CN108120702A - A kind of super resolution fluorescence lifetime imaging method and device based on parallel detecting - Google Patents
A kind of super resolution fluorescence lifetime imaging method and device based on parallel detecting Download PDFInfo
- Publication number
- CN108120702A CN108120702A CN201711241950.3A CN201711241950A CN108120702A CN 108120702 A CN108120702 A CN 108120702A CN 201711241950 A CN201711241950 A CN 201711241950A CN 108120702 A CN108120702 A CN 108120702A
- Authority
- CN
- China
- Prior art keywords
- mrow
- mover
- fluorescence lifetime
- rightarrow
- super resolution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- 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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
Landscapes
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Optics & Photonics (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Microscoopes, Condenser (AREA)
Abstract
The present invention discloses a kind of super resolution fluorescence lifetime imaging device based on parallel detecting, and including light source, the exciting light that light source is sent is focused on sample by microcobjective and collected the flashlight that sample is sent, wherein, receiving the detection system of flashlight includes：Fiber optic bundle, interior multifiber beam receive the flashlight simultaneously；Detector array has the multiple detectors for connecting every optical fiber respectively, obtains corresponding light intensity signal；Single Photon Counting device array has and connects each detector and the Single Photon Counting device synchronous with light source pulse respectively, for calculating fluorescence lifetime and realizing that super resolution fluorescence lifetime is imaged.Invention additionally discloses a kind of super resolution fluorescence lifetime imaging methods based on parallel detecting.The present invention utilizes parallel APD and parallel TCSPC, not only realizes the promotion of imaging resolution, also significantly improves service life image taking speed.
Description
Technical field
The invention belongs to super-resolution microscopic fluorescence service life imaging field, more particularly, to a kind of super based on parallel detecting
Resolved fluorometric service life imaging method and device.
Background technology
The effect of light and substance is most basic mechanism in nature, especially single atom and single photon
Interaction is to realize light and the most common element of material effect.And in this field, super-resolution fluorescence micro-imaging is always
It is the emphasis of biomedical research, by the special marking to biomolecule, fluorescence optical microscope is in observation subcellular structure
Play important role.
Fluorescence information tool is there are four basic physical dimension, including strength information, wavelength information (absorption spectrum and transmitting
Spectrum), life information and polarization information.The current main focus utilization of super-resolution microtechnic in intensity and wavelength, including
Absorption and emission spectra using fluorescence, realize intensity on " switch " modulate (STED technologies, PALM/STORM technologies etc.) or
Person includes SIM technologies or saturation SIM technologies using the intensity modulated technology in structure.And in terms of polarization, also have at present not
Few technology realizes polarization demodulation, obtains raising of the polarization information so as to fulfill resolution ratio.In addition, life information is
A very important information in terms of biological image microstructures, fluorescence lifetime imaging is the spectrum of a more and more extensive application
Technology, it is capable of providing the location information of specific fluorogen and the micro-environmental variation information of fluorescence molecule, including many biologies
Physics, biochemical parameters such as ion concentration, pH value, oxygen concentration, refractive index, stickiness and temperature etc., particularly, fluorescence lifetime
Imaging can also apply to the detection to the fluorescence resonance energy transfer (FRET) of fluorescence molecule pair, can be used in studying albumen
The mutual power of matter and cell acts on.The major advantage of fluorescence lifetime imaging is its imaging results to fluorescence intensity and focusing
Situation is simultaneously insensitive, therefore need not obtain fluorescence intensity and can obtain required life information.Fluorescence lifetime imaging simultaneously
Can focus on together, STED technologies are combined, new research means are provided for biomedical research.
Current fluorescence lifetime imaging mode mainly has two major classes, and one kind is time domain service life imaging method, and another kind of is frequency
Domain service life imaging method, but the essence of two methods is identical.Frequency domain service life imaging method is more suitable for service life distribution in more
The sample of exponential damping, and time lifetime imaging is then suitable for more flexible, big dynamic range and the sample of long-life.Base
One kind that lifetime measurement is time domain service life imaging method is carried out in confocal system and TCSPC counting devices, it is capable of providing
Preferable service life imaging resolution, while the long short life sample of various dynamic ranges can be flexibly measured, but it is this
Method is relatively low to the detection and counting efficiency of photon due to being influenced be subject to detector and counter dead journey time, therefore is imaged
Speed is very slow, this is also the shortcomings that this method is maximum.
