CN105973853A - Super-resolution microscopy method based on dual-mode competition stimulation and super-resolution microscopy device based on dual-mode competition stimulation - Google Patents
Super-resolution microscopy method based on dual-mode competition stimulation and super-resolution microscopy device based on dual-mode competition stimulation Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
Abstract
The invention discloses a super-resolution microscopy method based on dual-mode competition stimulation and a super-resolution microscopy device based on dual-mode competition stimulation. The method comprises the following steps of: irradiating the surface of a fluorescent sample by use of two types of modulated light beams emitted from a same light source, wherein one light beam is focused to form high-energy hollow light spot on the sample after passing through a spatial light modulator, so as to generate a saturation effect, and the other light beam is modulated into pulsed light by an acousto-optic modulator so as to be focused to form low-energy solid light spots on the sample; and collecting signal lights emitted from each scanning point of the sample, and extracting the signal lights which have same frequency with the pulsed light, wherein the extracted signal lights serve as effective signal lights for acquiring super-resolution images. The device is simple, and convenient to operate; by use of the competitive mechanism of the solid light spots and the hollow light spots during a process of stimulating sample fluorescent, the resolution ratio for saturated fluorescence to stimulate an ultra-diffraction limit is realized by only using one laser light source.
Description
Technical field
The invention belongs to super-resolution field, particularly relate to one and utilize the saturated principle of fluorescence to realize super diffraction limit in far field
The super-resolution microscopic method of resolution and device.
Background technology
Due to the impact of optical system diffraction, there is restriction in the attainable resolution of conventional far-field optical microscopy.Root
Theoretical according to Abbe diffraction limit, light beam size full width at half maximum of become hot spot after microcobjective focuses on is expressed asWherein λ is microscopical operation wavelength, and NA is the numerical aperture of microcobjective used.Therefore, conventional far-field optics
Microscopical limiting resolution has been typically restricted to about half-wavelength.
In order to break through the restriction of optical diffraction limit, improving the resolution of microscopic system, researchers propose many
Planting super-resolution optical microscopic method, wherein fluorescence excitation receives the biggest concern.Some the more commonly used methods have: be stimulated
Penetrate loss microscopy (STED:Stimulated Emission Depletion Microscopy), Structured Illumination fluorescence shows
Micro-art (SIM:Structured Illumination Microscopy), random light field reconstruction microscopy (STORM:
Stochastic Optical Reconstruction Microscopy) etc..
It is micro-that this several method all can realize fluorescence super-resolution in far field, have also been obtained and accordingly should in reality is tested
With, but the most also remain deficiency.Wherein, the resolution of STED microscopy is determined by the luminous power of added loss light, because of
This is when realizing high-resolution, and its required luminous power is very strong, is easily caused the bleaching of fluorescence molecule.SIM microscopy is to light
Although the requirement of power is the highest, but owing to it needs raster scanning, image taking speed is relatively slow, and imaging system is the most complex.
The image taking speed of STORM microscopy is the slowest, is also difficult at present apply in the middle of the detection in real time of active somatic cell.
During conventional fluorescent is micro-, the fluorescence intensity of electromagnetic radiation is directly proportional to excitating light strength；When excitation light intensity not
Disconnected increase, the fluorescence molecule number of excited state will reach saturated, and proportional relation is the most no longer set up and the point spread function of fluorescence
Number also can change, and may be used for obtaining finer structural information.
Summary of the invention
The invention provides a kind of super-resolution microscopic method and device, it is possible to use the saturated principle of fluorescence realizes super in far field
The resolution of diffraction limit.This kind of method and apparatus has that image taking speed is fast, device simple, resolution high, can be very
It is applied to well among the detection of fluorescence and non-fluorescence sample.
