CN109211871A - A kind of stimulated emission depletion fluorescence lifetime super-resolution imaging device - Google Patents
A kind of stimulated emission depletion fluorescence lifetime super-resolution imaging device Download PDFInfo
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- 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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- 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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- 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
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- 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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Abstract
The invention discloses a kind of stimulated emission depletion fluorescence lifetime super-resolution imaging devices, are imaged for living cells, solve the problems, such as that prior art living cells imaging precision is low, comprising: loss laser provides part；Loss laser is set, the loss laser adjusting component of part output end is provided；Loss laser is set, the excitation laser offer part of part side is provided；The isochronous controller that excitation laser provides part and is lost between laser offer part is set；Excitation laser is set, the excitation laser adjusting component of part output end is provided；The spiral phase plate that loss laser adjusts component side is set；The slide of spiral phase plate side is set；Second dichroic mirror of slide side is set；First dichroic mirror of the second dichroic mirror side is set；The fluorescent acquisition component of first dichroic mirror side is set；The fluorescence imaging component of second dichroic mirror side is set；The reference fluorescence signal collection component that excitation laser adjusts component side is set；To improve the precision of living cells imaging.
Description
Technical field
The present invention relates to optical microscopy imaging technical field more particularly to a kind of stimulated emission depletion fluorescence lifetime super-resolution
Imaging device.
Background technique
Light has duality principle, and it is a kind of electromagnetic wave that the fluctuation of light, which is embodied in it, has electric field, magnetic field and propagation side
To orthogonal property；And interference phenomenon can be generated in communication process, forms light and dark interference fringe.Therefore one
A ideal point light source is unable to get ideal image point, but one has due to the presence of diffraction phenomena by optical system imaging
A certain size Fu Lang and fraunhofer-diffraction picture.And the corpuscular property of light is embodied on its discrete energy, these discrete energy are exactly
Photon one by one.Object of the photon as physical presence can carry out energy exchange with other substances in communication process, and this
The basis of energy being changing into for spectroscopy.
At the end of the 20th century, Germany scientist Stefan W.Hell proposes stimulated emission depletion microtechnic (Stimulated
Emission Depletion, STED), so that the resolution ratio of optical microscopy is improved an order of magnitude, and typical STED is super
Two-beam is needed in resolution system: a branch of is exciting light, and another beam is loss light.The core concept of STED super resolution technology is benefit
The excitation state fluorescent molecule of border area in hot spot is excited with stimulated emission selectively loss, to reduce shining for effective fluorescence
Range compresses effective PSF scale to improve systemic resolution.
But in the microscopical use process of STED, in order to improve imaging resolution, need to apply sample high
Laser power (laser power > 100mW), and excessively high loss laser power meeting lesioned sample, and make fluorescent molecule that light drift occur
Bai Xiaoying, thus it is lower using imaging precision of the STED microscopy in terms of living cells.
Summary of the invention
The main purpose of the present invention is to provide a kind of stimulated emission depletion fluorescence lifetime super-resolution imaging devices, it is intended to solve
The lower technical problem of imaging precision certainly in the prior art in terms of living cells.
To achieve the above object, the present invention provides a kind of stimulated emission depletion fluorescence lifetime super-resolution imaging device, comprising:
Laser is lost, part is provided, for providing loss laser；The loss laser that the loss laser provides part output end is arranged in adjust
Component for adjusting the linear polarization and laser intensity of the loss laser, and broadens the loss laser；Setting
Part is provided in the excitation laser that the loss laser provides part side, for providing excitation laser；It is arranged in the excitation laser
Isochronous controller between part and the loss laser offer part is provided；The excitation laser is set, swashing for part output end is provided
It sends out laser and adjusts component, for adjusting the polarization characteristic and intensity of the excitation laser, and control excitation laser and loss laser
Between pulse spacing, and separate the excitation laser；Setting adjusts component far from the loss laser in the loss laser
The spiral phase plate of part side is provided；The glass that the spiral phase plate adjusts component side far from the loss laser is set
Piece；The second dichroic mirror that the slide adjusts component side far from the loss laser is set, the of second dichroic mirror
One light-emitting window is used for transmission loss laser, and the second light-emitting window of second dichroic mirror is used for reflected fluorescent light；Setting is described the
Two dichroic mirrors are far from the slide side and are located at the first dichroic mirror being lost in the optical path of laser and excitation laser simultaneously, use
In transmission loss laser, excitation laser is reflected, and finely tunes the transmission direction of excitation laser；It is arranged in first dichroic mirror side
Fluorescent acquisition component；The fluorescence imaging component of second light-emitting window side is set；It is arranged in the excitation laser
Adjust the reference fluorescence signal collection component of component side.