The content of the invention
The present invention provides a kind of super resolution fluorescence lifetime micro imaging methods and device based on parallel detecting, utilize
Parallel avalanche photodide (APD) array and parallel time correlated single photon counter (TCSPC) array are combined, altogether
In the case of focusing on spot scan, the fluorescence signal that is ejected of fluorescent samples is collected with fiber optic bundle, and is passed through in fiber optic bundle
Fluorescence signal collection into each individual APD, the acquisition for realizing life information is counted using TCSPC by each optical fiber, then
Using the related algorithm of parallel detecting, realize the super resolution fluorescence lifetime imaging to sample, improving intensity imaging resolution ratio
While, the precision of service life imaging is improved, realizes significantly improving for service life imaging rate.This kind of method and apparatus have imaging
The features such as speed is fast, high resolution, device are simple and convenient to operate can be advantageously applied to the super-resolution fluorescence of biological cell
Among service life imaging.
Specific technical solution of the present invention is as follows：
A kind of super resolution fluorescence lifetime imaging device based on parallel detecting, the scanning including light source, carrying sample to be tested
Platform is equipped with successively between the light source and scanning platform：
For ensureing the constant polarization maintaining optical fibre in laser emitting polarization direction；
For the collimation lens collimated to the light beam that lasing light emitter is sent；
Light beam for light source to be sent changes into 1/2 wave plate of pure linearly polarized light and quarter wave plate and by linear polarization
Light is converted to the quarter wave plate of circularly polarized light；
It is used to implement and laser beam is expanded and the 4f systems of spot scan, including scanning mirror and field lens；
For exciting light to be focused on to the microcobjective on sample；
It is used to implement the electronic piezotable of scanning；
For reflected excitation light, the dichroic mirror of transmission fluorescence；
And it is equipped with to control the controller of laser light source and platform and collects the flashlight that the sample to be tested is sent
Detection system.
The flashlight sent in the present invention by detection system reception sample, the detection system include：
For the narrow band filter slice for filtering off ambient noise, improving imaging signal-to-noise ratio；
For adjusting the 4f lens systems of signal spot size to be detected；
For signal beams to be focused on to the condenser lens in the fiber optic bundle for collecting flashlight；
For signal photon to be transferred to the fiber optic bundle of avalanche photodide (APD) array；
Based on the time correlation single photon calculated by the APD array and progress photon counting, fluorescence lifetime of detectable signal light
Number device (TCSPC) array.
Super resolution fluorescence lifetime imaging method provided by the invention based on parallel detecting, comprises the following steps：
1) laser beam that laser is sent is collimated after polarization maintaining optical fibre；
2) light beam is become pure linearly polarized light, is become using a quarter wave plate by 1/2 wave plate and quarter wave plate
Circularly polarized light；
3) light beam is entering microscope stand after Polarization Modulation by dichroic mirror and speculum, be scanned through mirror and
After the 4f systems of field lens composition expand, fluorescent samples are excited in sample surfaces by the immersion oil object lens post-concentration of large-numerical aperture
Generate fluorescence；
4) after the fluorescence ejected is returned along original optical path, through dichroic mirror, by narrow band filter slice and corresponding 4f
System realize fluorescence signal filtering and expand, finally converge to optical fiber beam port, fiber optic bundle port distribution as shown in Fig. 2,
Fluorescence signal is collected using fiber optic bundle, and every optical fiber in fiber optic bundle be connected to it is each in detector array
APD, at this time the light intensity signal received by each APD be：
Wherein,Scanned position vector on representative sample,The position vector of the object on object space is represented,It represents
Position vector where detector,Represent the intensity distribution of object space, effective point spread function
It can be expressed as
Wherein,Excitation point spread function is represented,Represent detection point spread function.
The light intensity signal that this method is received using APD array, the method recombinated using photon realize imaging resolution
Raising.In formula (2), some detector in detector array is not when in conjugate focus, and at this moment it is imaged effective
PSF will shift, then imaging shift, by the imaging results of offset move to originally should center,
Translation coefficient is represented with q, i.e.,：
It is stacked up again by detect each detector by the light intensity signal recombinated, just obtains final surpass
Resolution imaging result：
On the other hand, each APD in detector array is connected with a TCSPC, and TCSPC is carried out with light source pulse
It is synchronous, by calculating the arrival time of the photon that APD is received within each synchronizing cycle, so as to fulfill the calculating in service life.
The principle of the invention is as follows：
Fluorescence microscope has become modern biology and medicine private tutoring or scarce instrument at present, to intracellular different
Structure catch different fluorescence spectrum information dyestuff can effectively help modern science to the dependent dynamics of living cells into
Row research.Fluorescence lifetime imaging is then a kind of imaging method of wherein maximally efficient research intraor extracellular microenvironment, it into
Camera reason determines that it is not influenced be subject to fluorescence intensity, it is capable of providing the location information of specific fluorogen and fluorescence point
The micro-environmental variation information of son, including many biophysics, biochemical parameters such as ion concentration, pH value, oxygen concentration, refraction
Rate, stickiness and temperature etc., particularly, fluorescence lifetime imaging can also apply to turn the fluorescence resonance energy of fluorescence molecule pair
(FRET) detection moved, can be used in studying protein and cell the effect microscopic system imaging of mutual power resolution ratio by
The influence of optical system diffraction.