A kind of super-resolution microscopic method excited based on double mode competition, comprises the following steps:
1) regulate the polarization situation of collimated laser beam, make P-polarization component light intensity much larger than S-polarization component light intensity
(at least two orders of magnitude of difference)；
2) by through step 1) laser beam that processes is divided into two bunch polarized light, respectively P light and S light；
3) described P light is carried out phase-modulation, and be the pulsed light of predeterminated frequency by S light modulation；
4) P light and the described pulsed light through phase-modulation is combined into light beam, is converted into a left side further according to polarization characteristic
Rounding polarized light and right-circularly polarized light；
5) translate into the light beam of left circularly polarized light and right-circularly polarized light and project sample so that sample to be scanned,
Make the P light through phase-modulation pool hollow light spot on sample during scanning, make described pulsed light pool on sample
Solid hot spot；
6) receive the flashlight that sample sends when scanning, and be demodulated the flashlight received obtaining and described arteries and veins
Wash the optical signal of correspondence off for the image obtaining respective scanned position.
When testing sample (i.e. sample) is fluorescent samples, described flashlight is that described circularly polarized light projects through microcobjective
After the fluorescence that inspires on sample；When testing sample is non-fluorescence sample, described flashlight is that described circularly polarized light is through aobvious
Through the reflection light beam of sample surfaces after the projection of speck mirror.
X on testing sample, y-axis direction is determined by two-dimensional scan direction.
As preferably, the super-resolution microscopic method of the present invention utilize 0～2 π vortex phase plates P light is carried out phase place tune
System.
Present invention also offers a kind of super-resolution microscope equipment excited based on double mode competition, treat including light source, carrying
The sample stage of test sample product, is sequentially provided with between described light source and sample stage:
For changing 1/2 wave plate that described light source sends the polarization characteristic of light beam,
Light beam after being changed by polarization characteristic is divided into the polarization beam apparatus of two bunch polarized light, and two described bunch are inclined
The light that shakes is respectively P light and S light,
For described P light being carried out the spatial light modulator of phase-modulation, obtain for described S light is carried out intensity modulated
The acousto-optic modulator of the pulsed light of predeterminated frequency,
For two pieces of quarter wave plates that described polarized light polarization characteristic is changed,
For the P light through spatial light modulator and the S light through acousto-optic modulator are merged into a branch of polarization coupling
Device,
Light polarization of shaking in the light beam changing polarization beam combiner outgoing and the two pieces of quarter wave plates set gradually,
For the light beam sequentially passing through two pieces of quarter wave plates and being converted into left circularly polarized light and right-circularly polarized light is carried out light
Road deflection makes it project sample, and makes the P light through phase-modulation pool hollow light spot on sample, makes described pulse
Light pools solid hot spot with the scanning galvanometer system being scanned sample and setting gradually, scanning lens, field on sample
Mirror and microcobjective,
For gathering the detection system of the flashlight that sample sends,
For the flashlight received being demodulated obtaining the optical signal corresponding with described pulsed light for obtaining phase
Answer the lock-in amplifier of image at scan position,
And for controlling described spatial light modulator and the controller of scanning galvanometer system and for controlling acousto-optic modulation
Device frequency and the signal generator of lock-in amplifier frequency.
In the present invention, circularly polarized light projects on described testing sample by microcobjective, specific as follows:
Scanning galvanometer system carries out optical path-deflecting to the light beam through two quarter wave plates；
Scanning lens and field lens are respectively used to be focused the light beam of scanning galvanometer system exit and collimate.
Receiving, by detection system, the flashlight that sample sends during this is the brightest, this detection system includes:
Being arranged in the beam splitter between spatial light modulator and scanning galvanometer system, described beam splitter is glimmering at testing sample
Dichroscope should be selected during light sample, polarization splitting prism should be selected when testing sample is non-fluorescence sample；
The band pass filter of the veiling glare in the flashlight filtering off beam splitter outgoing, described band pass filter is to be measured
Can omit when sample is non-fluorescence sample；
For the detector of the light intensity signal of detectable signal light beam, described detector selects photomultiplier tube (PMT)；
Signal beams after filtering focuses on the condenser lens on detector；
For described signal beams carries out the spatial filter of space filtering, it is positioned at the focal plane of described condenser lens
Place, described spatial filter can use pin hole or multimode fibre, and according to pin hole, the diameter of pin hole used should be less than a Chinese mugwort
In spot diameter.
