CN106980174B - A kind of comprehensive fluorescence super-resolution microscopic imaging device - Google Patents
A kind of comprehensive fluorescence super-resolution microscopic imaging device Download PDFInfo
- Publication number
- CN106980174B CN106980174B CN201710114306.3A CN201710114306A CN106980174B CN 106980174 B CN106980174 B CN 106980174B CN 201710114306 A CN201710114306 A CN 201710114306A CN 106980174 B CN106980174 B CN 106980174B
- Authority
- CN
- China
- Prior art keywords
- light
- galvanometer
- polarization
- imaging device
- microscopic imaging
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- 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
-
- 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
-
- 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
-
- 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 for carrying out beam propagation angle and position by 4f- galvanometer system, beam is closed by two pieces of beam splitters, it is transmitted to the back focal plane of microcobjective in the form of converging by 4f system again, it is incident on sample by microcobjective in a manner of four beam directional lights finally, and is interfered；By the position adjusting of shutter, vortex phase plate and scanning galvanometer in four bundles light road, latticed, horizontal stripe shape and longitudinal stripe shape interference structure optical illumination mode can be formed, and structure photoperiod, phase and direction arbitrarily can quickly adjust.The present invention can be achieved including Structured Illumination micro-imaging, three-dimensional structure optical illumination micro-imaging, the full functions such as interior angle reflected fluorescent light micro-imaging and Fourier's shift frequency iteration micro-imaging of annular.
Description
Technical field
The present invention relates to micro-imaging technique fields, fill more particularly to a kind of comprehensive fluorescence super-resolution micro-imaging
It sets.
Background technique
Cell is the movable basic structure of all life and functional unit, and people can be helped by probing into intracellular vital movement
Understand the vital movement of mankind itself.Fluorescence super-resolution micro-imaging technique is deep etc. with lossless, high specificity, penetration depth
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 one after another, and has important scientific valence
Value and practical significance.
Currently, the super-resolution microtechnic of mainstream 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 has oneself unique
Working principle, independent system, independent mutually, this results in the micro- product of existing super-resolution to be often difficult to have, if needed
It realizes multiple functions, needs to access disparate modules on same micro-platform, system complexity, operation difficulty has been significantly greatly increased
And cost.
The features such as SIM microscope is due to wide visual field, low illumination optical power, smaller phototoxicity is super in living body biological cell
There is biggish application prospect in resolution imaging field.At present the SIM microscopic structure of mainstream mainly pass through physical grating or
Spatial light modulator (SLM) generates ± 1 grade of diffracted beam, and light beam is being loaded onto sample by lens system and microcobjective
Surface, forms periodical interference fringe in a manner of interference, and then carries out Structured Illumination to sample, by Mechanical Moving or
The patten loaded on SLM is adjusted, and is carried out phase shift and striped rotation, is finally obtained the necessary number needed for super resolution image restores
According to.For physical grating and SLM, the former needs accurate displacement servo-system to translate and rotate it, and the latter is limited
In SLM material property (majority is liquid crystal), they obtain the time of a frame image generally in tens of ms magnitudes, report at present most
High shooting speed is converted into super resolution image frame number about in 6 frames or so, lives for changing faster biology in 100 frames or so
Body motion process is still insufficient.
Fourier's shift frequency iteration micro-imaging (Fourier ptychographic microscopy, FPM) also belongs to width
Field microtechnic, illuminates sample by specific structure light or speckle, using the shift frequency iteration different from SIM image restoration
Algorithm, reduces the pseudomorphism in image restoration, and recovering quality will be substantially better than SIM.
Cyclic annular utilizing total internal reflection fluorescence microscope (Ring-TIRF) is suddenly to be died using light total reflection in the generation of medium another side
The characteristic of wave, excites fluorescent molecule to observe the very thin region of fluorescence calibration sample, the dynamic range of observation usually 200nm with
Under.Cyclic annular TIRF forms TIRF illumination imaging using a cyclic annular aperture, its advantage resides in reduced laser speckle influence, fastly
The color difference that the time for reducing 3D imaging is imaged in the multi-angle of speed and single angle imaging generates, and it is disconnected to provide progress image 3D
The possibility that layer is rebuild, makes system have Z axis nano-precision chromatography function.
Summary of the invention
The object of the present invention is to provide a kind of comprehensive fluorescence super-resolution microscopic imaging devices, 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 system, 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 functions such as Fourier's shift frequency iteration micro-imaging (FPM).