But current fluorescence lifetime imaging also there is some it is apparent the problem of, for utilizing confocal system, use
The problem of system of TCSPC count measurement fluorescence lifetimes, most important problem is exactly image taking speed.Since APD and TCSPC are dead
The limitation of journey time causes photon pile up effect so that the photon being really recorded is much smaller than authentic and valid flashlight
Son so as to seriously affect the accuracy of fluorescence lifetime, and in order to ensure the accuracy that fluorescence lifetime calculates, then needs use very weak
Excitation light intensity and prolonged sweep time compensate, cause the fluorescence lifetime imaging speed very slow.And this method
Using parallel APD array and parallel TCSPC arrays, APD has very high quantum efficiency in itself, to the detection efficient of fluorescence very
Height, and under identical sweep time, the mode of parallel detecting can weaken photon pile up effect, when some APD is subject to dead journey
Between influence when can not detect photon, other APD can receive photon, therefore the present invention can be detected and more had
Signal is imitated, improves the accuracy of service life imaging.And on the premise of the accuracy of fluorescence lifetime imaging is ensured, it is visited using parallel
The method of survey collects more photons due to that can weaken pile up effect within the equal time, can significantly improve fluorescence
The speed of service life imaging, solves the problems, such as that fluorescence lifetime imaging is slow-footed.And in terms of imaging resolution, the present invention is using simultaneously
The method of row detection photon restructuring, the position that the image translation of each APD offsets obtained is returned to its script is overlapped, obtains
Obtain the higher imaging results of resolution ratio.Simultaneously for the first time using APD array as detector, using its high temporal resolution with
Quantum efficiency can further improve the precision and signal-to-noise ratio of service life imaging.
Although this method is based on confocal system, by the way of counting using parallel detecting and parallel, using parallel
APD and parallel TCSPC, not only realizes the promotion of imaging resolution, also significantly improves service life image taking speed.Compared with existing
There is technology, the present invention has technique effect beneficial below：
(1) realized for the first time using parallel APD array and TCSPC arrays and the parallel detecting of photon is counted, realize the fluorescence longevity
Life image taking speed significantly improves；
(2) the photon reassembly algorithm of parallel detecting is realized using APD array for the first time, realizes the resolution of fluorescence lifetime imaging
Rate significantly improves, and signal-to-noise ratio is also further promoted；
(3) simple improvement has been carried out on the basis of confocal system, device is simple, easy to operate.
Description of the drawings
Fig. 1 is the structure diagram of the super resolution fluorescence lifetime imaging device based on parallel detecting of the present embodiment；
Fig. 2 is the fiber optic bundle end view that detector array is connected in the present embodiment；
Fig. 3 is that fluorescence intensity imaging resolution compares figure in the case of being copolymerized burnt and parallel detecting in the present embodiment；Wherein,
Fig. 3 (a) be in the case that common copolymerization is burnt to the imaging results of 100nm fluorescent grains, and Fig. 3 (b) be using the present invention into
As result；
Fig. 4 is single-point residence time identical in the present embodiment, copolymerization coke and Fluorescence lifetime distribution in the case of parallel detecting
The comparison figure of histogram；Wherein, for the present invention, focus method detects number of photons comparison diagram to Fig. 4 (a) together, and Fig. 4 (b) is pair
The service life statistic histogram of fluorescent grain；
Fig. 5 be in the present embodiment when the copolymerization burnt single-point residence time is 2.5 times of parallel detecting single-point residence time,
The comparison figure of respective Fluorescence lifetime distribution histogram, wherein, Fig. 5 (a) fluorescence lifetime statistic histograms, Fig. 5 (b) is standard
The fluorescence lifetime statistic histogram of change.
Specific embodiment
With reference to embodiment and attached drawing, the present invention will be described in detail, but the present invention is not limited to this.
Super resolution fluorescence lifetime imaging device based on parallel detecting as shown in Figure 1, including：Laser light source 1, polarization-maintaining
Optical fiber 2, collimation lens 3,4,1/2 wave plate 5 of quarter wave plate, quarter wave plate 6, dichroic mirror 7, speculum 8, scanning mirror 9, field lens 10 are high
Numerical value microcobjective 11, sample 12, scanning platform 13, speculum 14, narrow band filter slice 15, lens 16, aperture 17, lens 18,
Convergent lens 19, fiber optic bundle 20, APD array 21, TCSPC arrays 22 and computer and controller 23.