As preferably, between described light source and 1/2 wave plate, it is sequentially provided with the list for described laser beam is filtered
Mode fiber and the collimating lens of collimation.
Heretofore described spatial light modulator has variable modulation functionWherein, ρ is on light beam
Certain point and the distance of optical axis,Angle for position polar coordinate vectors in beam orthogonal optical axis section Yu x-axis.
Further preferably, described spatial light modulator is vortex phase plate.
Described lock-in amplifier and acousto-optic modulator share same signal generator.
This signal generator is for determining that the beam frequencies that lock-in amplifier is regulated and controled with acousto-optic modulator is identical.Preset frequency
Rate sets according to actual test sample, and when reality is applied, regulating this signal generator can be so that lock-in amplifier and acousto-optic
Working at this predeterminated frequency of manipulator.
Numerical aperture NA=1.4 of described microcobjective.
The frequency that described signal generator is carried on acousto-optic modulator and lock-in amplifier is equal.
The principle of the invention is as follows:
Due to the impact of optical system diffraction, the illuminating bundle of parallel incidence, after microcobjective focuses on, is treating test sample
A formed hot spot not preferable point on product, but a diffraction spot with certain size.In diffraction spot range of exposures
Interior sample all can send corresponding flashlight, so that details of sample cannot be resolved in the range of this, thus limits
The resolution of microscopic system.Therefore, the restriction of Gonna breakthrough optical diffraction limit, improves the resolution of microscopic system, how to subtract
Little at scanning element the light-emitting area of useful signal light become for key.
In the methods of the invention, when the output frequency of signal generator is f, the light beam after ovennodulation gathers through microscope
Jiao's one pulse frequency of formation on sample is the solid hot spot (i.e. the frequency of pulsed light is f) of f.The size of this solid hot spot with
In normal optical microscopy, illuminating bundle used focuses on the equivalently-sized of become diffraction spot.Modulation function when spatial light modulator
ForTime, Deby's integral Debye unit can be calculated, after modulation light beam through microcobjective focus on after on sample become hot spot
Hollow light spot for a loaf of bread ring.
Solid hot spot and hollow light spot come from same light source, arrive imaging surface, wherein do not draw after different modulation
Enter any other device changing spot size specially.After vortex position phasor is modulated, the size of hollow light spot compares solid light
Speckle is big, but its Center Dark Spot region is less than solid speckle.
The light intensity of hollow light spot, much larger than the light intensity of solid hot spot, is competed solid hot spot at this in its range of exposures and is irradiated
The flashlight that sample sends, the signal making detector collect is that solid speckle core and hollow speckle irradiate sample and send
Light.Using f as the reference signal frequency of lock-in amplifier, the flashlight received through lock-in amplifier, only pulse frequency
Signal for f can be exported, and the flashlight that the most solid speckle core irradiation sample sends is exported.Obviously should
The efficient lighting area at each scanning element that signal is corresponding is by the useful signal at each scanning element corresponding less than complete solid speckle
Light light-emitting area.
Therefore, compared with normal optical microscopic method, present invention decreases the light-emitting area of useful signal light, such that it is able to
Realize the resolution of super diffraction limit, and need not can directly obtain high-resolution figure by the process of data and calculating
Picture.
Relative to prior art, the present invention has a following useful technique effect:
(1) microscopic method by the competition stimulated radiation of same coloured light saturated hollow speckle is proposed first；
(2) micro-compared with original fluorescent radiation difference, resolution is improved；
(3) device is simple, easy to operate.
Accompanying drawing explanation
Fig. 1 is the structural representation of the super-resolution microscope equipment of the present embodiment；
Fig. 2 is become the normalization curve of light distribution of solid hot spot by the present embodiment pulsed light；
Fig. 3 by the present embodiment the normalization curve of light distribution of one-tenth bread ring hollow light spot；
Fig. 4 is the hot spot of useful signal light and the normalization light of flashlight hot spot in normal optical microscopy in the present embodiment
It is distributed by force comparison curves；
Fig. 5 is that the light distribution in same sample scanning gained image is compared by the present embodiment with normal optical microscopic method
Curve.
Detailed description of the invention
Describe the present invention in detail below in conjunction with embodiment and accompanying drawing, but the present invention is not limited to this.
As it is shown in figure 1, the super-resolution microscope equipment of the present embodiment, including: laser instrument 1, single-mode fiber 2, collimating lens 3,
1/2 wave plate 4, polarization beam apparatus 5, spatial light modulator 6, acousto-optic modulator 7, polarization beam combiner 8, quarter wave plate 9, quarter wave plate
10, dichroscope 11, scanning galvanometer 12, scanning lens 13, field lens 14, microcobjective 15, sample stage 16, band pass filter 17, poly-
Focus lens 18, pin hole 19, detector 20.
Wherein, single-mode fiber 2, collimating lens 3,1/2 wave plate 4, polarization beam apparatus 5, spatial light modulator 6 are sequentially located at sharp
On the optical axis of light device 1 outgoing beam；The light transmission shaft direction of described 1/2 wave plate 4 is adjusted so that light beam is divided by rear P polarization
Amount intensity, much larger than S-polarization component intensity, differs two orders of magnitude.
Wherein, acousto-optic modulator 7 be positioned at through polarization beam apparatus 5 modulate after light beam optical axis on.
Wherein, polarization coupling mirror 8 be positioned at through spatial light modulator 6 modulate after light beam optical axis on.
Wherein, quarter wave plate 9, quarter wave plate 10, dichroscope 11 are sequentially located at the emergent light of polarization beam combiner 8 to be reflected mirror inclined
On optical axis after discharging the retained water；Scanning galvanometer 12 be positioned at through dichroscope 11 reflect after light beam optical axis on.
Wherein, scanning lens 13, field lens 14, microcobjective 15, sample stage 16 is sequentially located at scanning galvanometer 12 outgoing beam
Optical axis on.
Wherein, band pass filter 17, condenser lens 18, pin hole 19, detector 20 are sequentially located at through dichroscope 11 back reflection
On the optical axis of mirror reflection light beam；Described pin hole 19 is positioned at the focal plane of condenser lens 18.
Wherein, controller is connected with spatial light modulator 6 and scanning galvanometer system 7 respectively, is used for loading spatial light and adjusts
The phase modulation of device 6 processed and the scanning of scanning galvanometer system 7.
In said apparatus, numerical aperture NA=1.4 of microcobjective 15；A diameter of 0.73 Airy disk of pin hole 19 used
Diameter, detector 20 is photomultiplier tube (PMT).
The method using the device shown in Fig. 1 to carry out super-resolution micro-is as follows:
The laser beam sent from laser instrument 1, is first introduced into single-mode fiber 2, from the laser light of single-mode fiber 2 outgoing
Bundle, completes collimation through collimating lens 3.Light beam after collimation incides 1/2 wave plate 4 and changes polarization situation, makes P polarization divide
Amount light intensity, much larger than S-polarization component light intensity, is divided into two bunch polarized light by polarization beam apparatus 5 afterwards.P light is irradiated to spatial light and adjusts
On device 6 processed modulated, S light deflection after modulated by acousto-optic modulator 7.
Utilize controller that spatial light modulator 6 is controlled, make the phase modulation function of spatial light modulator 6 switch
For:
Wherein, ρ is certain point and the distance of optical axis on light beam,For position polar coordinate vectors and x in beam orthogonal optical axis section
The angle of axle.
Therefore, after spatial light modulator 6 carries out phase-modulation, the electric vector intensity of outgoing beam can be by following formula table
Show:
Wherein,Exist for inciding the light beam in spatial light modulator 6The electric vector intensity at place,Exist for the outgoing beam after spatial light modulator 6 phase-modulationThe electric vector intensity at place, i is imaginary number list
Position.
And the light after acousto-optic modulator 7 becomes the pulsed light with certain frequency, concrete frequency can be according to test
Sample regulates.
Respectively by two bunch polarized light of spatial light modulator 6 and acousto-optic modulator 7 outgoing by polarization beam combiner 8 again
It is combined into light beam, through quarter wave plate 9 and quarter wave plate 10, makes P light be output as left circularly polarized light.
Light after modulation incides on scanning galvanometer 12, and the light beam of scanned galvanometer 12 outgoing is scanned lens 13 successively and gathers
Burnt, field lens 14 collimates, after project on the testing sample being positioned on sample stage 16 through microcobjective 15.
Incident illumination can be determined by Deby's integral Debye unit in optical field distribution formed by the near focal point of microcobjective 15, specific as follows:
In formula,It is the coordinate under the cylindrical coordinate with the focal position of microcobjective 15 as initial point,RepresentThe electric vector intensity at place, i is imaginary unit, and C is normaliztion constant, and θ is light beam aperture
Angle, φ is position polar coordinate vectors and the angle of x-axis, A in beam orthogonal Z axis section1(θ, φ) is the distribution of amplitudes of incident illumination,
A2(θ, φ) characterizes the structure of microcobjective 15,Then illustrating the polarization information of incident illumination, k=2 π/λ, n is medium folding
Penetrate rate.
Calculated by above formula it is found that now incident P light is become after microcobjective 15 focuses on testing sample
Hot spot is a loaf of bread ring hollow light spot, and its concrete optical field distribution normalized curve is as shown in Figure 2.And the S light of incidence is through aobvious
After speck mirror 15 focusing, on testing sample, become hot spot is a solid hot spot, and its concrete optical field distribution normalized curve is such as
Shown in Fig. 3.
The flashlight of testing sample institute outgoing is collected by microcobjective 15, pass sequentially through afterwards field lens 14, scanning lens 13,
Scanning galvanometer 12, by dichroscope 11, is finally reflected.Signal beams filters off veiling glare by band pass filter 17, after warp
Condenser lens 18 focuses on and passes through pin hole 19 and carries out space filtering, is finally received by detector 20.
Scanning galvanometer 12 is regulated, it is achieved for the two-dimensional scan of testing sample by controller.
When the output frequency of signal generator is f, the light beam after ovennodulation focuses on through microscope and is formed on sample
One pulse frequency is the solid hot spot of f.Illuminating bundle used focuses on the size of this solid hot spot with normal optical microscopy
Become diffraction spot equivalently-sized.When the modulation function of spatial light modulator isTime, Deby's integral Debye unit calculating can
, after modulation light beam through microcobjective focus on after on sample become hot spot be the hollow light spot of a loaf of bread ring.
The light intensity of hollow light spot is much larger than the light intensity of solid hot spot, its concrete optical field distribution contrast normalized curve such as Fig. 4
Shown in.Hollow speckle is competed solid hot spot at this in the range of irradiating and is irradiated the flashlight that sample sends, and makes detector collect
Flashlight be that the most of hollow speckle of solid speckle core and edge irradiates the light that sends of sample.Using f as phase-locked amplification
The reference signal frequency of device, the flashlight received can obtain defeated through the signal that lock-in amplifier, only pulse frequency are f
Going out, the flashlight that the most solid speckle core irradiation sample sends is exported.Obviously each scanning element that this signal is corresponding
The efficient lighting area at place is by the useful signal light light-emitting area at each scanning element corresponding less than complete solid speckle, the most permissible
Obtain super-resolution micro-image.
Useful signal light hot spot and the normalization light intensity of flashlight hot spot in standard copolymerization Jiao's microscopic method in the present embodiment
Distribution curve compares as shown in Figure 5.As seen from Figure 5, in the present invention, the more conventional copolymerization of the spot size of useful signal light is burnt
In microscopic method, flashlight spot size has reduced, and therefore the inventive method can realize the resolution of super diffraction limit.
Claims (8)
1. the super-resolution microscopic method excited based on double mode competition, it is characterised in that comprise the following steps:
1) regulate the polarization situation of collimated laser beam, make P-polarization component light intensity much larger than S-polarization component light intensity；
2) by through step 1) laser beam that processes is divided into two bunch polarized light, respectively P light and S light；
3) described P light is carried out phase-modulation, and be the pulsed light of predeterminated frequency by S light modulation；
4) P light and the described pulsed light through phase-modulation is combined into light beam, is converted into left-handed circle further according to polarization characteristic
Polarized light and right-circularly polarized light；
5) translate into the light beam of left circularly polarized light and right-circularly polarized light and project sample so that sample to be scanned, scanning
Time make the P light through phase-modulation pool hollow light spot on sample, make described pulsed light pool on sample solid
Hot spot；
6) receive the flashlight that sample sends when scanning, and be demodulated the flashlight received obtaining and described pulsed light
Corresponding optical signal is for the image obtaining respective scanned position.
2. the super-resolution microscopic method excited based on double mode competition as claimed in claim 1, it is characterised in that utilize 0～2
π vortex phase plate carries out phase-modulation to P light.
3. the super-resolution microscope equipment excited based on double mode competition, including light source, the sample stage of carrying testing sample, its
It is characterised by, is sequentially provided with between described light source and sample stage:
For changing 1/2 wave plate that described light source sends the polarization characteristic of light beam,
Light beam after being changed by polarization characteristic is divided into the polarization beam apparatus of two bunch polarized light, two described bunch polarized light
It is respectively P light and S light,
For described P light being carried out the spatial light modulator of phase-modulation,
For described S light is carried out the acousto-optic modulator that intensity modulated obtains the pulsed light of predeterminated frequency,
For two pieces of quarter wave plates that described polarized light polarization characteristic is changed,
For the P light through spatial light modulator and the S light through acousto-optic modulator are merged into a branch of polarization beam combiner,
Light polarization of shaking in the light beam changing polarization beam combiner outgoing and the two pieces of quarter wave plates set gradually,
It is inclined for the light beam sequentially passing through two pieces of quarter wave plates and being converted into left circularly polarized light and right-circularly polarized light is carried out light path
Turning makes it project sample, and makes the P light through phase-modulation pool hollow light spot on sample, makes described pulsed light exist
Pool on sample solid hot spot with scanning galvanometer system sample being scanned and setting gradually, scanning lens, field lens and
Microcobjective,
For gathering the detection system of the flashlight that sample sends,
For the flashlight received being demodulated obtaining the optical signal corresponding to described pulsed light for obtaining corresponding sweeping
Retouch the lock-in amplifier of the image of position,
And for controlling described spatial light modulator and the controller of scanning galvanometer system and for controlling acousto-optic modulator frequency
Rate and the signal generator of lock-in amplifier frequency.
4. the super-resolution microscope equipment excited based on double mode competition as claimed in claim 3, it is characterised in that described light source
And it is sequentially provided with the collimating lens of the single-mode fiber for described laser beam is filtered and collimation between 1/2 wave plate.
5. the super-resolution microscope equipment excited based on double mode competition as claimed in claim 3, it is characterised in that described space
Photomodulator has variable modulation functionWherein, ρ is certain point and the distance of optical axis on light beam,For light beam
Position polar coordinate vectors and the angle of x-axis in vertical optical axis section.
6. the super-resolution microscope equipment excited based on double mode competition as claimed in claim 5, it is characterised in that described space
Photomodulator is vortex phase plate.
7. the super-resolution microscope equipment excited based on double mode competition as claimed in claim 3, it is characterised in that described lock
Phase amplifier and acousto-optic modulator share same signal generator.
8. super-resolution microscope equipment as claimed in claim 3, it is characterised in that numerical aperture NA=of described microcobjective
1.4。
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