CN102033058A - Super resolution fluorescence lifetime imaging method and system - Google Patents
Super resolution fluorescence lifetime imaging method and system Download PDFInfo
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- CN102033058A CN102033058A CN 201010551748 CN201010551748A CN102033058A CN 102033058 A CN102033058 A CN 102033058A CN 201010551748 CN201010551748 CN 201010551748 CN 201010551748 A CN201010551748 A CN 201010551748A CN 102033058 A CN102033058 A CN 102033058A
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Abstract
The invention is applied to the fields of optics, biology, chemistry and the like and provides a super resolution fluorescence lifetime imaging method. The method comprises the following steps of: sparsely activating an optical switch dye molecule marked in a sample; exciting the activated optical switch dye molecule in the sample; collecting photons transmitted by the activated optical switch dye molecule and recording a fluorescent image of the optical switch dye molecule; carrying out the centroid positioning on the optical switch dye molecule in the fluorescent image; counting the photons received at the centroid positioning site and determining a fluorescence lifetime of the activated optical switch dye molecule; and constructing the super resolution fluorescence lifetime image by combining the centroid positioning result with the fluorescence lifetime of the obtained optical switch dye molecule. By combining the super resolution fluorescence microtechnique based on unimolecule positioning with the fluorescence lifetime imaging based on time relevant single photon counting, the invention realizes the super resolution fluorescence lifetime imaging, breaks through the traditional optical diffraction limit and has higher scientific significance and application value.
Description
Technical field
The invention belongs to optics, biology, chemical field, relate in particular to a kind of super-resolution fluorescence life-span formation method and imaging system.
Background technology
That fluorescence microscopy has is harmless, noncontact, high specific, highly sensitive, high live body is friendly and can provide outstanding advantage such as function information, is life science always, especially the important tool of RESEARCH ON CELL-BIOLOGY.In recent years, along with development of life science, fluorescence microscopy has also been proposed more and more higher requirement, the continuous development of laser technology, fluorescence probe labelling technique, novel fluorescence Detection Techniques and imaging means, greatly promote the development of fluorescence microscopy, become the important motivity that promotes the life science development.In addition, fluorescence microscopy is also obtaining a very large progress aspect the contrast mechanism of imaging.
At present, the research of life science has entered molecular level, in order to understand the molecule mechanism of vital movement and disease progression better, need interact between research intracellular protein position and function relationship and the protein molecule on the molecular level etc., this has higher requirement to fluorescence microscopy.The restriction of breakthrough diffraction limit, the far field fluorescence microscopy that development has the nanometer spatial discrimination has become one of the forward position focus in international micro-imaging field.The development of super-resolution (SR) imaging technique also provides huge opportunity for the new development of fluorescence lifetime imaging (FLIM).Research and development super-resolution fluorescence life-span imaging (SR-FLIM) technology, can significantly improve the measuring accuracy and the bearing accuracy of FLIM fluorescence lifetime, utilization is based on the FRET (fluorescence resonance energy transfer) (FRET) of SR-FLIM, can study interaction between the protein molecule at molecular scale, obtain between the molecule interactional efficient and accurate location thereof.
Yet, being subjected to the restriction of optical diffraction limit, the spatial resolution of fluorescence microscopy can only reach about 200 nanometers, is difficult to satisfy the needs of life science.And for fluorescence lifetime imaging, its spatial resolution is subjected to the influence of diffraction limit particularly serious.The fluorescence lifetime value of each pixel in traditional diffraction limited FLIM image can not reflect the fluorescence lifetime of its pairing molecule, can't accomplish the fluorescence lifetime imaging of molecular scale.
The imaging of super-resolution fluorescence life-span is an emerging research field, seeks to have highly original know-why and scheme, and takes the lead in solving the difficult problem that the super-resolution fluorescence life-span is imaged on existence in the life science application, has great scientific meaning and using value.
Summary of the invention
The invention provides a kind of super-resolution fluorescence life-span formation method, be intended to break through the optical diffraction limit restriction, realize the imaging of super-resolution fluorescence life-span.