Collimation lens 3, quarter wave plate 4,1/2 wave plate 5 and quarter wave plate 6 are sequentially located at the optical axis of 1 outgoing beam of laser light source
On.Excitation beam reflected by dichroic mirror 7 after using speculum microscopic system, scanning mirror 9, field lens 10 and microcobjective
11 are located on the optical axis after the reflection of speculum 8, wherein, scanning mirror 9 and field lens 10 constitute a 4f system so that incident
The numerical aperture of the beam size matching object lens of exciting light, and it is directional light to ensure in object lens entrance pupil position.Excitation
Fluorescence is returned along original optical path caused by sample, enters detection road, narrow band filter slice 15, lens 16, hole through dichroic mirror 7
On optical axis after door screen 17, lens 18, convergent lens 19 and the reflection (as shown in Figure 2) positioned at speculum 14 of fiber optic bundle 20.APD
21 reception optical fiber beam of array collect photon signal and be connected with TCSPC arrays 22 realize photon counting, TCSPC arrays 22 and
Excitation light source is connected with synchronous signal line realizes the measurement of impulsive synchronization realization fluorescence lifetime, computer and controller 23 and laser
Light source 1, scanning platform 13 are connected with TCSPC arrays, for controlling entire imaging process.
In the present embodiment, the numerical aperture NA=1.4 of microcobjective 11.
Super resolution fluorescence lifetime imaging method under parallel detecting is realized using device shown in FIG. 1, process is as follows：
The laser beam sent from laser light source 1 is completed to collimate after the outgoing of polarization maintaining optical fibre 2 through collimation lens 3.Through
The light beam crossed after collimation passes through the polarised light for being adjusted to pure line of 4 and 1/2 wave plate 5 of quarter wave plate, and linearly polarized light is using fast
Linearly polarized light can be converted to circularly polarized light by axis with linear polarization into during 45 degree of angles, so as to be conducive to the excitation to fluorescence.
Dichroic mirror 7 will be adjusted to the excitation light reflection of circularly polarized light, and exciting light is reflected into using speculum 8 by scanning
The 4f systems that mirror 9 and field lens 10 form, although the platform scanner rather than vibration mirror scanning that are used in this example, the 4f systems
Can carry out excitation beam to expand makes it match the numerical aperture of object lens, while ensures to be directional light at object lens entrance pupil.Enter
It penetrates laser and the fluorescent samples 12 being fixed on scanning platform 13, the fluorescence ejected is excited after being focused on by microcobjective 11
It returns along original optical path, through detection system is entered after dichroic mirror 7, ambient noise is filtered by narrow band filter slice 15
Afterwards, the 4f systems formed by lens 16 and lens 18 carry out shrink beam, realize the reduction of detection numerical aperture, allow and finally understood
The fluorescence signal hot spot that poly- lens 19 are assembled becomes larger to match the size of fiber optic bundle 20, and wherein aperture 17 is used for filtering
Off-axis and defocus signal, fiber optic bundle 20 is formed by 61 optical fiber combinations, and fiber end face is as shown in Fig. 2, this method can be with
Realize 61 road parallel detectings.Fluorescent photon is collected by fiber optic bundle into detector APD array 21, recycling and laser light source
The counting processing of synchronous TCSPC arrays 22, can finally generate rapid super-resolution fluorescence lifetime imaging result.
The main advantage of the present invention is the raising of fluorescence lifetime imaging resolution ratio and dramatically speeding up for image taking speed.Figure
3-5 is the result figure of verification this method superiority, is the analysis chart to the scanning result of 100nm particles, in the present embodiment
Fiber optic bundle has only used wherein 7 optical fiber, therefore has also only used the parallel APD array in 7 tunnels and TCSPC arrays, is total to as what is compared
It is the diameter simple optical fiber identical with used fiber optic bundle (7 optical fiber) total size used in focusing system.Such as Fig. 3
The imaging results figure of 100nm fluorescent grains is shown, Fig. 3 (a) is to 100nm fluorescent grains in the case that common copolymerization is burnt
Imaging results, and Fig. 3 (b) is using the imaging results of the present invention, and significantly carrying for imaging signal-to-noise ratio is realized by APD array
Height, while the algorithm of photon restructuring also allows the resolution ratio of imaging to significantly improve.
