WO2014026615A1 - 一种基于荧光共振能量转移的超分辨成像方法 - Google Patents

一种基于荧光共振能量转移的超分辨成像方法 Download PDF

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WO2014026615A1
WO2014026615A1 PCT/CN2013/081497 CN2013081497W WO2014026615A1 WO 2014026615 A1 WO2014026615 A1 WO 2014026615A1 CN 2013081497 W CN2013081497 W CN 2013081497W WO 2014026615 A1 WO2014026615 A1 WO 2014026615A1
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fret
super
resolution imaging
excitation light
imaging method
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PCT/CN2013/081497
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English (en)
French (fr)
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樊春海
黄庆
程亚
陈建芳
邓素辉
梁乐
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中国科学院上海应用物理研究所
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Publication of WO2014026615A1 publication Critical patent/WO2014026615A1/zh

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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
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

Definitions

  • the invention belongs to the field of super-resolution imaging technology, and in particular relates to a super-resolution imaging method based on Fluorescence Resonance Energy Transfer (FRET). Background technique
  • STED uses two combined lasers, that is, one beam is focused into a normal diffraction-limited focal spot, which emits the fluorescent molecules in the focal spot to the excited state; and the second beam is selected.
  • the emission wavelength range of the fluorescent molecule it is focused into a circular annular focal spot whose center is completely coincident with the center of the focal spot of the first beam but is hollow. Since the fluorescent molecule excited by the first excitation light to the excited state can be quenched from the excited state to the ground state by the second light, it is also called a quenching beam.
  • the intensity distribution of the quenching beam is zero at the focal point, in principle, as long as the quenching light is sufficiently strong, the volume occupied by the fluorescent molecules excited by the first beam can be compressed to a very small extent. Because these fluorescent molecules are quenched outside the focus.
  • the technical problem to be solved by the present invention is that the existing super-resolution imaging technology requires a large number of modifications to the fluorescence microscope, which is very expensive, and the imaging software is also very complicated, providing a fluorescence-based resonance energy transfer (FRET). Super-resolution imaging method.
  • FRET fluorescence-based resonance energy transfer
  • a super-resolution imaging method based on fluorescence resonance energy transfer which comprises the following steps:
  • the excitation light threshold of the fluorescence resonance energy transfer of the FRET molecule described in the step 1) can be imaged by laser scanning confocal microscopy.
  • the first fluorophore When the first fluorophore is in an excited state, its energy is transferred to the adjacent second fluorophore, causing excitation of the second fluorophore.
  • the excitation light intensity of the first fluorophore When the excitation light intensity of the first fluorophore is low, the irradiated first fluorophore substantially undergoes a FRET effect without autofluorescence.
  • FRET saturation occurs in the first fluorescent group near the center of the excitation light focal spot, and the first fluorescent group is forced to emit fluorescence.
  • the appropriate laser value is chosen to be slightly larger than the excitation light intensity threshold that causes FRET saturation, the laser focal spot center can be controlled.
  • the first fluorophore in the domain emits light. Thus, a light-emitting point smaller than the diffraction limit scale is obtained, thereby obtaining the resolution ability beyond the optical diffraction limit.
  • the probes described in the present invention are conventional probes in the art, wherein the probes preferably include, but are not limited to, nucleic acids, antibodies, proteins (eg, enzymes, receptors), small organic molecules (such as nucleotides).
  • the probes preferably include, but are not limited to, nucleic acids, antibodies, proteins (eg, enzymes, receptors), small organic molecules (such as nucleotides).
  • One or more of inorganic molecules such as calcium ions
  • the probes can be combined with the target substance (such as a sample to be tested) ) can be combined.
  • the combination of the probe and the target substance is specific.
  • the probe of the present invention preferably comprises: one or more of a deoxyribonucleotide oligomer, a ribonucleic acid oligomer and a polypeptide oligomer.
  • the FRET molecule pair labeled on the probe of the present invention comprises a first fluorophore (donor) and a second fluorophore (receptor), and the first fluorophore is capable of generating fluorescence resonance energy to the second fluorophore Transfer (FRET).
  • the FRET molecule pairs of the invention preferably have a higher FRET efficiency (preferably > 85%), less fluorescence signal crosstalk between the donor and the acceptor, higher donor fluorescence quantum yield and less fluorescent photobleaching.
  • the problem is the pair of FRET molecules.