To achieve the above object, the present invention provides following schemes:
A kind of comprehensive fluorescence super-resolution microscopic imaging device, including light source, in addition:
With the first polarization beam apparatus being arranged in optical path, and be located at the first polarization beam apparatus reflection and thoroughly
The second polarization beam apparatus and third polarization beam apparatus in optical path are penetrated, the incident beam for issuing light source is 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, it is incident on sample for four beam directional lights after beam will to be closed, and interfere, forms 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 mode of 2 beam light, 3 beam light and 4 beam optical interference patterns or 1 beam light.
It is preferred: there is the polarizer between the light source and the first polarization beam apparatus, enter for issue light source
Irradiating light beam becomes linearly polarized light, and the linearly polarized light is divided into the identical p light of intensity and s after through the first polarization beam apparatus
Light；
With the one 1/2 wave plate and the 2nd 1/2 wave plate in the optical path for being separately positioned on the p light and s light, it is used for institute
The second polarization beam apparatus and third polarization beam apparatus are respectively enterd after p light and s light the rotation 45° angle stated, forms four beam intensity phases
Equal light beams.
Preferably, the 4th 1/2 wave is arranged in the optical path that second polarization beam apparatus transmits the p-polarization light to be formed
Piece becomes s polarised light for p-polarization light to be rotated by 90 °；
3rd 1/2 wave plate is set in the s polarised light optical path that the third polarization beam apparatus reflects to form, for s is inclined
Vibration light, which is rotated by 90 °, becomes p-polarization light.
Preferably, there is the 4f- galvanometer module being located on four bundles light beam optical path, for modulating each light beam aobvious
The Exit positions of speck mirror back focal plane, to change shape, the period of interference fringe.
Preferably, the 4f- galvanometer module includes for the first galvanometer galvanometer of the direction x scanning, for the side y
It is described to the second galvanometer galvanometer of scanning, first reflective/transmission-type 4f system and second reflective/transmission-type 4f system
The front focal plane of first reflective/transmission-type 4f system should be overlapped with the first galvanometer galvanometer, second reflective/transmission-type 4f system
The back focal plane of system should be overlapped with the second galvanometer galvanometer, the back focal plane of first reflective/transmission-type 4f system and the second reflection
Formula/transmission-type 4f system front focal plane should be overlapped, and the first galvanometer galvanometer and the second galvanometer galvanometer are in conjugate position.
Preferably, the first reflective/transmission-type 4f system and second reflective/transmission-type 4f system be from
Axis paraboloidal mirror or achromatic lens.
In the present invention, the beam splitter includes: the first beam splitter, for the second polarization beam apparatus beam splitting to be obtained two beams
Light beam closes beam；Second beam splitter closes beam for third polarization beam apparatus beam splitting to be obtained two light beams；4th polarization beam apparatus,
Beam is closed for the light beam to the first beam splitter and the outgoing of the second beam splitter.
Further, vortex phase plate is respectively set on the emitting light path of first beam splitter and the second beam splitter,
For rotating to light beam polarization direction, light beam is made to generate interference in sample surfaces with s polarization mode.
Preferably, the adjusting light in the transmission of the second polarization beam apparatus and third 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.
Preferably, the adjusting optical path difference mechanism is to be coated with the straight of metallic reflective coating by what piezoelectric position moving stage drove
Angle prism.
Fluorescence super-resolution microscopic imaging device of the invention, can not only realize SIM, 3D-SIM, FPM and Ring-TIRF
Microtechnic, and can be realized by same Optical devices and the different super-resolution microtechnics of biological sample are imaged, 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.
Detailed description of the invention
It in order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, below will be to institute in embodiment
Attached drawing to be used is needed to be briefly described, it should be apparent that, the accompanying drawings in the following description is only some implementations of the invention
Example, for those of ordinary skill in the art, without any creative labor, can also be according to these attached drawings
Obtain other attached drawings.
Fig. 1 is the comprehensive fluorescence super-resolution microscopic imaging device structural schematic diagram of the present invention；
Fig. 2 is 4f- galvanometer modular structure schematic diagram of the present invention；
Fig. 3 is another structural schematic diagram of 4f- galvanometer 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 mode of wide field
Incoming position figure, Fig. 6 (b) are the incident beam location drawing under Ring-TIRF mode, and Fig. 6 (c) is the tripping in of SIM/TIRF-SIM mode
The irradiating light beam location drawing, Fig. 6 (d) are the incident beam location drawing under TIRF-SIM mode, and Fig. 6 (e) is incident light under 3D-SIM mode
Beam position figure, Fig. 6 (f) are the incident beam location drawing under FPM mode；
Fig. 7 is part-structure light effect schematic diagram caused by apparatus of the present invention, wherein Fig. 7 (a) is interference fringe striped
Axial distribution map, Fig. 7 (b) are latticed interference fringe picture.