Further, it includes: that the loss laser is arranged in provide the of part output end that the loss laser, which adjusts component,
Two half-wave plates；Second half-wave plate is set and provides the second Glan-Foucault laser prism of part side far from the loss laser；If
It sets in broadening glass bar of second Glan-Foucault laser prism far from second half-wave plate side.
Further, the loss laser adjusts component further include: is arranged in the broadening glass bar far from described second
The third lens of Glan-Foucault laser prism side；Single mode polarization-maintaining of the third lens far from the broadening glass bar side is set
Optical fiber；Fourth lens of the single-mode polarization maintaining fiber far from the third lens side are set；It is arranged in the 4th lens
Third half-wave plate far from the single-mode polarization maintaining fiber side；It is arranged in the third half-wave plate far from the 4th lens side
Third Glan-Foucault laser prism.
Further, it includes: that excitation laser is arranged in provide the single-mode optics of part output end that the excitation laser, which adjusts component,
It is fine；First lens of single mode optical fiber output end side are set；It is arranged in first lens far from the single mode optical fiber
First half-wave plate of side；The corner reflector of first half-wave plate far from first lens side be set, described first
Half-wave plate is located in the optical path of incident light of the corner reflector；The first Glan in the corner reflector reflected light optical path is set
Laser prism, for excitation laser to be divided into two bundles.
Further, the fluorescent acquisition component includes: to be arranged in the first dichroic mirror transmitted light path and reflection
Galvanometer scanner in optical path, excitation laser and loss laser for counterweight poststack synchronize scanning, realize to sample
Face battle array imaging；5th lens of the light-emitting window side of the galvanometer scanner are set；It is arranged in the 5th lens far from institute
State the 6th lens of galvanometer scanner side；The high power numerical aperture of 6th lens far from the 5th lens side is set
Diameter object lens；Objective table of the high power NA objective far from the 6th lens side is set.
Further, the fluorescence signal collection component that refers to includes: setting in excitation laser adjusting component side
Laser detector；The Single Photon Counting device of the laser detector output end, the time correlation list are set
Photon counter output end is mutually coupled with the fluorescence imaging component.
Further, the fluorescence imaging component includes: the 7 be arranged on the reflected light path of second dichroic mirror
Lens；The filter of 7th lens far from second dichroic mirror side is set；Setting is in the filter far from described the
The multimode fibre of seven lens sides；The photomultiplier tube of the multimode fibre output end, the photomultiplier tube output are set
End is mutually coupled with the fluorescence imaging component.
Further, the loss laser is adjusted in component, the excitation laser adjusts in component and the spiral phase
Reflecting mirror is provided between position plate and the slide.
Further, automatically controlled baffle is provided between the spiral phase plate and the slide.
Further, the slide is a quarter slide.
The present invention provides a kind of stimulated emission depletion fluorescence lifetime super-resolution imaging device, and beneficial effect is: by making
With excitation laser and laser is lost, and excitation laser and loss laser are adjusted, can selectively be damaged using stimulated emission
The excitation state fluorescent molecule for consuming excitation laser hot spot border area, after loss laser is superimposed upon excitation laser hot spot, excitation swashs
Overlap-add region around light hot spot is acted on by strong stimulated radiation, and excitation state fluorescent molecule can rapidly radiation energy return to
Ground state, it may thus be appreciated that the fluorescent molecule service life transformation of the overlap-add region around excitation laser hot spot is larger；And central area is sharp
The radiation effects that hair state fluorescent molecule is subject to is smaller, or not raying effect, fluorescence is issued still in the form of spontaneous radiation, therefore
The fluorescent molecule lifetime change very little of central area is known, thus in the fluorescence signal for extracting the generation of fluorescent molecule illuminated message
When, the fluorescence lifetime information of fluorescent molecule while the fluorescence intensity for obtaining fluorescent molecule, can be obtained, so that realizing has
The fluorescence lifetime imaging of super resolution information improves the precision of living cells imaging.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those skilled in the art without creative efforts, can also basis
These attached drawings obtain other attached drawings.
Fig. 1 is the structural schematic diagram of stimulated emission depletion of embodiment of the present invention fluorescence lifetime super-resolution imaging device.
Specific embodiment
In order to make the invention's purpose, features and advantages of the invention more obvious and easy to understand, below in conjunction with the present invention
Attached drawing in embodiment, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described reality
Applying example is only a part of the embodiment of the present invention, and not all embodiments.Based on the embodiments of the present invention, those skilled in the art
Member's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
Referring to Fig. 1, being a kind of stimulated emission depletion fluorescence lifetime super-resolution imaging device, comprising: loss laser provides
Part, loss laser adjust component, excitation laser provide part, isochronous controller, excitation laser adjust component, spiral phase plate VPP,
Slide QWP, the second dichroic mirror DM2, the first dichroic mirror DM1, fluorescent acquisition component, image-forming assembly and reference fluorescence signal are received
Collect component.
Laser is lost and provides part for providing loss laser, in the present embodiment, it is femtosecond laser that loss laser, which provides part,
Device Laster1, and the loss optical maser wavelength exported is 760 nanometers；Laser is lost and adjusts component setting in loss laser offer part
The output end side for exporting laser, for the linear polarization and laser intensity of regulation loss laser, and to the loss laser
It is broadened；Excitation laser provides any side that loss laser offer part is arranged in part, but does not adjust component with loss laser
It overlaps, for generating excitation laser, in the present embodiment, it is picosecond laser Laster2, output that excitation laser, which provides part,
Excitation laser wavelength be 635 nanometers；Isochronous controller setting provides part in loss laser and excitation laser is provided between part, damage
Excitation laser can be triggered by isochronous controller and provide part sending excitation while issuing loss laser by consuming laser offer part
Laser enables loss laser to provide part and excitation laser provides the laser that part issues identical pulse frequency；Excitation laser tune
Section component is arranged in excitation laser and provides the output end of part output excitation laser, for adjusting the polarization characteristic of excitation laser and strong
Degree, and the pulse spacing for controlling excitation laser and being lost between laser, and separate the excitation laser；Spiral phase plate VPP is set
Set loss laser adjust component far from loss laser provide part side, loss laser can by spiral phase plate VPP will
Wavefront is converted to annular by Gaussian；The side that spiral phase plate VPP adjusts component far from loss laser is arranged in slide QWP,
In the present embodiment, slide QWP is a quarter slide, is converted into circular polarization by linear polarization for laser will to be lost；Second pair
The side that a quarter slide adjusts component far from loss laser is arranged in Look mirror DM2, and there are two go out by the second dichroic mirror DM2
Optical port, the first light-emitting window are used for transmission loss laser, and the second light-emitting window is used for reflected fluorescent light；First dichroic mirror DM1 setting is the
Two sides of the dichroic mirror DM2 far from slide QWP, and be located in loss laser and excitation laser optical path, the first dichroic mirror DM1 is used for
Transmission loss laser, and reflect excitation laser, and finely tune the transmission direction of excitation laser, enables excitation laser and loss laser
Enough preferably overlappings；The side of the first dichroic mirror DM1 is arranged in fluorescent collecting component, for acquiring the fluorescence letter of sample reflection
Number；The fluorescence signal imaging in the second light-emitting window side, for that will acquire is arranged in fluorescence imaging component；With reference to fluorescence signal collection
The side that excitation laser adjusts component is arranged in component, the reference for detecting in excitation laser, as the measurement sample service life
Fluorescence.
It includes: the second half-wave plate H2, the second Glan-Foucault laser prism G2 and broadening glass bar GR that loss laser, which adjusts component,；The
Two half-wave plate H2 setting provides part output end in loss laser, for guaranteeing that loss laser is linearly polarized light, and adjusts when needed
The polarization direction of whole loss laser；Second Glan-Foucault laser prism G2 is arranged in the second half-wave plate H2 and provides part far from loss laser
Side, under the cooperation with the second half-wave plate H2, the intensity of laser to be lost in control；Glass bar GR is broadened to be arranged in the second lattice
Blue laser prism G2 is far from the second side half-wave plate H2, for broadening the pulse that laser is lost to 1 picosecond.