  • the fluorophores described therein are conventional fluorophores in the art.
  • the fluorophore refers to: after absorbing visible light and ultraviolet light, it can convert ultraviolet light into visible light with a longer wavelength and reflect it, which is a bright and vivid color. For example, acid blush, fluorescent yellow, red mercury, and some disperse dyes. Most of the fluorophores are compounds containing a benzene ring or a heterocyclic ring and having a conjugated double bond.
  • the FRET molecule is a conventional FRET molecule pair in the art.
  • the FRET molecule pair of the present invention preferably comprises: CFP-YFP (cyan fluorescent protein-yellow fluorescent protein), Cy3_Cy5 (cyanine dye 3-5), Atto488-Atto540, FITORhodamine (fluorescein isothiocyanate-rhodamine) , BFP-GFP (Blue Fluorescent Protein - Green Fluorescent Protein), Atto488-Atto647N, Atto550- Atto647N, Atto488- Atto590, Atto550- Atto655, Atto590- Atto655, CFP- dsRED (Cyan Fluorescent Protein-Red Fluorescent Protein), Alexa488- Alexa555 (Alexaf luor488—Alexaf luor555); Alexa488— Cy3 (Alexaf luor4 88-cyanine dye 3), YFP-TRITC (yellow fluorescent protein
  • the distance between pairs of FRET molecules described in the present invention is preferably less than 10 nm, the molecular pair distance is more preferably 2 to 5 nm, and the distance is preferably less than 2.5 nm.
  • the preparation method of the high FRET efficiency fluorescent probe described therein is a conventional preparation method in the art.
  • the method for preparing a fluorescent probe according to the present invention preferably comprises the steps of first preparing a fluorescent nanosphere by a reverse microemulsion method, and then combining the pellet with an antibody.
  • the reverse microemulsion method preferably comprises the following steps: Osmotic reaction:
  • the surfactant is preferably added in an amount of 2 to 5%, and the surfactant is preferably added in an amount of 2%.
  • the surfactant is a surfactant conventional in the art, preferably ten. Dithiocarbamate.
  • the amount of the fluorescent dye donor and the acceptor to be added is preferably 4 to 10%, the amount thereof is preferably 4%, the amount of toluene added is preferably 4 to 10%, and the amount thereof is preferably 4%.
  • the water glass modulus is preferably 3. 1 ⁇ 3. 4, the Baume degree is preferably 40, and the ultrasonic mixing is uniform (ultrasonic power 50W, and the water glass is preferably prepared in a beaker.
  • the mixing time is 1 to 5 minutes), the turbid emulsion is obtained, and n-pentanol is added dropwise until the system is suddenly transparent, that is, the microemulsion A is obtained, wherein the percentages are all by mass.
  • the microemulsion A is poured into a three-necked flask, and the temperature of the water bath is preferably 30 to 35 ° C, preferably At 30 ° C, 1 ⁇ 5 ml of microemulsion B was added to the microemulsion A obtained by the permeation step by a peristaltic pump, and the dropping time was preferably 40 to 60 min. After the dropwise addition, the fluorescent nanosphere solution C was obtained.
  • the obtained solution C is subjected to dialysis treatment for use in the next experiment, and the dialysis time is preferably 8 to 24 hours, preferably 24 hours, and the dialysis bag size used is preferably 10,000 to 14,000 molecular weight, and the specification thereof is preferably 14000 molecular weight.
  • the method of binding a fluorescent nanoglobule of the present invention to an antibody is a conventional method in the art.
  • the method for binding the fluorescent nanosphere to the antibody preferably comprises the steps of: weighing a certain volume of the prepared fluorescent nanosphere solution C, washing with PBS buffer, adding an equal volume of the antibody solution, and reacting at room temperature.
  • the reaction time is preferably 30 to 60 min. After centrifugation and washing, antibody-modified fluorescent nanoparticles are obtained, which are dissolved in PBS buffer and stored at 4 ° C for use.
  • the method of labeling a sample to be tested with a high FRET-efficiency fluorescent probe is conventional in the art.
  • the labeling method preferably comprises the steps of: labeling the cells with the high FRET-efficiency fluorescent probe, and after sufficient washing, performing laser scanning confocal microscopy.
  • the method of labeling is a conventional technique in the art and is well known and grasped by those skilled in the art.
  • the labeling method binds the probe to the sample by immunohistochemistry in a cell biology technique, the immunohistochemical method preferably comprising the steps of: blocking, labeling the primary antibody, labeling the secondary antibody, wherein The secondary antibody is a secondary antibody that binds to the aforementioned fluorescent nanoglobules.
  • the step 2) is: using the excitation light intensity, the excitation light threshold of the fluorescence resonance energy transfer of the FRET molecule described in the step 1) can be imaged by laser scanning confocal microscopy.
  • the excitation light intensity employed therein is an excitation light threshold capable of causing fluorescence resonance energy transfer of the FRET molecule pair described in the step 1).