And in terms of service life image taking speed, under the identical simple scan time, as shown in figure 4, light in the present invention
The absolute light subnumber that parallel detecting corresponding to fine beam is detected is detected apparently higher than the corresponding focus method altogether of single optical fiber
The number of photons (shown in such as Fig. 4 (a)) arrived, it is smart after over-fitting so as to cause the burnt obtained fluorescence lifetime statistic histogram of copolymerization
Degree is far inferior to this method, is the service life statistic histogram to fluorescent grain in Fig. 4 (b), it can be seen that parallel detecting corresponds to
Fitted Gaussian curve standard deviation be less than copolymerization coke corresponding to matched curve (the fitted Gaussian curve of parallel detecting
Standard deviation is 0.2881, is copolymerized burnt respective standard difference as 0.3097), it means that under the identical simple scan time, this
The measured fluorescence lifetime imaging precision of invention is higher than confocal system.And in order to ensure confocal system and this method all
It obtains accurate fluorescence lifetime, then must assure that the number of photons of two methods detection is suitable, i.e., as shown in Fig. 5 (a), and
At this time measured by the fitted Gaussian curve corresponding to the fluorescence lifetime histogram measured by parallel detecting and common confocal system
Relatively, the standard deviation of respective matched curve is also very close to as shown in Fig. 5 (b), but the single-point for being copolymerized coke at this time is swept for curve
Retouch 2.5 times that the time is this method, it is meant that in the parallel detecting module that 7 APD and TCSPC is used to form, can be implemented as
As speed at least improves 2.5 times.
The foregoing is merely the preferable implementation examples of the present invention, are not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modifications, equivalent replacements and improvements are made should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of super resolution fluorescence lifetime imaging device based on parallel detecting, including light source, the exciting light that light source is sent is by showing
Speck mirror focuses on sample and collects the flashlight that sample is sent, which is characterized in that receiving the detection system of flashlight includes：
Fiber optic bundle includes multifiber beam and receives the flashlight simultaneously；
Detector array has the multiple detectors for connecting every optical fiber respectively, obtains corresponding light intensity signal；
Single Photon Counting device array has and connects each detector and the time correlation synchronous with light source pulse respectively
Single photon counter, for calculating fluorescence lifetime and realizing that super resolution fluorescence lifetime is imaged.
2. super resolution fluorescence lifetime imaging device as described in claim 1, which is characterized in that the light source and microcobjective
Between be sequentially arranged：
For ensureing the constant polarization maintaining optical fibre in exciting light outgoing polarization direction；
For the collimation lens collimated to exciting light；
For being converted to circularly polarized light by exciting light for 1/2 wave plate of linearly polarized light and the first quarter wave plate and by linearly polarized light
Second quarter wave plate；
For being expanded to exciting light and the 4f systems of spot scan.
3. super resolution fluorescence lifetime imaging device as described in claim 1, which is characterized in that have and place the sample and real
The electronic piezotable now scanned.
4. super resolution fluorescence lifetime imaging device as described in claim 1, which is characterized in that the detection system include according to
Secondary arrangement：
For the narrow band filter slice for filtering off ambient noise, improving imaging signal-to-noise ratio；
For adjusting the 4f lens systems of signal spot size；
With for by signal beams focus on collect flashlight fiber optic bundle in condenser lens.
5. super resolution fluorescence lifetime imaging device as described in claim 1, which is characterized in that the detector array is classified as
APD array.
6. a kind of super resolution fluorescence lifetime imaging method based on parallel detecting, which is characterized in that including：
The exciting light that light source is sent focuses on sample surfaces by microcobjective, and fluorescent samples is excited to generate fluorescence；
Fluorescence signal is collected using fiber optic bundle, the detector acquisition light intensity signal being connected with each every optical fiber；
Using synchronous with light source pulse and connect the Single Photon Counting device array of each detector respectively, the fluorescence longevity is calculated
Life realizes super resolution fluorescence lifetime imaging.
7. super resolution fluorescence lifetime imaging method as claimed in claim 6, which is characterized in that exciting light is converted to after collimation
Linearly polarized light is reconverted into after circularly polarized light and focuses on sample surfaces by microcobjective.
8. super resolution fluorescence lifetime imaging method as claimed in claim 6, which is characterized in that received by each detector
Light intensity signal is：
<mrow>
<mi>I</mi>
<mrow>
<mo>(</mo>
<mover>
<mi>r</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>;</mo>
<mover>
<mi>s</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>)</mo>
</mrow>
<mo>=</mo>
<mo>&Integral;</mo>
<mi>O</mi>
<mrow>
<mo>(</mo>
<mover>
<mi>r</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>-</mo>
<msup>
<mover>
<mi>r</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>&prime;</mo>
</msup>
<mo>)</mo>
</mrow>
<msub>
<mi>PSF</mi>
<mrow>
<mi>e</mi>
<mi>f</mi>
<mi>f</mi>
</mrow>
</msub>
<mrow>
<mo>(</mo>
<msup>
<mover>
<mi>r</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>&prime;</mo>
</msup>
<mo>;</mo>
<mover>
<mi>s</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>)</mo>
</mrow>
<mi>d</mi>
<msup>
<mover>
<mi>r</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>&prime;</mo>
</msup>
</mrow>
Wherein,Scanned position vector on representative sample,The position vector of the object on object space is represented,Represent detector
The position vector at place,Represent the intensity distribution of object space, effective point spread functionIt can represent
For
<mrow>
<msub>
<mi>PSF</mi>
<mrow>
<mi>e</mi>
<mi>f</mi>
<mi>f</mi>
</mrow>
</msub>
<mrow>
<mo>(</mo>
<msup>
<mover>
<mi>r</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>&prime;</mo>
</msup>
<mo>;</mo>
<mover>
<mi>s</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>)</mo>
</mrow>
<mo>=</mo>
<msub>
<mi>PSF</mi>
<mrow>
<mi>e</mi>
<mi>x</mi>
<mi>c</mi>
</mrow>
</msub>
<mrow>
<mo>(</mo>
<msup>
<mover>
<mi>r</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>&prime;</mo>
</msup>
<mo>)</mo>
</mrow>
<msub>
<mi>PSF</mi>
<mi>det</mi>
</msub>
<mrow>
<mo>(</mo>
<msup>
<mover>
<mi>r</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>&prime;</mo>
</msup>
<mo>+</mo>
<mover>
<mi>s</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>)</mo>
</mrow>
</mrow>
Wherein,Excitation point spread function is represented,Represent detection point spread function.
9. super resolution fluorescence lifetime imaging method as claimed in claim 8, which is characterized in that some detector is not in confocal
When on point, imaging shifts, and the imaging results of offset is moved to the center that should locate, i.e.,：
<mrow>
<msub>
<mi>I</mi>
<mrow>
<mi>p</mi>
<mi>r</mi>
</mrow>
</msub>
<mrow>
<mo>(</mo>
<mover>
<mi>r</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>;</mo>
<mover>
<mi>s</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>)</mo>
</mrow>
<mo>=</mo>
<mi>I</mi>
<mrow>
<mo>(</mo>
<mover>
<mi>r</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>-</mo>
<mi>q</mi>
<mo>&CenterDot;</mo>
<mover>
<mi>s</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>;</mo>
<mover>
<mi>s</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>)</mo>
</mrow>
</mrow>
Wherein, q represents translation coefficient.
10. super resolution fluorescence lifetime imaging method as claimed in claim 9, which is characterized in that detect each detector
By restructuring light intensity signal and temporal information stack up, just obtain final super-resolution service life imaging results：
<mrow>
<msub>
<mi>I</mi>
<mrow>
<mi>f</mi>
<mi>i</mi>
<mi>n</mi>
<mi>a</mi>
<mi>l</mi>
</mrow>
</msub>
<mo>=</mo>
<munder>
<mo>&Sigma;</mo>
<mi>s</mi>
</munder>
<msub>
<mi>I</mi>
<mrow>
<mi>p</mi>
<mi>r</mi>
</mrow>
</msub>
<mrow>
<mo>(</mo>
<mover>
<mi>r</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>,</mo>
<mover>
<mi>s</mi>
<mo>&RightArrow;</mo>
</mover>
<mo>)</mo>
</mrow>
<mo>.</mo>
</mrow>
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711241950.3A CN108120702B (en) | 2017-11-30 | 2017-11-30 | Super-resolution fluorescence lifetime imaging method and device based on parallel detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711241950.3A CN108120702B (en) | 2017-11-30 | 2017-11-30 | Super-resolution fluorescence lifetime imaging method and device based on parallel detection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108120702A true CN108120702A (en) | 2018-06-05 |
| CN108120702B CN108120702B (en) | 2020-08-11 |
Family
ID=62229713
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711241950.3A Active CN108120702B (en) | 2017-11-30 | 2017-11-30 | Super-resolution fluorescence lifetime imaging method and device based on parallel detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108120702B (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109283674A (en) * | 2018-10-08 | 2019-01-29 | 西北大学 | A kind of fluorescence difference microscopes optical path device |
| CN110109239A (en) * | 2019-04-30 | 2019-08-09 | 华中科技大学 | A kind of while multilayer imaging mating plate illumination micro imaging method and system |
| CN111024658A (en) * | 2019-11-27 | 2020-04-17 | 浙江大学 | Fluorescent molecule directional positioning method and device |
| WO2020140283A1 (en) * | 2019-01-04 | 2020-07-09 | 深圳大学 | Imaging system of stimulated emission loss microscope |
| CN111537478A (en) * | 2020-04-24 | 2020-08-14 | 华东师范大学 | Super-resolution optical microscopic imaging system based on frequency division multiplexing |
| CN111796295A (en) * | 2020-06-04 | 2020-10-20 | 深圳奥锐达科技有限公司 | Collector, manufacturing method of collector and distance measuring system |
| CN112113939A (en) * | 2020-07-31 | 2020-12-22 | 浙江大学 | Fluorescence lifetime imaging method and device based on spectral technology |
| CN112903640A (en) * | 2021-01-19 | 2021-06-04 | 雷振东 | Photon recoil imaging confocal detection system and method |
| WO2022111602A1 (en) * | 2020-11-27 | 2022-06-02 | 浙江大学 | Super-resolution microscopic imaging method and apparatus based on shared-path parallel fluorescence emission difference |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102033058A (en) * | 2010-11-19 | 2011-04-27 | 深圳大学 | Super resolution fluorescence lifetime imaging method and system |
| CN104614318A (en) * | 2015-01-28 | 2015-05-13 | 浙江大学 | Rapid super-resolution micro-imaging method and device |
| CN105467572A (en) * | 2016-01-18 | 2016-04-06 | 北京大学 | Multiphoton subpulse STED-SPIM microscopic system realized by single wavelength |
| CN105510290A (en) * | 2015-12-22 | 2016-04-20 | 浙江大学 | Non-linear super-resolution microscopic method and device adopting photon recombination |
| CN105699056A (en) * | 2016-02-22 | 2016-06-22 | 中国科学院上海光学精密机械研究所 | Device and method for detecting intensity and phase transmission distribution of colored glass filter |
| CN206057175U (en) * | 2016-08-03 | 2017-03-29 | 复旦大学 | A kind of cancer diagnosis system based on the autofluorescence life-span |
| CN106770095A (en) * | 2016-11-30 | 2017-05-31 | 浙江大学 | A kind of super-resolution micro imaging method and device based on the modulation of non-linear hot spot |
| CN106841141A (en) * | 2017-01-26 | 2017-06-13 | 浙江大学 | A kind of fiber optic loop battle array resonance type piezoelectric scanning method and device based on photon restructuring |
| CN107167455A (en) * | 2017-05-23 | 2017-09-15 | 北京理工大学 | Light splitting pupil laser differential confocal CARS micro-spectrometer method and devices |
-
2017
- 2017-11-30 CN CN201711241950.3A patent/CN108120702B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102033058A (en) * | 2010-11-19 | 2011-04-27 | 深圳大学 | Super resolution fluorescence lifetime imaging method and system |
| CN104614318A (en) * | 2015-01-28 | 2015-05-13 | 浙江大学 | Rapid super-resolution micro-imaging method and device |
| CN105510290A (en) * | 2015-12-22 | 2016-04-20 | 浙江大学 | Non-linear super-resolution microscopic method and device adopting photon recombination |
| CN105467572A (en) * | 2016-01-18 | 2016-04-06 | 北京大学 | Multiphoton subpulse STED-SPIM microscopic system realized by single wavelength |
| CN105699056A (en) * | 2016-02-22 | 2016-06-22 | 中国科学院上海光学精密机械研究所 | Device and method for detecting intensity and phase transmission distribution of colored glass filter |
| CN206057175U (en) * | 2016-08-03 | 2017-03-29 | 复旦大学 | A kind of cancer diagnosis system based on the autofluorescence life-span |
| CN106770095A (en) * | 2016-11-30 | 2017-05-31 | 浙江大学 | A kind of super-resolution micro imaging method and device based on the modulation of non-linear hot spot |
| CN106841141A (en) * | 2017-01-26 | 2017-06-13 | 浙江大学 | A kind of fiber optic loop battle array resonance type piezoelectric scanning method and device based on photon restructuring |
| CN107167455A (en) * | 2017-05-23 | 2017-09-15 | 北京理工大学 | Light splitting pupil laser differential confocal CARS micro-spectrometer method and devices |
Non-Patent Citations (6)
| Title |
|---|
| DAY-UEI LI ET AL.: "Real-time fluorescence lifetime imaging system with a 32 × 32 0.13μm CMOS low dark-count single-photon avalanche diode array", 《OPTICS EXPRESS》 * |
| RINNENTHAL ET AL.: "Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation", 《PLOS ONE》 * |
| SHAOCONG LIU ET AL.: "Imaging scanning fluorescence emission difference microscopy based on a detector array", 《IMAGING AND APPLIED OPTICS CONGRESS》 * |
| SIMON P. POLAND ET AL.: "A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging", 《BIOMEDICAL OPTICS EXPRESS》 * |
| 王轶凡: "基于荧光时空调控的超分辨显微方法与系统研究", 《中国博士学位论文全文数据库信息科技辑》 * |
