one-dimensional step of piezoelectric position moving stage 8 (a) 10 and the second one-dimensional piezoelectric position moving stage
The process of the step of 8 (b) stepping 1, until the second one-dimensional piezoelectric position moving stage 8 (b) stepping number of times also reaches 10 steps, sweeps line by line so as to realize
Process is retouched, and utilizes 100 data under CCD camera sync pulse jamming to respective phase；
Finally, 100 resulting data, corresponding super-resolution image is obtained by FPM shift frequency iterative algorithm invertings.
TIRF-FPM patterns are：
Basically identical with FPM in TIRF-FPM operations, difference is that DC voltage value A is bigger than in the case of FPM, to ensure
Incident beam is in the TIRF regions on entrance pupil face, so as to produce the illumination of evanescent wave interference grid.
The embodiment of each in this specification is described by the way of progressive, and what each embodiment was stressed is and other
Between the difference of embodiment, each embodiment identical similar portion mutually referring to.
Specific case used herein is set forth to the principle and embodiment of the present invention, and above example is said
The bright method and its core concept for being only intended to help to understand the present invention；Simultaneously for those of ordinary skill in the art, foundation
The thought of the present invention, will change in specific embodiments and applications.In summary, this specification content is not
It is interpreted as limitation of the present invention.
Claims (10)
1. a kind of comprehensive fluorescence super-resolution microscopic imaging device, including light source, it is characterised in that：
With the first polarization beam apparatus being arranged in light path, and the reflection positioned at the first polarization beam apparatus and transmitted light respectively
The second polarization beam apparatus and the 3rd polarization beam apparatus on road, it is equal that the incident beam for light source to be sent is divided into four beam intensities
Light beam；
With the beam splitter being arranged on four bundles light beam optical path, for closing beam to four bundles light Shu Jinhang；
With a microcobjective, for the four beam directional lights closed after beam to be incident on into sample, and interfere, form structure light
Illumination；
Also there is the optics being separately positioned on four bundles light beam optical path to open the door, for selectively opened/corresponding light beam of closing, shape
Into 2 beam light, 3 beam light and 4 beam optical interference patterns or the Both wide field illumination or TIRF light illumination modes of 1 beam light.
2. comprehensive fluorescence super-resolution microscopic imaging device as claimed in claim 1, it is characterised in that：With positioned at the light
The polarizer between source and the first polarization beam apparatus, the incident beam for light source to be sent is changed into linearly polarized light, and described line is inclined
The light that shakes is divided into intensity identical p light and s light after by the first polarization beam apparatus；
With the one 1/2 wave plate being separately positioned in the light path of the p light and s light, for described p light and s light to be rotated
The second polarization beam apparatus and the 3rd polarization beam apparatus are respectively enterd after 45° angle, the equal light beam of four beam intensities is formed.
3. comprehensive fluorescence super-resolution microscopic imaging device as claimed in claim 2, it is characterised in that：In the described second polarization
Beam splitter, which is transmitted, sets the 2nd 1/2 wave plate in the light path for the p-polarization light to be formed, s polarizations are become for p-polarization light to be rotated by 90 °
Light；
3rd 1/2 wave plate is set in the s polarised light light paths that the 3rd polarization beam apparatus is reflected to form, for by s polarised lights
It is rotated by 90 ° and becomes p-polarization light.
4. comprehensive fluorescence super-resolution microscopic imaging device as claimed in claim 1, it is characterised in that：With respectively positioned at four
4f- galvanometer modules in light beams light path, for each light beam of system microcobjective back focal plane Exit positions, to change interference
The shape of striped, cycle.
5. comprehensive fluorescence super-resolution microscopic imaging device as claimed in claim 4, it is characterised in that：Described 4f- galvanometers
Module includes the first galvanometer galvanometer for x scanning directions, the second galvanometer galvanometer for y scanning directions, the first reflection
Formula/transmission-type 4f systems and second reflective/transmission-type 4f systems, the front focal plane of described first reflective/transmission-type 4f systems
It should be overlapped with the first galvanometer galvanometer, the back focal plane of second reflective/transmission-type 4f systems should be with the second galvanometer galvanometer
Overlapping, the back focal planes of first reflective/transmission-type 4f systems is reflective with second/and the front focal planes of transmission-type 4f systems should overlap,
First galvanometer galvanometer and the second galvanometer galvanometer are in conjugate position.
6. comprehensive fluorescence super-resolution microscopic imaging device as claimed in claim 5, it is characterised in that：The first described reflection
Formula/transmission-type 4f systems and second reflective/transmission-type 4f systems are off axis paraboloidal mirror or achromatic lens.
7. comprehensive fluorescence super-resolution microscopic imaging device as claimed in claim 1, it is characterised in that：Described beam splitter bag
Include：
First beam splitter, beam is closed for the second polarization beam apparatus beam splitting to be obtained into two light beams；
Second beam splitter, beam is closed for the 3rd polarization beam apparatus beam splitting to be obtained into two light beams；
4th polarization beam apparatus, for closing beam to the first beam splitter and the light beam of the second beam splitter outgoing.
8. comprehensive fluorescence super-resolution microscopic imaging device as claimed in claim 7, it is characterised in that：In first beam splitting
Vortex phase plate is set respectively on the emitting light path of mirror and the second beam splitter, for being rotated to light beam polarization direction, makes light
Beam produces interference with s polarization modes in sample surfaces.
9. comprehensive fluorescence super-resolution microscopic imaging device as claimed in claim 1, it is characterised in that：In the second polarization beam splitting
Regulation optical path difference mechanism in the transmission of device and the 3rd polarization beam apparatus or/and reflected light path, for adjusting the light between light beam
Path difference is to change the phase of interference fringe.
10. comprehensive fluorescence super-resolution microscopic imaging device as claimed in claim 9, it is characterised in that：Described regulation light
Path difference mechanism is the right-angle prism for being coated with metallic reflective coating driven by piezoelectric position moving stage.
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