Laser is lost and adjusts component further include: the third lens L3, single-mode polarization maintaining fiber PMF, the 4th lens L4, third half-wave
Piece H3 and third Glan-Foucault laser prism G3；Broadening glass bar GR is arranged in far from the second Glan-Foucault laser prism G2's in the third lens L3
Side；Side of the third lens L3 far from broadening glass bar GR, in the present embodiment, single mode is arranged in single-mode polarization maintaining fiber PMF
Polarization maintaining optical fibre PMF length is 100 meters, and laser is lost for further broadening, the pulse width that laser is lost is made to reach 200 skins
Second；Side of the single-mode polarization maintaining fiber PMF far from the third lens L3 is arranged in 4th lens L4；Third half-wave plate H3 is arranged the 4th
Side of the lens L4 far from single-mode polarization maintaining fiber PMF；Third Glan-Foucault laser prism G3 is arranged in third half-wave plate H3 far from the 4th
The side of lens L4.
Excitation laser adjust component include: single mode optical fiber SMF, the first lens L1, the first half-wave plate H1, corner reflector RR and
First Glan-Foucault laser prism G1；Single mode optical fiber SMF is arranged in excitation laser and provides the output end of part, for providing excitation laser
The excitation laser projected in part carries out mode adjusting；The output of single mode optical fiber SMF output excitation laser is arranged in first lens L1
Hold side；The first side of the lens L1 far from single mode optical fiber SMF is arranged in first half-wave plate H1；Corner reflector RR is arranged first
Side of the half-wave plate H1 far from the first lens L1, for extending or shortening the optical path of excitation laser, so that control swashs in time
The pulse spacing sending out laser and being lost between laser.
Fluorescent acquisition component includes: galvanometer scanner Scanner, the 5th lens L5, the 6th lens L6, high power numerical value
Aperture objective OL and objective table 3Dstage；Galvanometer scanner Scanner is arranged in the first dichroic mirror DM1 transmitted light path and reflection
In optical path, excitation laser and loss laser for counterweight poststack synchronize scanning, realize the face battle array imaging to sample；5th
The light-emitting window side of galvanometer scanner Scanner is arranged in lens L5；6th lens L6 is arranged in the 5th lens L5 far from galvanometer
The side of scanner Scanner；The 6th side of the lens L6 far from the 5th lens L5 is arranged in high power NA objective OL, tool
Body, the enlargement ratio of high power NA objective OL is 100 times, numerical aperture 1.4, for focusing the excitation laser of overlapping
With loss laser, and the fluorescence signal that print fires back is collected；Objective table 3Dstage is arranged in high power NA objective OL
Side far from the 6th lens L6.
It include: laser detector Detector and time correlated single photon counter with reference to fluorescence signal collection component
TCSPC；The side that excitation laser adjusts component is arranged in laser detector Detector, for detecting the pulse of excitation laser,
And using the pulse of excitation laser as the reference signal of fluorescence lifetime；Single Photon Counting device TCSPC is arranged in laser
The output end side of detector Detector, and the output end of Single Photon Counting device TCSPC and fluorescence imaging group
Part mutually couples, and is excited after laser excitation for measuring each fluorescent molecule that laser detector Detector is collected into sharp
Send out the time of state.
The second lens L2 is additionally provided between first Glan-Foucault laser prism G1 and laser detector Detector.
Fluorescence imaging component includes: the 7th lens L7, filter Filter, multimode fibre MMF and photomultiplier tube PMT；The
Seven lens L7 are arranged on the reflected light path of the second dichroic mirror DM2；Filter Filter is arranged in the 7th lens L7 far from second pair
The side of Look mirror DM2 needs to collect the fluorescence signal of wave band, and filters and need to collect wave band with the spuious of wave section for transmission
Light；Side of the filter Filter far from the 7th lens L7 is arranged in multimode fibre MMF, for transmitting the fluorescence signal being collected into
Photomultiplier tube PMT is given, the fibre core of multimode fibre MMF receives the fluorescence signal that the 7th lens L7 is focused as aperture, and excludes
The influence of stray light；Photomultiplier tube PMT is arranged in the output end of multimode fibre MMF, and photomultiplier tube PMT and fluorescence at
As component mutually couples, in the present embodiment, fluorescence imaging component is computer computer, and photomultiplier tube PMT will be for that will receive
The fluorescence signal collected amplifies.
Loss laser is adjusted in component, excitation laser adjusts in component and spiral phase plate VPP and a quarter glass
Reflecting mirror is provided between piece, reflecting mirror changes the transmission direction of light beam, specifically, swash in loss for reflecting incident light
It is the first reflecting mirror M1, the first reflecting mirror M1 setting in third half-wave plate H3 and the 4th lens L4 that light, which adjusts the reflecting mirror in component,
Between；Adjusting the reflecting mirror in component in excitation laser is the second reflecting mirror M2, the second reflecting mirror M2 setting in the first dichroic mirror
Between DM1 and the first Glan-Foucault laser prism G1；The reflecting mirror being arranged between spiral phase plate VPP and a quarter slide is third
Reflecting mirror M3.
Automatically controlled baffle E1 is provided between spiral phase plate VPP and a quarter slide, when automatically controlled baffle E1 closure,
Loss laser can be blocked, it is at this time co-focusing imaging that only excitation laser, which can be radiated on sample, when the flaps are opened,
It is be overlapped on sample with excitation laser that laser is lost, forms STED super-resolution imaging.
Stimulated emission depletion fluorescence lifetime super-resolution imaging device provided by the invention, the course of work or principle are as follows:
For this imaging device there are two laser light source, the loss laser for being respectively as follows: launch loss laser provides part, and transmitting excitation laser
Excitation laser provide part；Loss laser passes through the second half-wave plate H2 and the second Glan-Foucault laser prism G2 first, so that damage
The linear polarization and laser intensity for consuming laser are regulated, it is ensured that the loss laser into light path system is linearly polarized light.Exhibition
For wide glass bar GR for broadening the loss laser of femtosecond, the pulse width after broadening is about 1 picosecond, and laser is lost
Light beam is coupled into 100 meters of single-mode polarization maintaining fiber PMF after the third lens L3 focusing, and the pulse width of laser will be lost into one
Step broadening is to about 200 picoseconds；Laser is lost after single mode polarization-maintaining light, third half-wave plate is entered after the 4th lens L4 is expanded
H3 and third Glan-Foucault laser prism G3 is again adjusted the polarization characteristic and intensity of loss laser.The light of laser is lost
Beam passes through after spiral phase plate VPP, and wavefront is changed into annular by Gaussian；It is changed into after a quarter slide by linear polarization
Circularly polarized light.Loss light enters scanning system after being successively transmitted through the second dichroic mirror DM2 and the first dichroic mirror DM1.Through the 5th
Spot diameter is amplified to the aperture of equal or slightly larger than high power NA objective OL by lens L5 and the 6th lens L6, and will be entered
On the parallel light focusing to sample penetrated.
Excitation laser provides part by excitation laser and generates, and swashing for identical repetition rate is issued after the laser signal that is depleted triggering
Send out laser.It is coupled by single mode optical fiber SMF, by the first lens L1 amplification and successively the first half-wave plate H1 of incidence and the first Glan swash
Light prism G1, so that the polarization characteristic of excitation laser and intensity are adjusted, it is ensured that the excitation laser into light path system is line
Polarised light.Exciting light changes the light path of optical path by corner reflector RR, to control between excitation laser and loss laser
Pulse spacing.Excitation laser light is divided into two bundles by the first Glan-Foucault laser prism G1, and reflected light is visited by laser detector Detector
It surveys, the reference signal as the measurement fluorescent service life.After transmitted light is reflected by a reflector, at the first dichroic mirror DM1 and damage
Consumption laser meets.The transmitted light and loss laser of excitation laser are spatially overlapped latter same into fluorescent collecting component.
In this stimulated emission depletion fluorescence lifetime super-resolution imaging device, excitation laser provides part by excitation laser and mentions
For providing excitation laser to part by external connection and providing part with loss laser and be connected, provide the control system of part by loss laser
System triggering excitation laser provides part and exports impulse type excitation laser.The pulse spacing of laser and excitation laser is lost to super-resolution
Effect has and obviously influences, and ideal STED super resolution image in order to obtain is controlled by adjusting corner reflector RR
Pulse spacing between two beam laser pulses, so that laser pulse preceding, is lost in rear, excitation laser and damage in excitation laser pulse
Pulse spacing between consumption laser is maintained at 200ps or so, and specific numerical value can be by actual super-resolution imaging effect come really
It is fixed.Excitation laser and loss laser are overlapped on galvanometer scanner Scanner, and lens the 5th lens L5 and the 6th lens L6 is by light
Spot is amplified to the pore size of high power NA objective OL, and the laser after coincidence is focused by high power NA objective OL
Afterwards, fluorescent samples are excited.After fluorescence signal is collected by high power NA objective OL, successively thoroughly by the 6th lens L6, the 5th
Multimode is coupled into after mirror L5, galvanometer scanner Scanner, the first dichroic mirror DM1, the second dichroic mirror DM2 and the 7th lens L7
Optical fiber MMF.The core end surface of multimode fibre MMF is transferred to photoelectricity times by optical fiber as aperture, by the fluorescence signal being collected into
Increase pipe PMT, carries out signal amplification, be finally imaged on computer computer.
Since the wavefront of STED laser is distributed in a ring, and center intensity is almost nil, and annular region intensity is larger, when
After loss laser is superimposed upon the hot spot of excitation laser, overlap-add region is made by strong stimulated radiation around the hot spot of excitation laser
With rapidly radiation energy returns to ground state to excitation state fluorescent molecule, therefore lifetime change is larger；And the excitation state of central area is glimmering
Optical molecule is not influenced by or by weaker, and fluorescence, therefore lifetime change very little are issued still in the form of spontaneous radiation.
It is converted using the data that Single Photon Counting device TCSPC is collected by phase figure analysis method from time-domain
, can be to avoid the mistake as brought by index analysis to frequency domain, it can also be by the fluorescence information of each pixel to visualize
Form show, therefore be a kind of highly useful fluorescence lifetime data analysing method.It is glimmering according to phase diagram analysis theories
The optical signal laser that is excited is modulated with identical frequency (ω), and changes will occur for amplitude and phase, therefore phase diagram
In frequency domain information contain phase delay (φ) and amplitude modulation rate (m).Under determining modulating frequency, formula g=m is utilized
All Single Photon Counting device TCSPC data collected are transformed into phase by × cos (φ) and s=m × sin (φ)
Space；Wherein, g and the behalf coordinate value (g, s) of phase space.In above-mentioned data, the fluorescent molecule of each location of pixels
Attenuation trend is all completely recorded.For one pack system sample, coordinate value g and s with fluorescence lifetime and modulating frequency and
Variation, it may be assumed that g=1/ (1+ ω2τ2) and s=ω τ/(1+ ω2τ2).According to above-mentioned formula, can be established in phase diagram one with
Centered on coordinate (0.5,0), radius be 0.5 half-round curve.Wherein in the reference axis of phase diagram, coordinate origin, i.e. coordinate
(0,0) represents the service life as infinity, and it is zero that coordinate (1,0), which represents the service life, and the fluorescence lifetime value of one pack system sample must fall in this
In a half-round curve, and position different on semicircle represents different fluorescence lifetime.
After fluorescence lifetime data are transformed into phase space, due to the phase delay and amplitude tune of collected fluorescent molecule
Information processed is different, and fluorescent molecule can be distributed in phase space with different coordinates.According to phase diagram analysis principle, in one pack system
In co-focusing imaging, phase diagram is formed after lifetime data is converted, passes through fuzzy c class means clustering algorithm (Fuzzy c-mean
Clustering algorithm) its phase center coordinate, i.e., the mean fluorecence service life of all fluorescent molecules, phase is calculated
Centre coordinate is located in half-round curve.Closer to phase center coordinate, fluorescent molecule is more concentrated, and fluorescent molecule number is more.And
Under STED imaging pattern, the excitation state fluorescent molecule of the different location radiation energy in the form of spontaneous radiation and two kinds of stimulated radiation respectively
Amount.If the intensity that the spot center region of laser is lost is zero, the excited state molecule of there, which is not damaged, depletes influence, longevity
Life value should be in close proximity to the average life span under total focusing mode.But influenced by factors such as aberrations, annular loss light
Central area can generate the intensity of a non-zero, this intensity can become larger with the enhancing of laser energy.At this point, excitation laser
The part fluorescent molecule in spot center region stimulated radiation can also occur, and then its service life is reduced, so in phase diagram almost
All fluorescent molecules are all far from the phase center coordinate of Confocal Images.Nevertheless, the loss intensity of light center or small
In the intensity of annular region, caused the result is that impaired deplete the excitation state fluorescent molecule for influencing lesser region, phase is sat
It marks closer to the burnt centre coordinate of copolymerization；And the impaired excitation state fluorescent molecule for depleting the region being affected, phase coordinate is more
Far from the burnt centre coordinate of copolymerization.And the impaired influence depleted of fluorescent molecule with super resolution information is smaller, their phase is sat
It marks closer to the burnt centre coordinate of copolymerization；And then lifetime change is larger for remaining fluorescent molecule, therefore further from total focusing center
Coordinate.
When handling STED-FLIM data, double exponential curve not only can be very good to fit fluorescence lifetime, can be with letter
Change data handling procedure.Therefore, regard the fluorescence lifetime under STED mode as long-life and short-life combination, they distinguish
Represent the weaker central area fluorescent molecule of stimulated radiation effect and the stronger annular region fluorescent molecule of stimulated radiation effect.
Phase center coordinate under STED mode represents the average life span of sample, therefore it can be regarded as a judgment criteria.
Phase space is divided into two parts by the line segment of connection STED image phase centre coordinate and coordinate (0,0) point, and it is left to be located at line segment
It is leading that the fluorescent molecule in upper region is that the long-life accounts for, substantially the spot center region from excitation laser；And it is located at line segment bottom right
It is leading that the fluorescent molecule in region is that short life accounts for, greatly mostly from the hot spot annular region and ambient noise of excitation laser.It will be located at
The region of line segment two sides regards " selection region " and " abandoning region " as, can achieve after the fluorescent molecule of " abandoning region " is removed
The purpose that resolution ratio improves.Not only " useless " fluorescent molecule can be removed by using this method and realize mentioning for resolution ratio
Height can also retain enough fluorescent molecules to form the preferable super resolution image of a width.Therefore in the present apparatus, phase is utilized
It is further that figure analysis method selection by stimulated radiation is influenced the imaging resolution that lesser fluorescent molecule may be implemented under low-power
It improves.
In several embodiments provided herein, it should be understood that disclosed device, it can be by others side
Formula is realized.For example, the apparatus embodiments described above are merely exemplary.Another point, it is shown or discussed it is mutual it
Between coupling or direct-coupling can be through some interfaces, the indirect coupling or communication connection of device or module can be electricity
Property, mechanical or other forms.
It should be noted that for embodiment above-mentioned, those skilled in the art should understand that, it is described in this description
Embodiment belongs to preferred embodiment, and related actions and modules might not all be necessary to the present invention.
It is right the above are to a kind of description of stimulated emission depletion fluorescence lifetime super-resolution imaging device provided by the present invention
In those skilled in the art, thought according to an embodiment of the present invention has change in specific embodiments and applications
Become place, to sum up, the contents of this specification are not to be construed as limiting the invention.
Claims (10)
1. a kind of stimulated emission depletion fluorescence lifetime super-resolution imaging device characterized by comprising
Laser is lost, part is provided, for providing loss laser；
The loss laser that the loss laser provides part output end is set and adjusts component, for adjusting the line of the loss laser
Polarization direction and laser intensity, and the loss laser is broadened；
The excitation laser that the loss laser provides part side is set, part is provided, for providing excitation laser；
The excitation laser is set, part and the isochronous controller being lost between laser offer part are provided；
The excitation laser is set, the excitation laser adjusting component of part output end is provided, for adjusting the inclined of the excitation laser
Characteristic of shaking and intensity, and the pulse spacing for controlling excitation laser and being lost between laser, and separate the excitation laser；
The loss laser is set and adjusts spiral phase plate of the component far from the loss laser offer part side；
The slide that the spiral phase plate adjusts component side far from the loss laser is set；
The second dichroic mirror that the slide adjusts component side far from the loss laser is set, the of second dichroic mirror
One light-emitting window is used for transmission loss laser, and the second light-emitting window of second dichroic mirror is used for reflected fluorescent light；
Second dichroic mirror is set far from the slide side and while being located in the optical path of loss laser and excitation laser
The first dichroic mirror, be used for transmission loss laser, reflect excitation laser, and finely tune the transmission direction of excitation laser；
The fluorescent acquisition component of first dichroic mirror side is set；
The fluorescence imaging component of second light-emitting window side is set；
The reference fluorescence signal collection component that the excitation laser adjusts component side is set.
2. stimulated emission depletion fluorescence lifetime super-resolution imaging device according to claim 1, which is characterized in that the damage
Consumption laser adjusts component
The loss laser is set, the second half-wave plate of part output end is provided；
Second half-wave plate is set and provides the second Glan-Foucault laser prism of part side far from the loss laser；
Broadening glass bar of second Glan-Foucault laser prism far from second half-wave plate side is set.
3. stimulated emission depletion fluorescence lifetime super-resolution imaging device according to claim 2, which is characterized in that the damage
It consumes laser and adjusts component further include:
The broadening the third lens of the glass bar far from second Glan-Foucault laser prism side are set；
Single-mode polarization maintaining fiber of the third lens far from the broadening glass bar side is set；
Fourth lens of the single-mode polarization maintaining fiber far from the third lens side are set；
The third half-wave plate of 4th lens far from the single-mode polarization maintaining fiber side is set；
Third Glan-Foucault laser prism of the third half-wave plate far from the 4th lens side is set.
4. stimulated emission depletion fluorescence lifetime super-resolution imaging device according to claim 1, which is characterized in that described to swash
Hair laser adjusts component
Excitation laser is set, the single mode optical fiber of part output end is provided；
First lens of single mode optical fiber output end side are set；
First half-wave plate of first lens far from the single mode optical fiber side is set；
The corner reflector of first half-wave plate far from first lens side is set, and first half-wave plate is located at described
In the optical path of incident light of corner reflector；
The first Glan-Foucault laser prism in the corner reflector reflected light optical path is set, for excitation laser to be divided into two bundles.
5. stimulated emission depletion fluorescence lifetime super-resolution imaging device according to claim 1, which is characterized in that the sample
Product fluorescent collecting component includes:
Galvanometer scanner on the first dichroic mirror transmitted light path and reflected light path is set, and the excitation for counterweight poststack swashs
Light and loss laser synchronize scanning, realize the face battle array imaging to sample；
5th lens of the light-emitting window side of the galvanometer scanner are set；
Sixth lens of 5th lens far from the galvanometer scanner side are set；
The high power NA objective of 6th lens far from the 5th lens side is set；
Objective table of the high power NA objective far from the 6th lens side is set.
6. stimulated emission depletion fluorescence lifetime super-resolution imaging device according to claim 1, which is characterized in that the ginseng
Examining fluorescence signal collection component includes:
The laser detector that the excitation laser adjusts component side is set；
The Single Photon Counting device of the laser detector output end, the Single Photon Counting device are set
Output end is mutually coupled with the fluorescence imaging component.
7. stimulated emission depletion fluorescence lifetime super-resolution imaging device according to claim 1, which is characterized in that described glimmering
Light image-forming assembly includes:
The 7th lens on the reflected light path of second dichroic mirror are set；
The filter of 7th lens far from second dichroic mirror side is set；
Multimode fibre of the filter far from the 7th lens side is set；
The photomultiplier tube of the multimode fibre output end, the photomultiplier tube output end and the fluorescence imaging group are set
Part mutually couples.
8. stimulated emission depletion fluorescence lifetime super-resolution imaging device according to claim 1, which is characterized in that the damage
Consumption laser is adjusted in component, the excitation laser is adjusted in component and is all provided between the spiral phase plate and the slide
It is equipped with reflecting mirror.
9. stimulated emission depletion fluorescence lifetime super-resolution imaging device according to claim 1, which is characterized in that the spiral shell
Automatically controlled baffle is provided between rotation phase-plate and the slide.
10. stimulated emission depletion fluorescence lifetime super-resolution imaging device according to claim 1, which is characterized in that described
Slide is a quarter slide.
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