| 顾兆泰: "超分辨荧光寿命显微方法及系统", 《中国优秀硕士学位论文全文数据库基础科学辑》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109283674A (en) * | 2018-10-08 | 2019-01-29 | 西北大学 | A kind of fluorescence difference microscopes optical path device |
| WO2020140283A1 (en) * | 2019-01-04 | 2020-07-09 | 深圳大学 | Imaging system of stimulated emission loss microscope |
| CN110109239A (en) * | 2019-04-30 | 2019-08-09 | 华中科技大学 | A kind of while multilayer imaging mating plate illumination micro imaging method and system |
| CN111024658A (en) * | 2019-11-27 | 2020-04-17 | 浙江大学 | Fluorescent molecule directional positioning method and device |
| CN111537478A (en) * | 2020-04-24 | 2020-08-14 | 华东师范大学 | Super-resolution optical microscopic imaging system based on frequency division multiplexing |
| CN111796295A (en) * | 2020-06-04 | 2020-10-20 | 深圳奥锐达科技有限公司 | Collector, manufacturing method of collector and distance measuring system |
| CN111796295B (en) * | 2020-06-04 | 2023-12-12 | 深圳奥锐达科技有限公司 | Collector, manufacturing method of collector and distance measuring system |
| CN112113939A (en) * | 2020-07-31 | 2020-12-22 | 浙江大学 | Fluorescence lifetime imaging method and device based on spectral technology |
| WO2022111602A1 (en) * | 2020-11-27 | 2022-06-02 | 浙江大学 | Super-resolution microscopic imaging method and apparatus based on shared-path parallel fluorescence emission difference |
| CN112903640A (en) * | 2021-01-19 | 2021-06-04 | 雷振东 | Photon recoil imaging confocal detection system and method |
| CN112903640B (en) * | 2021-01-19 | 2023-01-03 | 雷振东 | Photon recoil imaging confocal detection system and method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108120702B (en) | 2020-08-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108120702A (en) | A kind of super resolution fluorescence lifetime imaging method and device based on parallel detecting | |
| CN102735617B (en) | Super-resolution microscopic method and super-resolution microscopic device | |
| CN104597590B (en) | A kind of super-resolution fluorescence light spectrum image-forming microscope | |
| CN103543135B (en) | A kind of nano-precision hot spot alignment methods based on Fluorescence lifetime distribution and device | |
| CN103091297B (en) | Super-resolution microscope method and device based on random fluorescence bleaching | |
| CN102798622B (en) | Intensity difference based three-dimensional super-resolution microscopic method and device | |
| CN110208224B (en) | Living body single cell multifunctional spectrometer based on coaxial double waveguide optical fibers | |
| Paige et al. | A Comparison of Through‐the‐Objective Total Internal Reflection Microscopy and Epifluorescence Microscopy for Single‐Molecule Fluorescence Imaging | |
| CN105116529A (en) | Light-pad microscope for high-resolution 3D fluorescence imaging and 2D fluctuation spectroscopy | |
| CN104614318A (en) | Rapid super-resolution micro-imaging method and device | |
| CN107192702B (en) | Spectroscopic pupil laser confocal CARS (coherent anti-Raman scattering) microspectroscopy testing method and device | |
| CN110118726A (en) | A kind of method and apparatus of parallel detecting fluorescent emission difference micro-imaging | |
| CN102818768A (en) | Multifunctional biomedical microscope | |
| CN103616355B (en) | Super-resolution confocal optics microscope and secondary ion mass spectrum combined system | |
| CN102830102A (en) | Method and device for hollow focused light spot excitation-based confocal microscopy | |
| CN103163106A (en) | Super-resolution fluorescent lifetime imaging method and device based on stimulated emission lost | |
| CN109211871A (en) | A kind of stimulated emission depletion fluorescence lifetime super-resolution imaging device | |
| CN103940796A (en) | Novel multi-angle and multi-mode quick switching circular optical illumination microscopic imaging system | |
| CN109187438A (en) | Postposition is divided pupil confocal laser Brillouin-Raman spectra test method and device | |
| CN113267252A (en) | Staring type confocal microscopic morphology spectrum four-dimensional detection system | |
| CN112816396A (en) | Detection imaging system based on EMCCD and AFM | |
| CN112485232A (en) | Sub-ten-nanometer positioning direction-finding method and device based on one-dimensional dark spot time-sharing illumination | |
| US20190383740A1 (en) | Apparatus and method for measuring fluorescence lifetime | |
| CN102419250B (en) | Active polymer plane waveguide propagation constant measuring instrument based on fluorescence imaging | |
| Ruckstuhl et al. | Simultaneous near-field and far-field fluorescence microscopy of single molecules |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |