CN109520982A - A kind of fluorescence correlation spectroscopy measuring system - Google Patents
A kind of fluorescence correlation spectroscopy measuring system Download PDFInfo
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- CN109520982A CN109520982A CN201811382587.1A CN201811382587A CN109520982A CN 109520982 A CN109520982 A CN 109520982A CN 201811382587 A CN201811382587 A CN 201811382587A CN 109520982 A CN109520982 A CN 109520982A
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- optical fiber
- correlation spectroscopy
- measuring system
- fluorescence
- fluorescence correlation
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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
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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
- G01N21/645—Specially adapted constructive features of fluorimeters
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N2021/6484—Optical fibres
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- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Optics & Photonics (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention belongs to spectral detection system technical fields, in particular to a kind of fluorescence correlation spectroscopy measuring system, exciting light is limited in using tapered fiber and is ascended to heaven in volume, while collecting fluorescence signal using optical fiber and carrying out time correlation photon counting, to obtain fluorescence correlation spectroscopy；This method avoid confocal microscope, utilizing total internal reflection fluorescence microscope or the microscopical uses of two-photon fluorescence, reduce the measurement cost of fluorescence correlation spectroscopy, optical fiber can be goed deep into solution simultaneously, in reactor, in micro-fluidic chip, it is intracellular or in vivo, the space limitation for eliminating imaging device, can be widely applied to Reaction kinetics research, live cell assays, biological detection, medical diagnosis on disease and drug screening of life science, physics and chemical field etc..
Description
Technical field
The invention belongs to spectral detection system technical field, in particular to a kind of fluorescence correlation spectroscopy measuring system.
Background technique
With the development of life science and optical measuring technique, people have begun to be studied between substance on a molecular scale
Interaction and life process.The method of one of Single Molecule Detection is exactly the fluorescence correlation spectroscopy utilized.Utilize fluorescence
Correlation spectrum, which carries out Single Molecule Detection, can obtain the information such as diffusion constant, chemical kinetics parameters, can also be applied to
Life science, live cell assays, biological detection, medical diagnosis on disease and drug screening etc..
Fluorescence correlation spectroscopy (Fluorescence correlation spectroscopy, FCS) technology is a kind of utilization
The fluctuation of fluorescence intensity at any time carries out the fluorescence spectroscopy technique of analysis detection.Fluorescence correlation spectroscopy by measurement microcell (such as
Ascend to heaven (fL)) fluorescent molecule is generated due to Brownian movement or chemical reaction in volume fluorescence intensity fluctuation, to obtain fluorescence
The fluctuation signal of intensity, then auto-correlation computation is carried out to it, certain information of particle, such as the concentration of solution are obtained, particle
Diffusion coefficient etc..FCS is suitble to solution and cell measurement, and measures very sensitive, it can be achieved that unimolecule monitors.
Currently in order to use fluorescence correlation spectroscopy to carry out the information of detection molecules level, it will usually it is micro- to use fluorescence co-focusing
The large-scale instruments such as mirror, utilizing total internal reflection fluorescence microscope or two-photon fluorescence microscope, by microscopic system scanning, detect and
Manage data, display digital imaging results etc..Using the higher cost of these measurement methods, occupied space is larger, and inconvenient empty
Between on displacement.In addition, also having higher requirement to sample due to using microscope to detect, for example, to the place of sample to be tested
Reason, the specifying measurement standard of glass slide, placement of sample etc..Due to the limitation of these factors, to the measurement of fluorescence correlation spectroscopy
Bring inconvenience.
Summary of the invention
The present invention solves the above-mentioned technical problems in the prior art, provides a kind of fluorescence correlation spectroscopy measuring system.
To solve the above problems, technical scheme is as follows:
A kind of fluorescence correlation spectroscopy measuring system, including excitation light source, the first lens, the first colour filter, dichroscope, object
Mirror, optical fiber, optical fiber cone, the second colour filter, the second lens and detector；The end face of the optical fiber cone is provided with nano-pore；
The laser that the excitation light source issues enters object lens by the first lens, the first colour filter and dichroscope, then passes through object lens
It focuses and enters optical fiber and optical fiber cone, finally form confinement excitation in space in nano-pore；Excitation fluorescence in nano-pore passes through
Optical fiber, object lens, dichroscope, the second colour filter and the second lens enter detector.
Fluorescence correlation spectroscopy measuring system (is made of, the optical fiber centrum sets tapered fiber optical fiber and optical fiber cone
Set in one end of optical fiber) exciting light is limited in and is ascended to heaven in (fL) volume, then laser beam is converged on sample, it excites specific
The fluorescence in region is collected fluorescence signal by optical fiber, is counted by detector, such as Single Photon Counting device；Then
Enumeration data is transferred on computer, auto-correlation computation is carried out to data by computer software, obtains autocorrelator trace；Last root
According to the local environment of sample, exciting light condition and diffusion equation etc. obtain fluorescence fluctuation theoretical equation, and with theoretical equation come
It is fitted experiment value, to obtain relevant parameter.
Preferably, the optical fiber is single mode optical fiber or multimode fibre.
Preferably, the optical fiber can be glass optical fiber perhaps silica fibre perhaps polymer optical fiber or liquid-core optical fibre.
Preferably, the tip diameter of the optical fiber cone is between 10 microns to 1000 microns.
Preferably, the end face of the optical fiber cone is coated with metal coating.Preferably, the metal coating be gold, silver, aluminium,
Copper, nickel, in platinum any one or a few metal alloy.Preferably, the thickness of the metal coating is in 20 nanometers to 1 microns
Between.
Preferably, the diameter of the nano-pore is between 50 nanometers to 200 nanometers.Due to the dimensional effect of nano-pore, swash
Evanescent field cannot can only be formed by nano-pore in nano-pore by shining, thus can only tens nanometers of excitation nano hole bottom surface
Fluorescent dye in range realizes the excitation and measurement for being less than fluorescence fluctuation in fL volume.
Preferably, the nano-pore runs through entire metal coating, or through entire metal coating and gos deep into fiber core layer 100
Within nanometer.
Preferably, to be that rectangle is perhaps trapezoidal be also possible to other rules or irregular to the cross sectional shape of the nano-pore
Shape.
Preferably, exciting light can have all the way perhaps the corresponding colour filter of multichannel and dichroscope also use one or
It is multiple.
Preferably, the detector is Single Photon Counting device, time correlation photomultiplier tube, avalanche diode
Or EMCCD.
Preferably, the excitation light source can be a kind of wavelength or multi-wavelength, and corresponding light path uses one
Or it is multiple.
Compared with the existing technology, advantages of the present invention is as follows,
1) system of present invention measurement fluorescence correlation spectroscopy avoids the use of the instruments such as confocal microscope, uses
Tapered fiber and Single Photon Counting device collect fluorescence fluctuation signal, reduce the measurement of fluorescence correlation spectroscopy at
This.
2) optical measuring system, optical fiber and the Single Photon Counting device that the present invention uses are smaller in equal volume, cooperation
Computer software reduces measuring system for spatially displacement request, can answer in use, compared with portability is had more for microscope
It is measured for more occasions.
3) tapered fiber used herein replaces the glass slide on original microscope to be attached with sample, and adopts
Fluorescence signal is collected with optical fiber, reduces the processing requirement of original pair of sample, expands the range of measurement sample；For example, can
With will measure sample amplification into solution, in reactor, in micro-fluidic chip, it is intracellular or in vivo, eliminate original measurement
System is to limitation spatially.
Detailed description of the invention
Fig. 1 is fluorescence correlation spectroscopy measuring system schematic diagram of the present invention；
Wherein, 1, optical fiber cone；2, optical fiber；3, object lens；4, dichroscope；5, the first colour filter；6, the first lens；7, swash
Light emitting source；8, the second colour filter；9, the second lens；10, detector；11, nano-pore.
Specific embodiment
Embodiment 1:
As shown in Figure 1, a kind of fluorescence correlation spectroscopy measuring system, including excitation light source 7, the first lens 6, the first colour filter
5, dichroscope 4, object lens 3, optical fiber 2, optical fiber cone 1, the second colour filter 8, the second lens 9 and detector 10；The optical taper
The end face of body 1 is provided with nano-pore 11；The laser that the excitation light source 7 issues passes through the first lens 6, the first colour filter 5 and two
Enter object lens 3 to Look mirror 4, then focused by object lens 3 and enter optical fiber 2 and optical fiber cone 1, is finally formed in nano-pore 11 empty
Between confinement excite；Excitation fluorescence in nano-pore 11 is by optical fiber 2, object lens 3, dichroscope 4, and the second colour filter 8 and second is thoroughly
Mirror 9 enters detector 10.
Fluorescence correlation spectroscopy measuring system is by tapered fiber (by optical fiber 2 and optical fiber cone structure 1 at the optical fiber centrum 2
One end of optical fiber 1 is set) exciting light is limited in and is ascended to heaven in (fL) volume, then laser beam is converged on sample, it excites
The fluorescence of specific region is collected fluorescence signal by optical fiber, is counted by detector, such as Single Photon Counting device；
Then enumeration data is transferred on computer, auto-correlation computation is carried out to data by computer software, obtains autocorrelator trace；Most
The theoretical equation of fluorescence fluctuation is obtained according to the local environment of sample, exciting light condition and diffusion equation etc. afterwards, and with theory side
Journey is fitted experiment value, to obtain relevant parameter.
Single mode optical fiber or multimode fibre may be selected in the optical fiber 2.
Glass optical fiber perhaps silica fibre perhaps polymer optical fiber or liquid-core optical fibre may be selected in the optical fiber 2.
The tip diameter of the optical fiber cone 1 is between 10 microns to 1000 microns.
The end face of the optical fiber cone 1 is coated with metal coating.Preferably, the metal coating be gold, silver, aluminium, copper, nickel,
The alloy of any one or a few metal in platinum.Preferably, the thickness of the metal coating is between 20 nanometers to 1 microns.
The diameter of the nano-pore 11 is between 50 nanometers to 200 nanometers.Due to the dimensional effect of nano-pore 11, exciting light
Evanescent field cannot can only be formed in nano-pore 11 by nano-pore 11, thus can only 11 bottom surface tens of excitation nano hole receive
Fluorescent dye in rice range, realizes the excitation and measurement for being less than fluorescence fluctuation in fL volume.
The nano-pore 11 runs through entire metal coating, or through entire metal coating and gos deep into 100 nanometers of 2 sandwich layer of optical fiber
Within.
The cross sectional shape of the nano-pore 11, which is that rectangle is perhaps trapezoidal, is also possible to other rules or irregular shape.
Preferably, exciting light can have all the way the perhaps corresponding colour filter of multichannel and dichroscope 4 also using one or
It is multiple.
The detector 10 be Single Photon Counting device, time correlation photomultiplier tube or avalanche diode or
Person EMCCD.
The excitation light source 7 can be a kind of wavelength, and perhaps the corresponding light path of multi-wavelength is using one or more
It is a.
Embodiment 2:
The fluorescence correlation spectroscopy measurement and calculating of fluorescent dye solution:
It is related with the fluorescence of Single Photon Counting method measurement fluorescent dye FITC solution using Fig. 1 shown device
Spectrum.
Laser is transmitted through the optical fiber to optical fiber cone tip by 488nm laser, the effective detection zone in optical fiber centrum tip
Volume is the volume of exciting light and the volume overlapping for receiving cone, can achieve range of ascending to heaven, and FITC sample molecule is stimulated generation
Fluorescence single photon counter system is passed to by optical fiber, single photon counter system detects the number of photons in a period of time, discretization
It is stored in memory at digital signal, computer reads the information in its memory, calculates fluorescence from phase according to time related information
Close function
Its auto-correlation function is indicated with following formula
Wherein N is average mark subnumber, and F is resultant signal, and B is ambient noise, τdIt is diffusion time, s is in analysis volume
The ratio between transverse and longitudinal size, nTIt is the quantity of fluorescent molecule triplet, τTIt is the damping time constant of fluorescent molecule triplet.
Each individually FCS measurement can continue at least 10 times operations by averagely every 60 seconds and obtain.
Embodiment 3:
The use in conjunction of fluorescence correlation spectroscopy and micro-fluidic chip:
Utilize the fluorescence correlation light of 10mmol/L fluorescent dye FITC solution in Fig. 1 shown device measurement microfluidic channel
Spectrum.By in the channel of fiber optic tip insertion micro-fluidic chip for drawing cone, contact the nano-pore of fiber optic tip with FITC solution, benefit
Pass through the fluorescent dye in optical fiber excitation nano hole with 488nm exciting light.
The FITC solution of same concentration is placed in centrifuge tube, is deep into solution with optical fiber and carries out fluorescence correlation spectroscopy
Detection.Normalized curve obtained by two systems is compared, is fitted, can analyze the system structure parameter and diffusion of the two
Time basic difference, so that analyzing micro-fluidic chip duct wall has reflex or channel size to expand fluorescent dye fluorescence
Scattered influence.
Embodiment 4:
One pack system electrophoresis-fluorescence correlation spectroscopy detection:
In the use in conjunction of fluorescence correlation spectroscopy and micro-fluidic chip, chip channel Design of length is 2cm, will be diluted
Rhodamine almashite acid esters injection micro-fluidic chip channel in, DC voltage 200V, 400V are added outside the both ends of channel,
600V, 800V, obtaining electric field strength is respectively 100V/cm, 200V/cm, 300V/cm, 400V/cm.
The Brownian movement of FITC molecule and the ordered movement of FITC molecule under the electric field when without electric field are studied in channel,
The ordered movement and diffusion motion formula of fluorescence correlation spectroscopy can be indicated with following formula:
Wherein τDAnd τFCharacteristic time when being pure diffusion and orderly stream in microcell, N is mean fluorecence molecular amounts, and T is glimmering
Optical molecule triplet percentage composition, τtripletIt is fluorescent molecule triplet damping time constant, passes through τFTo be measured point can then be found out
The electrophoresis flow velocity of son.When fitting, (ω0/s0)2With characteristic diffusion times τDUsing voltage data measured is not added, it is determined as constant ginseng
Number, the experimental result of different voltages are fitted with formula (4).Since FCS is detected as probability as a result, can use repeatedly real
The method that result is averaged is tested to improve precision.It is found by measurement, with the increase of electric field strength, rhodamine almashite acid
Ester ion is constantly reduced by the time of detection zone, and the directed movement rate of ion is continuously increased.
It should be noted that above-described embodiment is only presently preferred embodiments of the present invention, there is no for the purpose of limiting the invention
Protection scope, the equivalent substitution or substitution made on the basis of the above all belong to the scope of protection of the present invention.
Claims (10)
1. a kind of fluorescence correlation spectroscopy measuring system, including excitation light source, the first lens, the first colour filter, dichroscope, object
Mirror, optical fiber, optical fiber cone, the second colour filter, the second lens and detector；It is characterized in that, the end face of the optical fiber cone is set
It is equipped with nano-pore；The laser that the excitation light source issues enters object lens by the first lens, the first colour filter and dichroscope, so
It is focused afterwards by object lens and enters optical fiber and optical fiber cone, finally form confinement excitation in space in nano-pore；Swashing in nano-pore
It fluoresces through optical fiber, object lens, dichroscope, the second colour filter and the second lens enter detector.
2. fluorescence correlation spectroscopy measuring system as described in claim 1, which is characterized in that the optical fiber is single mode optical fiber or more
Mode fiber；The optical fiber is glass optical fiber perhaps silica fibre perhaps polymer optical fiber or liquid-core optical fibre.
3. fluorescence correlation spectroscopy measuring system as described in claim 1, which is characterized in that the tip diameter of the optical fiber cone
Between 10 microns to 1000 microns.
4. fluorescence correlation spectroscopy measuring system as described in claim 1, which is characterized in that the end face of the optical fiber cone is coated with
Metal coating；The metal coating be gold, silver, aluminium, copper, nickel, in platinum any one or a few metal alloy.
5. fluorescence correlation spectroscopy measuring system as claimed in claim 4, which is characterized in that the thickness of the metal coating is 20
Nanometer is between 1 micron.
6. fluorescence correlation spectroscopy measuring system as described in claim 1, which is characterized in that the diameter of the nano-pore is received 50
Rice is between 200 nanometers.
7. fluorescence correlation spectroscopy measuring system as claimed in claim 4, which is characterized in that the nano-pore runs through entire metal
Coating, or through entire metal coating and go deep within 100 nanometers of fiber core layer.
8. fluorescence correlation spectroscopy measuring system as described in claim 1, which is characterized in that the cross sectional shape of the nano-pore is
Rectangle is trapezoidal.
9. fluorescence correlation spectroscopy measuring system as described in claim 1, which is characterized in that exciting light can have all the way or more
Road, corresponding colour filter and dichroscope also use one or more；The excitation light source is a kind of wavelength or a variety of waves
Long, corresponding light path uses one or more.
10. fluorescence correlation spectroscopy measuring system as described in claim 1, which is characterized in that the detector is time correlation
Single photon counter, time correlation photomultiplier tube, avalanche diode or EMCCD.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110579454A (en) * | 2019-08-19 | 2019-12-17 | 陈云 | fluorescent group identification method and device based on electric field modulation fluorescence correlation spectroscopy |
| CN111215161A (en) * | 2020-01-15 | 2020-06-02 | 北京中科生仪科技有限公司 | Optical detection system for nucleic acid amplification instrument |
| CN112666144A (en) * | 2020-12-21 | 2021-04-16 | 中国计量大学 | Fluorescent compound micro-flow detector based on tapered multimode optical fiber |
| CN112812954A (en) * | 2020-12-29 | 2021-05-18 | 中国科学院长春光学精密机械与物理研究所 | Gene sequencing chip |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110579454A (en) * | 2019-08-19 | 2019-12-17 | 陈云 | fluorescent group identification method and device based on electric field modulation fluorescence correlation spectroscopy |
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| CN111215161A (en) * | 2020-01-15 | 2020-06-02 | 北京中科生仪科技有限公司 | Optical detection system for nucleic acid amplification instrument |
| CN112666144A (en) * | 2020-12-21 | 2021-04-16 | 中国计量大学 | Fluorescent compound micro-flow detector based on tapered multimode optical fiber |
| CN112666144B (en) * | 2020-12-21 | 2023-07-18 | 中国计量大学 | Fluorescent compound micro-flow detector based on tapered multimode fiber |
| CN112812954A (en) * | 2020-12-29 | 2021-05-18 | 中国科学院长春光学精密机械与物理研究所 | Gene sequencing chip |
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Application publication date: 20190326 |