Symbol description:
Collimation lens 1, the polarizer 2, plane mirror 3, the first polarization beam apparatus 4 (a), the second polarization beam apparatus 4 (b),
Third 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),
First 4f- galvanometer module 6 (a), the 2nd 4f- galvanometer module 6 (b), the 3rd 4f- galvanometer module 6 (c), the 4th 4f- galvanometer module 6
(d), the first electronic shutter7 (a), the second electronic shutter7 (b), the electronic shutter7 (c) of third, the 4th is 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 is coated with metallic reflective coating
Right-angle prism 9 (a), second is coated with the right-angle prism 9 (b) of metallic reflective coating, and third is coated with the right-angle prism 9 of metallic reflective coating
(c), the 4th right-angle prism 9 (d) for being coated with metallic reflective coating, the one 1/2 wave plate 10 (a), the 2nd 1/2 wave plate 10 (b), the 3rd 1/
2 wave plates 10 (c), the 4th 1/2 wave plate 10 (d), the first D type reflecting mirror 11 (a), the 2nd D type reflecting mirror 11 (b), the reflection of the 3rd D type
Mirror 11 (c), the 4th D type reflecting mirror 11 (d), the first vortex phase plate 12 (a) and the second vortex phase plate 12 (b), the first f- θ are saturating
Mirror 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), third Guan Jing (Tube lens) 15, microcobjective 16, dichroscope
17, imaging len 18, CCD camera 19, the first galvanometer galvanometer 20 (a) and the second galvanometer galvanometer 20 (b), first reflective/
Transmission-type 4f system 21 and second is reflective/transmission-type 4f system 22.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Comprehensive fluorescence super-resolution microscopic imaging device of the 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 system, so that realizing includes that structure light is shone
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 functions such as Fourier's shift frequency iteration micro-imaging (FPM).
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, with reference to the accompanying drawing and specific real
Applying mode, the present invention is described in further detail.
As shown in Figure 1, comprehensive fluorescence super-resolution microscopic imaging device of the invention includes:
Incident beam passes through the polarizer 2 after collimating via collimation lens 1 and plane mirror 3 is reflected into the first polarization
Beam splitter 4 (a) is divided into the identical p light of intensity and s light, then again by the first half wave plate 10 (a) and the second half
Wave plate 10 (b) enters in the second polarization beam apparatus 4 (b) and third polarization beam apparatus 4 (c) after rotating 45° angle, forms 4 beam in total
The equal light beam of intensity；Then pass through the first 4f- galvanometer module 6 (a), the 2nd 4f- galvanometer module 6 (b), the 3rd 4f- galvanometer module 6
(c) and after the 4th 4f- galvanometer module 6 (d) carries out 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 the 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) is combined into
After closing beam, the back focal plane of microcobjective 16 is transmitted in the form of convergence by third Guan Jing (Tube lens) 15, finally by micro-
It is incident on sample by object lens in a manner of four beam directional lights, and is interfered, and Structured Illumination is formed.
The s polarised light reflected to form by third polarization beam apparatus 4 (c) should be rotated by 90 ° by the 3rd 1/2 wave plate 10 (c)
Become p-polarization light, by the p-polarization light that the transmission of the second polarization beam apparatus 4 (b) is formed, should be revolved by the 4th 1/2 wave plate 10 (d)
Turn 90 ° and becomes s polarised light.
It is electronic by the first electronic shutter7 (a) in four bundles light road, the second electronic shutter7 (b), third
Shutter7 (c) and the 4th electronic shutter7 (d) alternative open/close corresponding light beam, formed 2 beam light, 3 beam light with
And 4 beam optical interference pattern or 1 beam light Both wide field illumination or TIRF light illumination mode.
By the first vortex phase plate 12 (a) as shown in Figure 4 and the second vortex phase plate 12 (b) to light beam polarization direction into
Row rotation, to guarantee that light beam generates interference in sample surfaces with s polarization mode always, to form optimal fringe contrast.
As shown in figure 5, the polarization direction of incident light is answered with vortex phase plate fast axle relative positional relationship are as follows: incident light polarization
Direction should be vertical with 0 ° of fast axis direction of vortex phase plate, and it is inclined to thereby may be ensured that the polarization direction of emergent light is in s always
Vibration.
Pass through the first 4f- galvanometer module 6 (a), the 2nd 4f- galvanometer module 6 (b), the 3rd 4f- galvanometer module 6 (c) and the 4th
Galvanometer in 4f- galvanometer module 6 (d) carries out x to light beam, the modulation in the direction y so that the Exit positions of every Shu Guang can be at it is micro-
Any position of object lens back focal plane, to change the shape of interference fringe, period.
By the back-and-forth motion of the first one-dimensional piezoelectric position moving stage 8 (a) and the second one-dimensional piezoelectric position moving stage 8 (b), light beam is adjusted
Between optical path difference, 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 first f- θ lens 13 (a) of lens, the
Two f- θ lens 13 (b), the 3rd f- θ lens 13 (c) and the 4th f- θ lens 13 (d) back focal plane near, with guarantee four bundles light with
Convergence form passes through, so that light beam polarization direction is rotated by same fast axle to tangential polarization direction.
As shown in Fig. 2, the first 4f- galvanometer module 6 (a), the 2nd 4f- galvanometer module 6 (b), the 3rd 4f- galvanometer module 6 (c)
It should be anti-in conjunction with first by the first galvanometer galvanometer 20 (a) and the second galvanometer galvanometer 20 (b) with the 4th 4f- galvanometer module 6 (d)
The formula of penetrating/transmission-type 4f system 21 and second is reflective/and transmission-type 4f system 22 constitutes, wherein the first galvanometer galvanometer 20 (a) is negative
The scanning of the direction x is blamed, the second galvanometer galvanometer 20 (b) is responsible for the scanning of the direction y；
The front focal plane of first reflective/transmission-type 4f system 21 should be overlapped with the first galvanometer galvanometer 20 (a), and second is anti-
The formula of penetrating/transmission-type 4f system 22 back focal plane should be overlapped with the second galvanometer galvanometer 20 (b), first reflective/transmission-type 4f
The back focal plane of system 21 should be overlapped with the front focal plane of second reflective/transmission-type 4f system 22, guarantee the first galvanometer galvanometer 20
(a) conjugate position is in the second galvanometer galvanometer 20 (b).
In the present embodiment, if the first 4f- galvanometer module 6 (a), the 2nd 4f- galvanometer module 6 (b), the 3rd 4f- galvanometer module 6
It (c) and is reflective structure in the 4th 4f- galvanometer module 6 (d), then first reflective/transmission-type 4f system 21 and the second reflection
Formula/transmission-type 4f system 22 should select off axis paraboloidal mirror, as shown in figure 3, if the first 4f- galvanometer module 6 (a), the 2nd 4f- shake
It is transmission-type structure in mirror module 6 (b), the 3rd 4f- galvanometer module 6 (c) and the 4th 4f- galvanometer module 6 (d), then the first reflection
Formula/transmission-type 4f system 21 and second is reflective/and transmission-type 4f system 22 should select achromatic lens.
The fluorescence signal that sample is generated by Structured Illumination should be reflexed on imaging len 18 by dichroscope 17, and by
In the imaging to CCD camera 19 of imaging len 18；
Four bundles light road should be as symmetrical as possible, and to guarantee the coherence of four bundles light, light path difference slightly should be by second
The right-angle prism 9 (b) and third 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
It repays.
Comprehensive fluorescence super-resolution microscopic imaging device in the present embodiment includes following several operating modes:
Wide field mode are as follows:
The second electronic shutter7 (b), the electronic shutter7 (c) of third and the 4th electronic shutter7 (d) are closed, is opened
First electronic shutter7 (a), the galvanometer of the first 4f- galvanometer module 6 (a) are in zero-bit, and light beam is in microcobjective entrance pupil
Heart position is incident, as shown in Fig. 6 (a)；
Ring-TIRF mode are as follows:
The second electronic shutter7 (b), the electronic shutter7 (c) of third and the 4th electronic shutter7 (d) are closed, is opened
First electronic shutter7 (a), load Asin (ω t) is driven on the first galvanometer galvanometer 20 (a) of the first 4f- galvanometer module 6 (a)
Signal is moved, loads Acos (ω t) driving signal on the second galvanometer galvanometer 20 (b), so that incident beam is in such as Fig. 6 (b) institute
It is shown into the region TIRF in pupil face, in formula, A is voltage, and ω is frequency, passes through and changes A value, thus it is possible to vary TIRF angle；
SIM/TIRF-SIM mode are as follows:
The electronic shutter7 (c) of electronic third and the 4th electronic shutter7 (d) are closed, the first electronic shutter7 is opened
(a) and the second electronic shutter7 (b), firstly, the first of the first 4f- galvanometer module 6 (a) and the 2nd 4f- galvanometer module 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
The 0 ° of position as shown in Fig. 6 (c) is walked to form horizontal direction interference fringe using the first one-dimensional piezoelectric position moving stage 8 (a) respectively
Into 0, λ/6, the distance of 2 λ/6 to striped into 0,2 π/3, the phase shift of 4 π/3, and is utilized under CCD camera sync pulse jamming to respective phase
Photo；
Secondly, on the first galvanometer galvanometer 20 (a) of the first 4f- galvanometer module 6 (a) and the 2nd 4f- galvanometer module 6 (b)
DC driven signal A is loaded, is loaded in the second galvanometer galvanometer 20 (b)DC driven signal, so that incident light is in
The 60 ° of positions as shown in Fig. 6 (c) are walked to form 60 ° of direction interference fringes using the first one-dimensional piezoelectric position moving stage 8 (a) respectively
Into 0, λ/6, the distance of 2 λ/6 to striped into 0,2 π/3, the phase shift of 4 π/3, and is utilized under CCD camera sync pulse jamming to respective phase
Photo；
Again, on the first galvanometer galvanometer 20 (a) of the first 4f- galvanometer module 6 (a) and the 2nd 4f- galvanometer module 6 (b)
DC driven signal-A is loaded, is loaded in the second galvanometer galvanometer 20 (b)DC driven signal, so that incident light is in
The 120 ° of positions as shown in Fig. 6 (c), to form 120 ° of direction interference fringes, respectively using the first one-dimensional piezoelectric position moving stage 8 (a)
Stepping 0, λ/6, the distance of 2 λ/6 to striped into 0,2 π/3, the phase shift of 4 π/3, and utilize under CCD camera sync pulse jamming to respective phase
Photo；
Finally, obtained 9 data, obtain corresponding super-resolution image by SIM algorithm inverting.
TIRF-SIM mode are as follows:
Almost the same with SIM in TIRF-SIM operation, difference is in the case of DC voltage value A ratio SIM greatly, to guarantee
Incident beam is in the region TIRF on entrance pupil face, i.e. the 0 ° of position as shown in Fig. 6 (d), so that system intervention light is suddenly to die
Wave interference, obtained 9 data, Ying You TIRF-SIM algorithm inverting obtain corresponding super-resolution image.
3D-SIM mode are as follows:
Close the 4th electronic shutter7 (d), open the first electronic shutter7 (a), the second electronic shutter7 (b) and
The electronic shutter7 (c) of third, firstly, the first galvanometer of the first 4f- galvanometer module 6 (a) and the 2nd 4f- galvanometer module 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 module 6 (c)
Galvanometer be in zero-bit so that incident light is in the 0 ° of position as shown in Fig. 6 (e), 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, and 2 λ/10,
3 λ/10,4 λ/10 distance to striped into 0,2 π/5,4 π/5,6 π/5, the phase shift of 8 π/5, and utilize CCD camera sync pulse jamming to accordingly
Photo under phase；
Secondly, on the first galvanometer galvanometer 20 (a) of the first 4f- galvanometer module 6 (a) and the 2nd 4f- galvanometer module 6 (b)
DC driven signal A is loaded, is loaded in the second galvanometer galvanometer 20 (b)DC driven signal, so that incident light is in
The 60 ° of positions as shown in Fig. 6 (d) utilize the first one-dimensional piezoelectric position moving stage 8 to form the Three-beam Interfere striped in 60 ° of directions
(a) stepping 0 respectively, λ/10,2 λ/10,3 λ/10, the distance of 4 λ/10, to striped into 0,2 π/5,4 π/5,6 π/5, the phase shift of 8 π/5, 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 module 6 (a) and the 2nd 4f- galvanometer module 6 (b)
DC driven signal-A is loaded, is loaded in the second galvanometer galvanometer 20 (b)DC driven signal, so that incident light is in
The 120 ° of positions as shown in Fig. 6 (d) utilize the first one-dimensional piezoelectric position moving stage 8 to form the Three-beam Interfere striped in 120 ° of directions
(a) stepping 0 respectively, λ/10,2 λ/10,3 λ/10, the distance of 4 λ/10, to striped into 0,2 π/5,4 π/5,6 π/5, the phase shift of 8 π/5, and
Utilize the photo under CCD camera sync pulse jamming to respective phase；
Finally, obtained 15 data, obtain corresponding super-resolution image by 3D-SIM algorithm inverting.
FPM mode are as follows:
Firstly, opening the first electronic shutter7 (a) and the second electronic shutter7 (b), the first 4f- galvanometer module 6 (a)
With load DC driven signal A, the second galvanometer galvanometer on the first galvanometer galvanometer 20 (a) of the 2nd 4f- galvanometer module 6 (b)
20 (b) are in zero-bit, so that incident light is in the 0 ° of position as shown in Fig. 6 (e), to form horizontal direction interference fringe；Third
DC driven signal A is loaded on second galvanometer galvanometer 20 (b) of 4f- galvanometer module 6 (c) and the 4th 4f- galvanometer module 6 (d),
First galvanometer galvanometer 20 (a) is in zero-bit, so that incident light is in the 90 ° of positions as shown in Fig. 6 (e), to form Vertical Square
To interference fringe, and then form the latticed interference fringe as shown in Fig. 7 (b)；
Secondly, using the first one-dimensional piezoelectric position moving stage 8 (a) stepping 0 respectively, λ/20,2 λ/20 ... the distance of 9 λ/20, to level
Striped carries out 0, π/10, after the phase shift of 9 π/10 of 2 π/10 ..., using the second one-dimensional piezoelectric position moving stage 8 (b) stepping λ/20 distance, to vertical
Vertical bar line carries out the phase shift of π/10, repeats mobile first one-dimensional 10 step of piezoelectric position moving stage 8 (a) and the second one-dimensional piezoelectric position moving stage
The process of 8 (b) stepping, 1 step is swept until the second one-dimensional piezoelectric position moving stage 8 (b) stepping number also reaches 10 steps to realize line by line
Process is retouched, and utilizes 100 data under CCD camera sync pulse jamming to respective phase；
Finally, obtained 100 data, obtain corresponding super-resolution image by FPM shift frequency iterative algorithm inverting.
TIRF-FPM mode are as follows:
Almost the same with FPM in TIRF-FPM operation, difference is in the case of DC voltage value A ratio FPM greatly, to guarantee
Incident beam is in the region TIRF on entrance pupil face, to generate the illumination of evanescent wave interference grid.
Each embodiment in this specification is described in a progressive manner, the highlights of each of the examples are with other
The difference of embodiment, the same or similar parts in each embodiment may refer to each other.
Used herein a specific example illustrates the principle and implementation of the invention, and above embodiments are said
It is bright to be merely used to help understand method and its core concept of the invention；At the same time, for those skilled in the art, foundation
Thought of the invention, there will be changes in the specific implementation manner and application range.In conclusion the content of the present specification 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 optical path, and it is located at the reflection and transmitted light of the first polarization beam apparatus
The second polarization beam apparatus and third polarization beam apparatus of road, it is equal that the incident beam for issuing light source 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, it is incident on sample for four beam directional lights after beam will to be closed, and interfere, forms 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
At 2 beam light, 3 beam light and 4 beam optical interference patterns or the Both wide field illumination or TIRF light illumination mode of 1 beam light.
2. comprehensive fluorescence super-resolution microscopic imaging device as described in claim 1, it is characterised in that: have and be located at the light
The polarizer between source and the first polarization beam apparatus, the incident beam for issuing light source become linearly polarized light, and the line is inclined
Vibration light is divided into the identical p light of intensity and s light after through the first polarization beam apparatus；
With the one 1/2 wave plate and the 2nd 1/2 wave plate in the optical path for being separately positioned on the p light and s light, for will be described
The second polarization beam apparatus and third polarization beam apparatus are respectively enterd after p light and s light rotation 45° angle, forms the equal light of four beam intensities
Beam.
3. comprehensive fluorescence super-resolution microscopic imaging device as claimed in claim 2, it is characterised in that: in second polarization
Beam splitter, which transmits, is arranged the 4th 1/2 wave plate in the optical path for the p-polarization light to be formed, become s polarization for p-polarization light to be rotated by 90 °
Light；
3rd 1/2 wave plate is set in the s polarised light optical path that the third polarization beam apparatus reflects to form, is used for s polarised light
It is rotated by 90 ° and becomes p-polarization light.
4. comprehensive fluorescence super-resolution microscopic imaging device as described in claim 1, it is characterised in that: have and be located at four
4f- galvanometer module in light beams optical path, for modulating each light beam in the Exit positions of microcobjective back focal plane, to change interference
The shape of striped or period.
5. comprehensive fluorescence super-resolution microscopic imaging device as claimed in claim 4, it is characterised in that: the 4f- galvanometer
Module includes the first galvanometer galvanometer for the scanning of the direction x, the second galvanometer galvanometer for the scanning of the direction y, the first reflection
Formula/transmission-type 4f system and second reflective/transmission-type 4f system, the front focal plane of described first reflective/transmission-type 4f system
It should be overlapped with the first galvanometer galvanometer, the back focal plane of second reflective/transmission-type 4f system should be with the second galvanometer galvanometer
It being overlapped, the back focal plane of first reflective/transmission-type 4f system should be overlapped with the front focal plane of second reflective/transmission-type 4f system,
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: first reflection
Formula/transmission-type 4f system and second reflective/transmission-type 4f system are off axis paraboloidal mirror or achromatic lens.
7. comprehensive fluorescence super-resolution microscopic imaging device as described in claim 1, it is characterised in that: the beam splitter packet
It includes:
First beam splitter closes beam for the second polarization beam apparatus beam splitting to be obtained two light beams；
Second beam splitter closes beam for third polarization beam apparatus beam splitting to be obtained two light beams；
4th polarization beam apparatus closes beam for the light beam to the first beam splitter and the outgoing of the second beam splitter.
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 respectively set on the emitting light path of mirror and the second beam splitter, for rotating to light beam polarization direction, makes light
Beam generates interference in sample surfaces with s polarization mode.
9. comprehensive fluorescence super-resolution microscopic imaging device as described in claim 1, it is characterised in that: in the second polarization beam splitting
The transmission of device and third polarization beam apparatus or/and the adjusting optical path difference mechanism on 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: the adjusting light
Path difference mechanism is the right-angle prism for being coated with metallic reflective coating driven by piezoelectric position moving stage.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710114306.3A CN106980174B (en) | 2017-02-28 | 2017-02-28 | A kind of comprehensive fluorescence super-resolution microscopic imaging device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710114306.3A CN106980174B (en) | 2017-02-28 | 2017-02-28 | A kind of comprehensive fluorescence super-resolution microscopic imaging device |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106980174A CN106980174A (en) | 2017-07-25 |
| CN106980174B true CN106980174B (en) | 2019-04-16 |
Family
ID=59338102
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710114306.3A Active CN106980174B (en) | 2017-02-28 | 2017-02-28 | A kind of comprehensive fluorescence super-resolution microscopic imaging device |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106980174B (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108982456B (en) * | 2018-07-31 | 2020-08-18 | 浙江大学 | Three-dimensional living cell super-resolution microscopic imaging method and device based on evanescent wave illumination |
| CN108802989B (en) * | 2018-08-17 | 2020-06-02 | 华中科技大学 | Parallel multizone image device |
| EP3951466A4 (en) * | 2019-03-28 | 2023-01-04 | Hamamatsu Photonics K.K. | Confocal microscope unit and confocal microscope |
| CN110161008B (en) * | 2019-06-04 | 2021-10-29 | 山东理工大学 | Fluorescent particle tracing method and device with self-calibration of coaxial-axis degree and amplification factor |
| CN110220875B (en) * | 2019-06-10 | 2020-11-24 | 浙江大学 | Lattice light section fluorescence microscopic imaging equipment and method based on fluorescence difference method |
| CN114076750A (en) * | 2020-08-20 | 2022-02-22 | 深圳华大智造科技股份有限公司 | Super-resolution imaging device and method, biological sample identification system and identification method |
| CN113049550B (en) * | 2021-02-04 | 2022-05-27 | 浙江大学 | Longitudinal tomography method and device based on image reconstruction algorithm |
| CN113466192A (en) * | 2021-06-16 | 2021-10-01 | 浙大宁波理工学院 | Ultra-high-speed real-time super-resolution microscopic imaging method |
| CN114234954A (en) * | 2022-02-28 | 2022-03-25 | 深圳奥斯诺导航科技有限公司 | Double sensitization optical path integrated optical fiber gyroscope |
| CN115327757A (en) * | 2022-07-05 | 2022-11-11 | 浙江大学 | Method and device for realizing ultra-high-speed structured light illumination microscopic imaging |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102012009836A1 (en) * | 2012-05-16 | 2013-11-21 | Carl Zeiss Microscopy Gmbh | Light microscope and method for image acquisition with a light microscope |
| CN103472042A (en) * | 2013-08-30 | 2013-12-25 | 浙江大学 | Rapid super-resolution microscopy method and device based on fluorescent switch |
| CN103926225A (en) * | 2014-03-28 | 2014-07-16 | 浙江大学 | Fluorescence emitting differential microscopy method and device based on evanescent wave lighting |
| CN104748672A (en) * | 2015-03-05 | 2015-07-01 | 哈尔滨工业大学 | Interference-mount separating type nonlinear error correcting method and device for single-frequency laser interferometer |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7038848B2 (en) * | 2002-12-27 | 2006-05-02 | Olympus Corporation | Confocal microscope |
| JP5686684B2 (en) * | 2011-07-19 | 2015-03-18 | 株式会社日立製作所 | Optical device |
-
2017
- 2017-02-28 CN CN201710114306.3A patent/CN106980174B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102012009836A1 (en) * | 2012-05-16 | 2013-11-21 | Carl Zeiss Microscopy Gmbh | Light microscope and method for image acquisition with a light microscope |
| CN103472042A (en) * | 2013-08-30 | 2013-12-25 | 浙江大学 | Rapid super-resolution microscopy method and device based on fluorescent switch |
| CN103926225A (en) * | 2014-03-28 | 2014-07-16 | 浙江大学 | Fluorescence emitting differential microscopy method and device based on evanescent wave lighting |
| CN104748672A (en) * | 2015-03-05 | 2015-07-01 | 哈尔滨工业大学 | Interference-mount separating type nonlinear error correcting method and device for single-frequency laser interferometer |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106980174A (en) | 2017-07-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106980174B (en) | A kind of comprehensive fluorescence super-resolution microscopic imaging device | |
| CN208399380U (en) | A kind of Structured Illumination super-resolution micro imaging system | |
| CN107389631B (en) | High-speed multicolor multi-modal structured light illumination super-resolution microscopic imaging system and method thereof | |
| CN106124468B (en) | A kind of super-resolution fluorescence microscopy method and device based on photoactivation and Structured Illumination | |
| US7599069B2 (en) | Vector beam generator using a passively phase stable optical interferometer | |
| CN105784653A (en) | Wide-field super resolution fluorescence microscopic imaging device | |
| CN110954522B (en) | Multifocal super-resolution optical microscopic imaging method and device | |
| CN107505695B (en) | Structured Illumination device and its method for generating fringe structure light | |
| CN114460731B (en) | Multi-color structured light illumination super-resolution microscopic imaging method and device based on DMD | |
| JPWO2019044336A1 (en) | Holographic imaging device and data processing method used for the device | |
| CN107329245B (en) | Interference type structured light illumination microscope system and method based on radial polarization modulation | |
| CN110954523B (en) | Two-photon scanning structure light microscopic imaging method and device | |
| WO2024051079A1 (en) | Super-resolution microscopic imaging method and system based on active-structured illumination | |
| CN107966110A (en) | A kind of bimodal Digital holographic microscopy device | |
| Chen et al. | Widefield and total internal reflection fluorescent structured illumination microscopy with scanning galvo mirrors | |
| Rego et al. | Practical structured illumination microscopy | |
| CN112859315A (en) | Multicolor dual-mode structured light illumination microscopic imaging system and imaging method thereof | |
| US20240126059A1 (en) | Programmable multiple-point illuminator, confocal filter, confocal microscope and method to operate said confocal microscope | |
| US11947098B2 (en) | Multi-focal light-sheet structured illumination fluorescence microscopy system | |
| CN109870441B (en) | Frequency shift-based three-dimensional super-resolution optical section fluorescence microscopic imaging method and device | |
| WO2024007674A1 (en) | Method and apparatus for realizing ultra-high-speed structured illumination microscopy | |
| WO2023221400A1 (en) | Super-resolution single-objective light-sheet optical microscopy system and imaging system comprising same | |
| CN115598820A (en) | Double-objective three-dimensional structured light illumination super-resolution microscopic imaging device and method | |
| CN111504970B (en) | Mirror-assisted three-dimensional super-resolution microscopic imaging system and method | |
| CN107966757B (en) | Segmented half-wave plate and structured light illumination microscopic system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |