CN109633881A - A kind of microscopical imaging system of stimulated emission depletion - Google Patents
A kind of microscopical imaging system of stimulated emission depletion Download PDFInfo
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- CN109633881A CN109633881A CN201910009315.5A CN201910009315A CN109633881A CN 109633881 A CN109633881 A CN 109633881A CN 201910009315 A CN201910009315 A CN 201910009315A CN 109633881 A CN109633881 A CN 109633881A
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- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
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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
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- G01N21/6458—Fluorescence microscopy
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Abstract
The invention discloses a kind of microscopical imaging systems of stimulated emission depletion, including light unit is lost, excite light unit, imaging unit and counter unit, loss light unit generates annular loss light and controls the light intensity and polarization of annular loss light, excitation light unit is depleted after light unit triggers and issues Gaussian exciting light, Gaussian exciting light is divided into the first Gaussian exciting light and the second Gaussian exciting light by third Glan prism, imaging unit is inputted after annular loss light is Chong Die with the first Gaussian exciting light, imaging unit is scanned imaging to sample and collects fluorescence signal.Second Gaussian exciting light enters counter unit with fluorescence signal as reference signal together, for fluorescence intensity imaging and fluorescence lifetime imaging.When applying lower annular loss light energy, the fluorescence lifetime of sample increases with laser irradiation time, and increased fluorescence lifetime reduces the saturation intensity of fluorescent samples, and then further increasing for imaging resolution may be implemented.
Description
Technical field
The present invention relates to optical microscopy imaging field more particularly to a kind of microscopical imaging systems of stimulated emission depletion
System.
Background technique
In optical system, the image patch formed after the imaged system of object point is known as point spread function (Point Spread
Function,PSF).Point spread function describes imaging system to the analytic ability of object point, is to judge that optical system imaging is differentiated
The important evidence of rate height.It is limited by diffraction limit, when two object points are closer in object plane, they pass through optical system
System forms the image patch of two overlappings.The distance between image patch between object point at a distance from it is corresponding, when image patch is apart from each other, point
Spread function is separated from each other, then is easy to be resolved；When image patch close proximity, two PSF high superposeds, and then cannot be distinguished each
From image patch；And when the distance between image patch falls between, PSF partly overlaps, and image patch at this time is in distinguishable and not
Between distinguishable, be in a critical distance, less than this apart from when image patch superposition after will become indistinguishable.The critical distance
Determination generallys use Rayleigh criterion, i.e., is overlapped as differentiation and is marked with the first dark ring of another image patch using the center of an image patch
Standard, two image patch centre distances are equal to Airy radius, the size of Airy radius and the numerical aperture of optical wavelength and object lens at this time
Diameter is related.
At the end of the 19th century, roentgen Ernst K.Abbe is according to the sine condition in geometric optics, it is determined that optics
Half or so of the microscopical resolution limit in visible wavelength.This means that can be differentiated by traditional optical microscopy
Individual cells and intracellular organelle ingredient out, but can not the smaller object of resolution size, such as smaller size of disease
Malicious, single protein or other small-molecule substances.1994, Germany scientist Stefan W.Hell, which is proposed, to be stimulated
Loss microtechnic (Stimulated Emission Depletion, STED) is penetrated, the resolution ratio of optical microscopy is improved
An order of magnitude, this outstanding work make him obtain Nobel chemistry Prize in 2014.In principle, all optics is super
Resolution techniques are unfolded around how to obtain a smaller point spread function.Stefan W.Hell is proposed
Stimulated emission depletion microtechnic be a pure physics super resolution technology.It is needed in one typical STED super-resolution system
Two-beam: a branch of is exciting light, and another beam is loss light.The core concept of STED super resolution technology is selected using stimulated emission
Property loss excitation hot spot in border area excitation state fluorescent molecule, to reduce the light emitting region of effective fluorescence, compression is effective
PSF scale improves systemic resolution.
Common STED microscope can only acquire fluorescence intensity signals, and have ignored the other information of fluorescent molecule.?
In STED microscopy, in order to improve imaging resolution, need to apply sample high laser power (> 100mW), and mistake
High loss laser power can lesioned sample, and make fluorescent molecule that photobleaching effect occur, thus limit its living cells at
The application of picture etc., is one of the disadvantage of STED microscope maximum.
Summary of the invention
The main purpose of the present invention is to provide a kind of microscopical imaging systems of stimulated emission depletion, can solve existing
Excessively high loss laser is used in order to improve imaging resolution in technology, thus the technical issues of influencing the sample service life.
To achieve the above object, the present invention provides a kind of microscopical imaging system of stimulated emission depletion, which is characterized in that
The system comprises loss light unit, excitation light unit, imaging unit and counter units；
The loss light unit is used to generate the annular loss light of predetermined pulse frequency and adjusts the control annular loss
The light intensity of light, it is described annular loss light laser wave before be annular；
The loss light unit is connect with the excitation light unit, and the excitation light unit is used in the loss light unit
Triggering under generate the Gaussian exciting light of the pulse frequency, and the Gaussian exciting light is divided into the excitation of the first Gaussian
Light and the second Gaussian exciting light are Gaussian, the Gaussian exciting light and institute before the laser wave of the Gaussian exciting light
State annular loss light between peak value of pulse between be divided into predetermined pulse spacing value；
Annular loss light and the first Gaussian exciting light converge it is Chong Die after input the imaging unit, it is described at
As unit is used to carry out sample planar array scanning imaging and collect the fluorescence signal that the sample reflects, the fluorescence signal and institute
State the second Gaussian exciting light and inject the counter unit respectively, the counter unit be used for based on the fluorescence signal and
The second Gaussian exciting light carries out fluorescence intensity imaging and fluorescence lifetime imaging.
The present invention provides a kind of microscopical imaging system of stimulated emission depletion, and loss light unit generates in the imaging system
Annular loss light and the light intensity for adjusting control annular loss light, make the performance number of annular loss light be maintained at lower level.It is low
The annular loss luminous energy of power increases the fluorescence lifetime in sample, and with the increase of laser irradiation time, the image point of sample
Resolution also with fluorescence lifetime increase and be gradually increased.
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 a kind of structural schematic diagram of the microscopical imaging system of stimulated emission depletion in the embodiment of the present invention；
Fig. 2 is the structural schematic diagram that light unit 1 is lost in the embodiment of the present invention；
Fig. 3 is the structural schematic diagram that light unit 2 is excited in the embodiment of the present invention；
Fig. 4 is the structural schematic diagram of imaging unit 3 in the embodiment of the present invention；
Fig. 5 is the structural schematic diagram of counter unit 4 in the embodiment of the present invention；
Fig. 6 is Gaussian exciting light hot spot in the embodiment of the present invention, annular loss light hot spot and the two hot spot space overlap
Structural schematic diagram；
Fig. 7 is that fluorescent bead is swept in laser lengthy scan region and for the first time under co-focusing imaging mode in the embodiment of the present invention
Retouch life span comparison's schematic diagram in region；
Fig. 8 is fluorescent bead under STED imaging pattern in the embodiment of the present invention in laser lengthy scan region and first scan
Life span comparison's schematic diagram in region.
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.
Excessively high loss laser is used in order to improve imaging resolution due to existing in the prior art, to influence sample
The technical issues of service life.
In order to solve the above-mentioned technical problem, the present invention proposes a kind of microscopical imaging system of stimulated emission depletion, this at
Annular loss light is generated as light unit is lost in system and adjusts the light intensity of control annular loss light, makes the power of annular loss light
Value is maintained at lower level.The annular loss luminous energy of low-power increases the fluorescence lifetime in sample, and when with laser irradiation
Between increase, the image resolution ratio of sample also with fluorescence lifetime increase and be gradually increased.
Referring to Fig. 1, for a kind of structural representation of the microscopical imaging system of stimulated emission depletion in the embodiment of the present invention
Figure, wherein the transmission direction of arrow expression laser.
The system includes loss light unit 1, excitation light unit 2, imaging unit 3 and counter unit 4；
Loss light unit 1 is used to generate the annular loss light of predetermined pulse frequency and adjusts the light of control annular loss light
It by force, is annular before the laser wave of annular loss light；
Loss light unit 1 is connect with excitation light unit 2, and excitation light unit 2 is used under the triggering of loss light unit 1 generate
The Gaussian exciting light of pulse frequency, and Gaussian exciting light is divided into the first Gaussian exciting light and the excitation of the second Gaussian
Light, is Gaussian before the laser wave of Gaussian exciting light, the peak value of pulse interval between Gaussian exciting light and annular loss light
For predetermined pulse spacing value；
Annular loss light and the first Gaussian exciting light converge it is Chong Die after input imaging unit 3, imaging unit 3 is for sample
Product carry out planar array scanning imaging and collect the fluorescence signal of sample reflection, and fluorescence signal and the second Gaussian exciting light are injected respectively
Counter unit 4, counter unit 4 are used to carry out fluorescence intensity imaging and glimmering based on fluorescence signal and the second Gaussian exciting light
The imaging of light service life.
Preferably, annular loss light wavelength be 760 nanometers, the first Gaussian exciting light, the second Gaussian exciting light and
The wavelength of Gaussian exciting light is 635 nanometers.
Preferably, it is divided into predetermined pulse spacing value between the peak value of pulse between Gaussian exciting light and annular loss light, it should
Pulse spacing value is maintained at the range of 200ps or so, just can guarantee annular loss light more thoroughly by Gaussian exciting light in this way
The excitation state electronics of generation returns to ground state in the form of stimulated radiation.
It should be noted that common STED microscope can only acquire fluorescence intensity signals, the present invention uses fluorescence lifetime
Microscope (Fluorescence Lifetime Imaging Microscopy, FLIM) describes fluorescence lifetime sky with FLIM
Between be distributed, can be applied in the imaging of fixed cell and living cells, to disclose more biological informations (such as intracellular microenvironment
Variation).The microscopical imaging system of stimulated emission depletion of the invention is that FLIM technology and STED technology is combined to believe from fluorescence
The information of fluorescence intensity and fluorescence lifetime is extracted in number, and then realizes the fluorescence lifetime imaging with super resolution information, i.e. STED-
FLIM image.The microscopical imaging system of stimulated emission depletion in the present invention is can be collected simultaneously fluorescence intensity and fluorescence lifetime
The super-resolution imaging system of information, it is intended to the influence of Study of Laser intensity and irradiation time to fluorescence lifetime and imaging resolution,
Belong to STED super-resolution micro imaging system, System spatial resolution can be significantly improved.
Further, which further includes the second reflecting mirror 5, third reflecting mirror 6, the first dichroic mirror 7 and the second dichroic mirror
8；
First Gaussian exciting light successively passes through the reflection of the second reflecting mirror 5, the reflection of third reflecting mirror 6 and the first dichroic mirror 7
Reflection, annular loss light successively passes through the transmission of the second dichroic mirror 8 and the transmission of the first dichroic mirror 7, by the transmission of the first dichroic mirror 7
Annular loss light with by the first Gaussian exciting light that the first dichroic mirror 7 reflects converge it is Chong Die after injection imaging unit 3；
Fluorescence signal successively after the transmission 7 of the first dichroic mirror and the second dichroic mirror 8 are reflected, injects counter unit 4.
It should be noted that the second reflecting mirror 5 and third reflecting mirror 6 change the propagation side of light beam for reflecting incident light
To.First dichroic mirror 7 is applied not only to the first Gaussian exciting light of reflection, transmits annular loss light, is also used to finely tune the first Gauss
The transmission direction of type exciting light makes the first Gaussian exciting light and annular loss light can be good at converging overlapping.Second is double-colored
Mirror 8 is for transmiting annular loss light, while reflected fluorescence signal.
Further, referring to Fig. 2, for the structural schematic diagram of light unit 1 is lost in the embodiment of the present invention, wherein arrow
Indicate the transmission direction of light.It includes that femto-second laser 11, first adjusts unit 12, pulse broadening unit 13, that light unit 1, which is lost,
Two adjust unit 14 and spiral phase plate 15；
Femto-second laser 1 is used to generate the femtosecond pulse type loss light of pulse frequency, and femtosecond pulse type loss light emission enters the
One adjusts unit 12, adjusts after unit 12 carries out linear polarization adjusting and light intensity regulating through first and exports First Line polarization loss light,
First Line polarization loss light emission enters pulse broadening unit 13, exports predetermined pulse after pulse broadening unit 13 carries out pulse broadening
Light is lost in the broadening of width, and broadening loss light emission enters the second adjusting unit 14, adjusts unit 14 through second and carry out linear polarization adjusting
Light is lost with the second linear polarization is exported after light intensity regulating, the second linear polarization loss light exports annular damage after spiral phase plate 15
It depletes；
Femto-second laser 11 is connect with excitation light unit 2, and femto-second laser 11 is also used to trigger excitation light unit 2 and generates height
This type exciting light.
It should be noted that spiral phase plate 15 (Vortex Phase Plate, VPP) be used to be lost the wavefront of light by
Gaussian conversion circularizes.
Further, the first adjusting unit 12 includes the first half-wave plate 121 and the first Glan prism 122；
Femtosecond pulse type loss light emission enters the first half-wave plate 121, is adjusted to default first polarization side through the first half-wave plate 121
To linearly polarized light after inject the first Glan prism 122, through the first Glan prism 122 adjust light intensity after export the first linear polarization damage
It depletes.
It should be noted that the first half-wave plate 121 is used to femtosecond pulse type loss light being adjusted to linearly polarized light and adjust
Its polarization direction, the first Glan prism 122 of arranging in pairs or groups, controls the intensity of laser.
Further, pulse broadening unit 13 includes glass bar 131, the first lens 132, single-mode polarization maintaining fiber 133 and the
Two lens 134；
First Line polarization loss light emission enters glass bar 131, injects the first lens after glass bar 131 carries out pulse broadening
132, single-mode polarization maintaining fiber 133 is injected after the focusing of the first lens 132, pulse is carried out through single-mode polarization maintaining fiber 133 and broadens again
After inject the second lens 134, through the second lens 134 focusing after output broadening loss light.
It should be noted that glass bar 131 (Glass Rod, GR) is used to broaden the pulse of First Line polarization loss light
To about 1 picosecond, single-mode polarization maintaining fiber 133 (Polarization Maintaining Fiber, PMF) is 100 meters of single mode polarization-maintainings
Optical fiber is lost light for further broadening, its pulse width is made to reach 200 picoseconds.
Further, the second adjusting unit 14 includes the second half-wave plate 141 and the second Glan prism 142；
Broadening loss light emission enters the second half-wave plate 141, and the line of default second polarization direction is adjusted to through the second half-wave plate 141
The second Glan prism 142 is injected after polarised light, and the second linear polarization loss light is exported after the second Glan prism 142 adjusts light intensity.
It should be noted that the second half-wave plate 141 is adjusted to linearly polarized light and adjusts its polarization for that will broaden loss light
Direction, the second Glan prism 142 of arranging in pairs or groups, controls the intensity of laser.
Preferably, femtosecond pulse type loss light is generated by femto-second laser 1, passes through the first half-wave plate 121 and first first
Glan prism 122, for the linear polarization and laser intensity of regulation loss light, it is ensured that the loss light into light path system is line
Polarised light.For glass bar 131 for broadening the loss pulsed light of femtosecond, the pulse width after broadening is about 1 picosecond.Light
Beam through the first lens 132 focusing after be coupled into 100 meters of single-mode polarization maintaining fibers, by the pulse width that light is lost further broaden to
About 200 picoseconds.Outgoing beam enters the second half-wave plate 141 and the second Glan prism 142 after the second lens 134 expand, further
Secondary polarization characteristic and intensity to loss light is adjusted.Light beam passes through after spiral phase plate 15, and wavefront is changed by Gaussian
For annular.
Further, referring to Fig. 3, being the structural schematic diagram for exciting light unit 2 in the embodiment of the present invention, wherein arrow
Indicate the transmission direction of light.
Exciting light unit 2 includes picosecond laser 21, single mode optical fiber 22, the third lens 23 and regulation unit 24；
Picosecond laser 21 is connect with loss light unit 1, and picosecond laser 21 is used under the triggering of loss light unit 1 produce
The picosecond pulse type exciting light of raw pulse frequency, picosecond pulse type exciting light are injected single mode optical fiber 22, are carried out through single mode optical fiber 22
The third lens 23 are injected after mode adjusting, regulation unit 24 is injected after the third lens 23 amplify, and are controlled through adjusting
It is excited before exporting laser wave after the progress of unit 24 linear polarization adjusting, pulse interval modulation and light intensity regulating for the Gaussian of Gaussian
Light.