The present invention is achieved in that a kind of super-resolution fluorescence life-span formation method, and described method comprises the steps:
The photoswitch dye molecule that is marked in the sample is carried out sparse activation;
The photoswitch dye molecule that is activated in the excited sample, the photon that the photoswitch dye molecule that collection is excited is launched and the fluoroscopic image of recording light switch dye molecule carry out the barycenter location to the photoswitch dye molecule in the fluoroscopic image;
The photon that receives at barycenter location place is counted, determined the fluorescence lifetime of the photoswitch dye molecule that is excited;
In conjunction with the barycenter positioning result and the fluorescence lifetime of the photoswitch dye molecule that obtains, make up super-resolution fluorescence life diagram picture.
The present invention also provides a kind of imaging system that is used to realize aforesaid super-resolution fluorescence life-span formation method, comprising:
First laser instrument is used for realizing the sparse activation to the photoswitch dye molecule that is marked on sample;
Second laser instrument is used for the photoswitch dye molecule that excited sample is activated;
Light combination mirror, what be used for that light that described first laser instrument is sent and described second laser instrument sent photosyntheticly becomes a branch of light; The light that described first laser instrument is sent is certain incident angle by first reflecting surface of described light combination mirror to be injected, and reflects at first reflecting surface; The light that described second laser instrument is sent is injected by second reflecting surface of described light combination mirror, and first reflecting surface by described light combination mirror after described light combination mirror transmission penetrates;
Be located at first lens, first optical filter, catoptron between described first laser instrument and the described light combination mirror successively;
Be located at second lens, second optical filter, scanning galvanometer between described second laser instrument and the described light combination mirror successively;
First object lens, second object lens;
Be located at successively first reflecting surface of described light combination mirror and described first object lens between the first pipe mirror, dichroic mirror;
The time correlation single photon counter is used for the photon that receives is counted;
Be located at the emission of first between described dichroic mirror and described time correlation single photon counter optical filter, the second pipe mirror, photomultiplier successively;
Electron multiplication type CCD camera is used for the fluoroscopic image of recording light switch dye molecule;
Be located at the emission of second between described second object lens and described electron multiplication type CCD camera optical filter, the 3rd pipe mirror;
Main frame, connect described time correlation single photon counter and described electron multiplication type CCD camera simultaneously, be used for fluoroscopic image in conjunction with the photoswitch dye molecule of described electron multiplication type CCD camera record, photoswitch dye molecule in the fluoroscopic image is carried out the barycenter location, and, make up super-resolution fluorescence life diagram picture according to the result that described time correlation single photon counter is counted the photon that receives at barycenter location place; Also connect described first laser instrument and described second laser instrument simultaneously, be used to control the work of described first laser instrument and described second laser instrument;
And
Delayer is connected between described main frame and the described electron multiplication type CCD camera, is used to realize the synchronous of described second laser instrument and described electron multiplication type CCD camera.
The present invention will realize the imaging of super-resolution fluorescence life-span based on the super-resolution fluorescence microtechnic of unimolecule location with based on the fluorescence lifetime imaging combination of time correlation single photon counting, break through the optical diffraction limit restriction, have scientific meaning and using value.
Description of drawings
Fig. 1 is the realization flow figure of the super-resolution fluorescence life-span formation method that provides of the embodiment of the invention;
Fig. 2 is the optical structure chart of the super-resolution fluorescence life-span imaging system that provides of the embodiment of the invention.
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer,, the present invention is further elaborated below in conjunction with drawings and Examples.Should be appreciated that specific embodiment described herein only in order to explanation the present invention, and be not used in qualification the present invention.
The basic ideas of the embodiment of the invention are to realize super-resolution fluorescence life-span imaging (SR-FLIM) based on the micro-STORM technology of super-resolution fluorescence of unimolecule location with based on the fluorescence lifetime imaging combination of time correlation single photon counting (TCSPC).
Described in detail below in conjunction with specific embodiments of the invention.
Fig. 1 shows the realization flow of the super-resolution fluorescence life-span formation method that the embodiment of the invention provides, and details are as follows:
In step S 101, the photoswitch dye molecule that is marked in the sample is carried out sparse activation.
Wherein, the molecule that will observe in photoswitch dye molecule and the sample needs can the specificity combination, can select red cyanine dye molecule for use, as Cy5 and Alexa 647, the mode by immunofluorescence dyeing with red cyanine dye molecular labeling in sample.Also can select for use the structure picture that utilizes the cis-trans isomeride to change the fluorescin cause invertible switch, as Dronpa series, the mode by transfection with fluorescent protein labeling in sample.
In the embodiment of the invention, specifically can be by the method exciting light switch dye molecule of whole audience face illumination.
In step S102, the photoswitch dye molecule that is activated in the excited sample, the photon that the photoswitch dye molecule that collection is excited is launched and the fluoroscopic image of recording light switch dye molecule carry out the barycenter location to the photoswitch dye molecule in the fluoroscopic image.
Can adopt the picopulse photoscanning to excite the fluorescence molecule that is activated, because the embodiment of the invention is will be based on the micro-STORM technology of super-resolution fluorescence of unimolecule location with based on the fluorescence lifetime imaging combination of time correlation single photon counting (TCSPC), therefore the photon that the photoswitch dye molecule that is excited is launched need be collected two parts: portion is used for the fluoroscopic image of recording light switch dye molecule, single light emitting molecule in the sample is carried out the barycenter location, realize laterally reaching nano level bearing accuracy; Another part then be used to realize to individual molecule the accurate classification of luminous son, by adopting the negative exponential function match, thereby can obtain the fluorescence lifetime accurately of each light emitting molecule, fitting formula is I (t)=I
0Exp (t/ τ), wherein, t is the time, τ is a fluorescence lifetime, I
0Be the fluorescence intensity of initial time (during t=0), I (t) is a t fluorescence intensity constantly.
The purpose of barycenter location is to realize laterally reaching nano level bearing accuracy.Though for microscopic system, the picture of a pointolite is the Airy disk by the decision of system point spread function, but the locus of pointolite can be obtained by the barycenter of its fluoroscopic image, the square root from the photon number of this point source that bearing accuracy (standard deviation) and system detect is inversely proportional to, be directly proportional with the standard deviation of system point spread function own, therefore can obtain bearing accuracy up to nanometer.And horizontal barycenter location just directly just can be realized with the Gaussian function match, axially the unimolecule location is micro-axially differentiates supplementary means in conjunction with some, for example transform point spread function, make disalignment carry the coordinate information of z axle to locational point spread function (PSF), for example utilize the cylindrical mirror astigmatism, spiral PSF also can bring up to axial resolution 50 nanometers even higher level.
In step S103, the photon that receives at barycenter location place is counted, determine the fluorescence lifetime of the photoswitch dye molecule that is excited.
The principle of calculating fluorescence lifetime according to count results described in the step S102, repeats no more as mentioned herein.
In step S104,, make up super-resolution fluorescence life diagram picture in conjunction with the barycenter positioning result and the fluorescence lifetime of the photoswitch dye molecule that obtains.
In conjunction with the fluorescence lifetime image that can reconstruct super-resolution, for example the point of each in the image has been represented the position of each molecule with life-span and locating information, and the color of this institute's respective pixel can be represented the fluorescence lifetime of this molecule.
Fig. 2 shows the optical structure chart of the super-resolution fluorescence life-span imaging system that the embodiment of the invention provides, and for convenience of description, only shows the part relevant with present embodiment.English tag definitions among Fig. 2 is as follows: Laser1: first laser instrument; Laser2: second laser instrument (emission picopulse exciting light); L1: first lens; L2: second lens; F1: first optical filter; F2: second optical filter; Scan Device: scanning galvanometer; M: catoptron; DM: dichroic mirror; BS: light combination mirror; TL1: the first pipe mirror; TL2: the second pipe mirror; TL3: the 3rd pipe mirror; O1: first object lens; O2: second object lens; S: sample; TCSPC: time correlation single photon counter; Delay: delayer; PMT: photomultiplier; EMCCD: electron multiplication type CCD camera; EF emission in 1: the first optical filter; EF2: the second emission optical filter.
The above-mentioned first laser instrument Laser1 is used for realizing the sparse activation to the photoswitch dye molecule that is marked on sample, and the second laser instrument Laser2 then is used for the photoswitch dye molecule that excited sample is activated.Light combination mirror BS is used for that light that the first laser instrument Laser1 is sent and the second laser instrument Laser2 sent photosyntheticly becomes a branch of light, wherein the light that sent of the first laser instrument Laser1 is certain incident angle by first reflecting surface (right flank of light combination mirror BS among Fig. 2) of light combination mirror BS and injects, and reflect at first reflecting surface, the light that the second laser instrument Laser2 is sent is injected by second reflecting surface (left surface of light combination mirror BS among Fig. 2) of light combination mirror BS, and first reflecting surface by light combination mirror BS after light combination mirror BS transmission penetrates.The first lens L1, the first optical filter F1, mirror M are located between the first laser instrument Laser1 and the light combination mirror BS successively, and the second lens L2, the second optical filter F2, scanning galvanometer Scan Device are located between the second laser instrument Laser2 and the light combination mirror BS successively.The first pipe mirror TL1, dichroic mirror DM are located between first reflecting surface and the first object lens O1 of light combination mirror BS successively.Time correlation single photon counter TCSPC is used for the photon that receives is counted.The first emission optical filter EF1, the second pipe mirror TL2, photomultiplier PMT are located between dichroic mirror DM and the time correlation single photon counter TCSPC successively.Electron multiplication type CCD camera EMCCD is used for the fluoroscopic image of recording light switch dye molecule.The second emission optical filter EF2, the 3rd pipe mirror TL3 are located between the second object lens O2 and the electron multiplication type CCD camera EMCCD.Main frame is relevant single photon counter TCSPC of tie-time and electron multiplication type CCD camera EMCCD simultaneously, be used for fluoroscopic image in conjunction with the photoswitch dye molecule of electron multiplication type CCD camera EMCCD record, photoswitch dye molecule in the fluoroscopic image is carried out the barycenter location, and, make up super-resolution fluorescence life diagram picture according to the result that time correlation single photon counter TCSPC counts the photon that receives at barycenter location place; Main frame also connects the first laser instrument Laser1 and the second laser instrument Laser2 simultaneously, controls the work of the first laser instrument Laser1 and the second laser instrument Laser2.Delayer Delay is connected between main frame and the electron multiplication type CCD camera EMCCD, is used to realize the synchronous of described second laser instrument and described electron multiplication type CCD camera.
With reference to Fig. 2, it is the CW semiconductor laser of 640 nanometers that Laser1 can select output wavelength for use, as the activating light source of switch molecule.In light path, the method for employing face illumination utilizes object lens O1 that exciting light is converged on the sample S.By control power of exciting light and activationary time etc., realize the sparse activation of switch molecule.Laser2 is that output wavelength is the picopulse semiconductor laser of 640 nanometers, as the excitation source of the switch molecule that is activated.In light path, the picopulse exciting light focuses on the sample S, does quick scanning by galvanometer Scan Device along sample, is used to excite the switch molecule that is activated.The light that sample S is sent is collected with relative two object lens O1 and O2, sends into PMT and EMCCD respectively.The former sends the photon signal that receives into TCSPC, is used for the counting of photon; The latter obtains the fluoroscopic image of sparse activating molecules and image is transferred to main frame, and main frame can obtain the accurate position of light emitting molecule by the barycenter location algorithm.Activate the switching of light path and excitation light path by host computer control, and TCSPC detects and the EMCCD imaging synchronously, specifically each cyclic process is: Laser1 sends laser so that the photoswitch dye molecule is carried out sparse activation, and--the photoswitch dye molecule that is activated in the Laser2 excited sample--EMCCD imaging---TCSPC counts.
Experimental procedure is as follows:
(1) utilizes powerful CW laser Laser1, sample S is carried out the illumination of whole audience face, controller by Laser1 or the neutral density filter (not shown among Fig. 2) of being located at described first optical filter front side or rear side are controlled illumination intensity, control lighting hours by shutter, realize sparse activation photoswitch dye molecule in the sample.
(2) utilize low power picosecond pulse laser Laser2, and sample is scanned the switch molecule that has been activated in the excited sample by scanning galvanometer Scan Device.
(3) since sample at whole spatial emission photon, the basic thinking that realizes this project super-resolution imaging is to carry out molecule location earlier, then with photon " distribution " to correct molecule.Therefore, to the fluorescence that activating molecules sent, collect by O1 and two object lens of O2 respectively.The photon that the O2 object lens are collected adopts common STORM formation method, and unimolecule is positioned analysis, obtains the super-resolution fluorescence micro-image; The photon of O1 object lens is received by photomultiplier PMT, sends into time correlation single photon counter TCSPC and carries out fluorescence lifetime measurement.
(4) repeating step 1-3 all is positioned and durability analysis finishes until all molecules.Obtain the super-resolution fluorescence life diagram picture of sample by image reconstruction.
The present invention will be based on the super-resolution fluorescence microtechnic of unimolecule location with based on the fluorescence lifetime imaging combination of time correlation single photon counting, realize the imaging of super-resolution fluorescence life-span, broken through the optical diffraction limit restriction, have scientific meaning and using value, will effectively promote the development of China at life science.
The above only is preferred embodiment of the present invention, not in order to restriction the present invention, all any modifications of being done within the spirit and principles in the present invention, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. a super-resolution fluorescence life-span formation method is characterized in that described method comprises the steps:
The photoswitch dye molecule that is marked in the sample is carried out sparse activation;
The photoswitch dye molecule that is activated in the excited sample, the photon that the photoswitch dye molecule that collection is excited is launched and the fluoroscopic image of recording light switch dye molecule carry out the barycenter location to the photoswitch dye molecule in the fluoroscopic image;
The photon that receives at barycenter location place is counted, determined the fluorescence lifetime of the photoswitch dye molecule that is excited;
In conjunction with the barycenter positioning result and the fluorescence lifetime of the photoswitch dye molecule that obtains, make up super-resolution fluorescence life diagram picture.
2. super-resolution fluorescence life-span formation method as claimed in claim 1 is characterized in that, described step of the photoswitch dye molecule in the sample being carried out sparse activation realizes by sample being carried out the illumination of whole audience face.
3. an imaging system that is used to realize super-resolution fluorescence life-span formation method as claimed in claim 1 is characterized in that, comprising:
First laser instrument is used for realizing the sparse activation to the photoswitch dye molecule that is marked on sample;
Second laser instrument is used for the photoswitch dye molecule that excited sample is activated;
Light combination mirror, what be used for that light that described first laser instrument is sent and described second laser instrument sent photosyntheticly becomes a branch of light; The light that described first laser instrument is sent is certain incident angle by first reflecting surface of described light combination mirror to be injected, and reflects at first reflecting surface; The light that described second laser instrument is sent is injected by second reflecting surface of described light combination mirror, and first reflecting surface by described light combination mirror after described light combination mirror transmission penetrates;
Be located at first lens, first optical filter, catoptron between described first laser instrument and the described light combination mirror successively;
Be located at second lens, second optical filter, scanning galvanometer between described second laser instrument and the described light combination mirror successively;
First object lens, second object lens;
Be located at successively first reflecting surface of described light combination mirror and described first object lens between the first pipe mirror, dichroic mirror;
The time correlation single photon counter is used for the photon that receives is counted;
Be located at the emission of first between described dichroic mirror and described time correlation single photon counter optical filter, the second pipe mirror, photomultiplier successively;
Electron multiplication type CCD camera is used for the fluoroscopic image of recording light switch dye molecule;
Be located at the emission of second between described second object lens and described electron multiplication type CCD camera optical filter, the 3rd pipe mirror;
Main frame, connect described time correlation single photon counter and described electron multiplication type CCD camera simultaneously, be used for fluoroscopic image in conjunction with the photoswitch dye molecule of described electron multiplication type CCD camera record, photoswitch dye molecule in the fluoroscopic image is carried out the barycenter location, and, make up super-resolution fluorescence life diagram picture according to the result that described time correlation single photon counter is counted the photon that receives at barycenter location place; Also connect described first laser instrument and described second laser instrument simultaneously, be used to control the work of described first laser instrument and described second laser instrument;
And
Delayer is connected between described main frame and the described electron multiplication type CCD camera, is used to realize the synchronous of described second laser instrument and described electron multiplication type CCD camera.
4. imaging system as claimed in claim 3 is characterized in that, also comprises the neutral density filter of being located at described first optical filter front side or rear side.
5. imaging system as claimed in claim 3 is characterized in that, described first laser instrument is the continuous light laser instrument.
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| WO2019086537A1 (en) | 2017-11-02 | 2019-05-09 | Centre National De La Recherche Scientifique | Device and method for super-resolution fluorescence microscopy and fluorescence lifetime measurement |
| CN108120702A (en) * | 2017-11-30 | 2018-06-05 | 浙江大学 | A kind of super resolution fluorescence lifetime imaging method and device based on parallel detecting |
| CN108333157A (en) * | 2018-01-23 | 2018-07-27 | 深圳大学 | biomolecule three-dimensional dynamic analysis method and system |
| CN111521596A (en) * | 2020-06-04 | 2020-08-11 | 深圳大学 | Fluorescence differential super-resolution imaging method and imaging system |
| WO2021243755A1 (en) * | 2020-06-04 | 2021-12-09 | 深圳大学 | Fluorescence difference super-resolution imaging method and imaging system |
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