It should be noted that single mode optical fiber 22 (Single Mode Fiber, SMF) is mainly used for picosecond laser 21
The exciting light of middle outgoing carries out mode adjusting.
Further, regulation unit 24 includes third half-wave plate 241, corner reflector 242 and third Glan prism
243；
Third half-wave plate 241 is injected through the amplified laser of the third lens 23, is adjusted to default the through third half-wave plate 241
By corner reflector 242 after the linearly polarized light of three polarization directions, corner reflector 242 is damaged for adjusting Gaussian exciting light and annular
Pulse spacing between depleting, the laser after corner reflector 242 carries out pulse interval modulation inject third Glan prism 243,
After third Glan prism 243 adjusts light intensity, the first Gaussian exciting light and the second Gaussian exciting light are exported respectively.
It should be noted that the optical path of exciting light can be changed in corner reflector 242 (Retro Reflector, RR), it is used for
The pulse spacing for controlling exciting light on time and being lost between light, in general, the optical path for changing exciting light is usually to extend excitation
The optical path of light or the optical path for shortening exciting light, meanwhile, third half-wave plate 241 is adjusted to linearly polarized light for that will broaden loss light
And its polarization direction is adjusted, third Glan prism 243 of arranging in pairs or groups controls the intensity of laser, meanwhile, third Glan prism 243 will be high
This type exciting light is divided into two-way, respectively the first Gaussian exciting light and the second Gaussian exciting light.
Exciting light is generated by picosecond laser 21, and the laser of identical repetition rate is issued after the laser signal that is depleted triggering.
It is coupled, is amplified by the third lens 23 and successively incidence third half-wave plate 241, corner reflector 242 and third by single mode optical fiber 22
Glan prism 243 adjusts the polarization characteristic and intensity of exciting light, it is ensured that the exciting light into light path system is linearly polarized light.Swash
It shines and changes the light path of optical path by corner reflector 242, thus the pulse spacing for controlling exciting light and being lost between light.
Further, referring to Fig. 4, being the structural schematic diagram of imaging unit 3 in the embodiment of the present invention, wherein arrow table
Show the transmission direction of light.
Imaging unit 3 includes the 4th lens 31, galvanometer scanning system 32, the 5th lens 33, a quarter slide 34, high number
It is worth aperture objective 35 and objective table 36, places sample on objective table 36；
Annular loss light and the first Gaussian exciting light converge it is Chong Die after successively pass through the 4th lens 31, galvanometer scanning system
32 and the 5th after lens 33, inject a quarter slide 34, the first Gaussian after a quarter slide 34 will converge overlapping
After exciting light and annular loss light are adjusted to circularly polarized light by linearly polarized light, loading is incident to after high-NA objective 35
Platform 36；
High-NA objective 35 is used to collect the fluorescence signal of sample reflection, and fluorescence signal successively passes through a quarter glass
Counter unit 5 is injected after piece 34, the 5th lens 33, galvanometer scanning system 32, the 4th lens 31；
Galvanometer scanning system 32 is used to synchronize the first Gaussian exciting light and annular loss light that converge after being overlapped
Scanning is realized and the planar array scanning of sample is imaged.
It should be noted that the enlargement ratio of high-NA objective 35 is 100 times, numerical aperture 1.4, for focusing
The the first Gaussian exciting light and annular loss light of overlapping, are collected simultaneously the reflected fluorescence signal of sample.A quarter glass
Piece 34 (Quarter-Wave Plate, QWP) is used to annular loss light being converted to circularly polarized light by linearly polarized light, to ensure ring
Light, which is lost, in shape has better optical quality.Wherein, annular loss light is through the 4th lens 31 and the 5th lens 33 by spot diameter
It is amplified to the aperture of equal or slightly larger than high power NA objective 35, and by the sample of incident parallel light focusing to objective table 36
On product.
Further, referring to Fig. 5, being the structural schematic diagram of counter unit 4 in the embodiment of the present invention, wherein arrow
Indicate the transmission direction of light.
Counter unit 4 includes the 6th lens 41, filter 42, multimode fibre 43, photomultiplier tube 44, the first reflecting mirror
45, the 7th lens 46, detector 47 and Single Photon Counting device 48；
Fluorescence signal is successively by the transmission of the 6th lens 41, after filter 42 filters out stray light and multimode fibre 43, incident light
Electric multiplier tube 44, input time correlated single photon counter 48 after photomultiplier tube 44 amplifies fluorescence signal；
Second Gaussian exciting light injects detector 47 after the reflection of the first reflecting mirror 45 and the transmission of the 7th lens 46,
The input time correlated single photon counter 48 after detector 47 is converted to electric signal；
Single Photon Counting device 48 is used to carry out fluorescence intensity based on the second Gaussian exciting light and fluorescence signal
Imaging and fluorescence lifetime imaging.
It should be noted that filter 42 is used for the fluorescence signal through the wave band collected, and filter other than this wave band
Stray light, multimode fibre 43, for the fluorescence signal being collected into be transferred to photomultiplier tube 44, the optical fiber of multimode fibre 43 is fine
Core can be used as aperture, receives the fluorescence signal that the 6th lens 41 focus, removes the influence of stray light side by side.Detector 47 is for detecting
Second Gaussian excitation light pulse, as the reference signal in measurement fluorescent service life, 48 energy of Single Photon Counting device
The fluorescence signal that photomultiplier tube 44 is collected into is divided into two-way, is imaged all the way for fluorescence intensity, another way is used for the fluorescence longevity
Life imaging.
For a better understanding of the present invention, referring to Fig. 6, for Gaussian exciting light hot spot, annular damage in the embodiment of the present invention
Deplete the structural schematic diagram of hot spot and the two hot spot space overlap.Wherein a indicates that Gaussian exciting light hot spot, b indicate ring in Fig. 6
Light hot spot is lost in shape, and c indicates the space overlap schematic diagram of Gaussian exciting light hot spot and annular loss light hot spot.
Further, Fig. 7 is please referred to, is fluorescent bead under co-focusing imaging mode in the embodiment of the present invention in laser long-time
Life span comparison's schematic diagram of scanning area and first scan region.The Confocal-FLIM formed under co-focusing imaging mode
In image, dashed centre line region is through the long-irradiated imaging region of laser, and rest part is scanning imagery region for the first time.
T indicates the average life span of whole region, and t1 indicates the service life of peripheral region, and t2 indicates the service life of central area.In service life image
In, the fluorescence lifetime of central area is higher than the fluorescence lifetime of peripheral region, illustrates the service life of fluorescent bead in prolonged laser
Change under irradiation to the long-life.Although will lead to the reduction of fluorescence intensity to photobleaching after sample progress long-time irradiation,
Excitating light strength only has microwatt magnitude, therefore the variation of intensity is not obvious.
Further, Fig. 8 is please referred to, is swept for a long time for fluorescent bead under STED imaging pattern in the embodiment of the present invention in laser
Retouch life span comparison's schematic diagram in region and first scan region.In the STED-FLIM image formed under STED imaging pattern, in
Heart dashed region is through the long-irradiated imaging region of laser, and rest part is scanning imagery region for the first time.T indicates entire
The average life span in region, T1 indicate the service life of peripheral region, and T2 indicates the service life of central area.It is long under STED imaging pattern
The laser irradiation of time can make the service life of fluorescent bead in scanning area that more obvious variation occur, and central area fluorescent bead is presented
Darker, fluorescence lifetime of the service life much higher than peripheral region out.Since loss laser energy is higher, long-time laser shines
The photobleaching rate for increasing fluorogen is penetrated, thereby reduces the number of photons of autofluorescence, therefore centre scan region is swept with periphery
The fluorescence intensity for retouching region is different.
STED super-resolution imaging technology needs use loss light and exciting light, and resolution ratio is with the increase that light energy is lost
And improve, it is indicated by formula are as follows: R=λ/[2NA (1+ISTED/IS)1/2], wherein IS=hc/ λ τ σ, λ are optical wavelength, and NA is object
The numerical aperture of mirror, ISTEDFor the energy that laser is lost, ISFor the saturation energy of sample, by sample used fluorescence lifetime (τ) and
The absorption cross-section (σ) that light is lost codetermines.Therefore, under fixed loss laser energy, the increase of fluorescence lifetime be will lead to
The reduction of saturation power, this also means that can realize the promotion of resolution ratio under lower laser energy.In Confocal image
In, due to the limitation of diffraction limit, many fluorescent bead close proximities and can not be distinguished.When the loss laser for applying lower-wattage
Afterwards, image resolution ratio is slightly improved compared to Confocal Images.Increase the irradiation time of laser, image point under STED imaging pattern
Resolution is gradually increased with the increase of fluorescence lifetime.It therefore in the present system, can be using the method for increase fluorescence lifetime low
The further promotion of STED imaging resolution is realized under power.
In embodiments of the present invention, light unit 1 is lost in the imaging system to generate annular loss light and adjust control annular
The light intensity of light is lost, the performance number of annular loss light is made to be maintained at lower level.When annular loss light energy is lower, sample
Fluorescence lifetime gradually increased with the increase of laser irradiation time, increased fluorescence lifetime reduces the saturation of fluorescent samples
Intensity, so the image resolution ratio of sample also with fluorescence lifetime increase and be gradually increased.
In the above-described embodiments, it all emphasizes particularly on different fields to the description of each embodiment, there is no the portion being described in detail in some embodiment
Point, it may refer to the associated description of other embodiments.
The above are to a kind of description of the microscopical imaging system of stimulated emission depletion provided by the present invention, for ability
The technical staff in domain, thought according to an embodiment of the present invention, there will be changes in the specific implementation manner and application range,
To sum up, the contents of this specification are not to be construed as limiting the invention.
Claims (10)
1. a kind of microscopical imaging system of stimulated emission depletion, which is characterized in that the system comprises loss light units, excitation
Light unit, imaging unit and counter unit；
The loss light unit is used to generate the annular loss light of predetermined pulse frequency and adjusts the control annular loss light
Light intensity, it is described annular loss light laser wave before be annular；
The loss light unit is connect with the excitation light unit, and the excitation light unit is used for the touching in the loss light unit
Give the Gaussian exciting light for generating the pulse frequency, and by the Gaussian exciting light be divided into the first Gaussian exciting light with
Second Gaussian exciting light is Gaussian, the Gaussian exciting light and the ring before the laser wave of the Gaussian exciting light
Predetermined pulse spacing value is divided between peak value of pulse between shape loss light；
Annular loss light and the first Gaussian exciting light converge it is Chong Die after input the imaging unit, the imaging is singly
Member is for carrying out planar array scanning imaging to sample and collecting the fluorescence signal of sample reflection, the fluorescence signal and described the
Two Gaussian exciting lights inject the counter unit respectively, and the counter unit is used for based on the fluorescence signal and described
Second Gaussian exciting light carries out fluorescence intensity imaging and fluorescence lifetime imaging.
2. imaging system according to claim 1, which is characterized in that the loss light unit includes femto-second laser,
One adjusts unit, pulse broadening unit, the second adjusting unit and spiral phase plate；
The femto-second laser is used to generate the femtosecond pulse type loss light of the pulse frequency, and light is lost in the femtosecond pulse type
It injects described first and adjusts unit, it is inclined to adjust output First Line after unit carries out linear polarization adjusting and light intensity regulating through described first
Vibration loss light, the First Line polarization loss light emission enter the pulse broadening unit, carry out pulse through pulse broadening unit
Light is lost in the broadening that predetermined pulse width is exported after broadening, and the broadening loss light emission enters described second and adjusts unit, through described
Second adjusting unit carries out linear polarization and adjusts and export the second linear polarization loss light, the second linear polarization loss after light intensity regulating
Light exports the annular loss light after the spiral phase plate；
The femto-second laser is connect with the excitation light unit, and the femto-second laser is also used to trigger the excitation light unit
Generate the Gaussian exciting light.
3. imaging system according to claim 2, which is characterized in that it is described first adjust unit include the first half-wave plate and
First Glan prism；
The femtosecond pulse type loss light emission enters first half-wave plate, is adjusted to default first polarization through first half-wave plate
First Glan prism is injected after the linearly polarized light in direction, exports described first after first Glan prism adjusts light intensity
Light is lost in linear polarization.
4. imaging system according to claim 2, which is characterized in that the pulse broadening unit includes glass bar, first
Lens, single-mode polarization maintaining fiber and the second lens；
The First Line polarization loss light emission enters the glass bar, injects described first after the glass bar carries out pulse broadening
Lens inject the single-mode polarization maintaining fiber after first lens focus, carry out pulse again through the single-mode polarization maintaining fiber
Second lens are injected after broadening, and the broadening loss light is exported after second lens focus.
5. imaging system according to claim 2, which is characterized in that it is described second adjust unit include the second half-wave plate and
Second Glan prism；
The broadening loss light emission enters second half-wave plate, is adjusted to default second polarization direction through second half-wave plate
Second Glan prism is injected after linearly polarized light, exports second linear polarization after second Glan prism adjusts light intensity
Light is lost.
6. imaging system according to claim 1, which is characterized in that the excitation light unit includes picosecond laser, list
Mode fiber, the third lens and regulation unit；
The picosecond laser is connect with the loss light unit, and the picosecond laser is used for the touching in the loss light unit
The picosecond pulse type exciting light for generating the pulse frequency is given, the picosecond pulse type exciting light injects the single mode optical fiber,
The third lens are injected after the single mode optical fiber carries out mode adjusting, after the third lens amplify described in injection
Regulation unit exports after the regulation unit carries out linear polarization adjusting, pulse interval modulation and light intensity regulating and swashs
It is the Gaussian exciting light of Gaussian before light wave.
7. imaging system according to claim 6, which is characterized in that the regulation unit include third half-wave plate,
Corner reflector and third Glan prism；
The third half-wave plate is injected through the amplified laser of the third lens, is adjusted to default the through the third half-wave plate
By the corner reflector after the linearly polarized light of three polarization directions, the corner reflector for adjust the Gaussian exciting light with
Pulse spacing between the annular loss light, the laser after the corner reflector carries out pulse interval modulation inject described the
Three Glan prisms export the first Gaussian exciting light and described the after the third Glan prism adjusts light intensity respectively
Two Gaussian exciting lights.
8. imaging system according to claim 1, which is characterized in that the imaging unit is swept including the 4th lens, galvanometer
System, the 5th lens, a quarter slide, high-NA objective and objective table are retouched, places the sample on the objective table；
Annular loss light and the first Gaussian exciting light converge it is Chong Die after successively pass through the 4th lens, the vibration
After scarnning mirror system and the 5th lens, a quarter slide is injected, overlapping will be converged through a quarter slide
After the first Gaussian exciting light and the annular loss light afterwards is adjusted to circularly polarized light by linearly polarized light, by the height
The objective table is incident to after NA objective；
The high-NA objective is used to collect the fluorescence signal of the sample reflection, and the fluorescence signal successively passes through described
The counter unit is injected after a quarter slide, the 5th lens, the galvanometer scanning system, the 4th lens；
The galvanometer scanning system be used for converge overlapping after the first Gaussian exciting light and it is described annular loss light into
Row synchronous scanning is realized and the planar array scanning of the sample is imaged.
9. imaging system according to claim 1, which is characterized in that the counter unit include the 6th lens, filter,
Multimode fibre, photomultiplier tube, the first reflecting mirror, the 7th lens, detector and Single Photon Counting device；
The fluorescence signal successively by the 6th lens transmission, after the filter filters out stray light and the multimode fibre,
The photomultiplier tube is injected, the photomultiplier tube inputs the time correlation single photon meter after amplifying the fluorescence signal
Number device；
The second Gaussian exciting light injects the spy after first reflecting mirror reflection and the 7th lens transmission
Device is surveyed, the Single Photon Counting device is inputted after the detector is converted to electric signal；
The Single Photon Counting device is used to carry out based on the second Gaussian exciting light and the fluorescence signal glimmering
Luminous intensity imaging and fluorescence lifetime imaging.
10. imaging system described in -9 any one according to claim 1, which is characterized in that anti-the system also includes second
Penetrate mirror, third reflecting mirror, the first dichroic mirror and the second dichroic mirror；
The first Gaussian exciting light is successively by second reflecting mirror reflection, third reflecting mirror reflection and described the
The reflection of one dichroic mirror, the annular loss light is successively by second dichroic mirror transmission and first dichroic mirror transmission, warp
The annular loss light and first Gaussian by first dichroic mirror reflection for crossing the first dichroic mirror transmission
Exciting light injects the imaging unit after converging overlapping；
The fluorescence signal successively after first dichroic mirror transmission and second dichroic mirror reflection, injects the counting
Device unit.
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