  • the excitation light threshold capable of causing the fluorescence resonance energy transfer of the FRET molecule pair described in the step 1) refers to the intensity of the excitation light for causing the FRET molecule to undergo fluorescence resonance energy transfer to FRET saturation, which is called a saturation excitation light threshold.
  • the present invention detects the saturation excitation light threshold of the FRET molecule pair by conventional methods in the art.
  • the saturation excitation light threshold detection method preferably comprises the steps of: separately detecting a donor fluorescent molecule and a fluorescent probe labeled with a pair of FRET molecules under a laser scanning confocal microscope to detect fluorescence of the donor fluorescent molecule
  • the curve of the excitation intensity changes, comparing the changes of the two curves, it can be found that the donor fluorescence of the FRET probe has a nonlinear optical response at a certain excitation intensity, that is, the donor fluorescence occurs near a certain excitation light threshold.
  • a significant increase in FRET saturation is achieved, which is called the saturation excitation light threshold.
  • the saturation excitation light threshold for the pair of FRET molecules is obtained by a preferred number of experiments.
  • the saturation excitation light threshold of the FRET molecule pair of the present invention is preferably 350 to 450 ⁇ .
  • the detection method is as follows: Chen, JF & Cheng, Y. Far-field superresolution imaging with dual-dye-doped nanoparticles. Optics Letters 34 , 1831-1833 (2009).
  • the method of laser scanning confocal microscopy imaging described therein is conventional in the art.
  • Super-resolution imaging can be achieved using conventional methods of operation under conventional laser scanning confocal microscopy in the art.
  • the super-resolution imaging method described herein utilizes a conventional conventional laser scanning confocal microscope laser to effect saturation fluorescence resonance energy transfer of the fluorescent probe of the present invention.
  • the super-resolution imaging method of the present invention preferably performs scanning imaging under excitation light intensity slightly above the saturation threshold to achieve super-resolution imaging capability of the system.
  • the intensity of the excitation light is preferably 1. 1 to 2 times higher than the saturation threshold, and the intensity of the excitation light is preferably 400 ⁇ ⁇ to 500 ⁇ ⁇ .
  • the intensity of the excitation light is generally 5 ⁇
  • the saturation excitation light threshold is 1. 05-5 times, preferably 1. 1-2 times.
  • the present invention enables super-resolution imaging of biological samples on a conventional laser scanning confocal microscope.
  • the laser scanning confocal microscope of the present invention is a conventional laser scanning confocal microscope system in the art.
  • the laser scanning confocal microscope system refers to an optical imaging system that uses a laser as a light source to add a laser scanning device and a conjugate focusing device to perform digital image acquisition and processing through computer control based on conventional fluorescence microscope imaging.
  • the laser source described therein preferably includes a single laser and a multi-laser system, wherein the laser used in the laser source preferably comprises: a semiconductor laser, an argon ion laser, a helium laser, and a UV laser (ultraviolet laser).
  • the laser scanning confocal microscope manufacturer and model of the present invention preferably comprises: ZessiLSM700, Leica TCSSP5II, Olypums FV500, Nikon CI, the manufacturer and model of the laser confocal microscope of the present invention are preferably: Leica TCS SP5II, Germany .
  • the super-resolution imaging method based on saturated fluorescence resonance energy transfer can be widely applied in the field of biological research.
  • the application preferably includes: three-dimensional super-resolution imaging of biological samples such as cells and microorganisms, such as detection of natural and oncogenic fusion proteins, detection of oncogenic fusion proteins in solid tumors, research The components between protein complexes and the distance and state between the various components; study the transcriptional complexes to study the mechanism of gene regulation, study the transcriptional activation caused by protein complexes, and study the interaction between living cells and cells.
  • the application examples preferably include: detecting the state of intracellular fusion proteins, molecular complexes, or organelles.
  • the cell or subcellular structure can be analyzed by flow cytometry or fluorescence microscopy at the single cell level while retaining the cell morphology intact.
  • the subcellular structure is a conventional subcellular structure in the art.
  • the subcellular structure of the present invention is preferably a microtubule system of HeLa cells.
  • the super-resolution imaging method based on fluorescence resonance energy transfer of the present invention utilizes nonlinear optical effects, and can perform different detection objects on a laser confocal microscope without any modification.
  • Super-resolution imaging detection analysis the super-resolution imaging method has the advantages of being fast and simple, and the method fully utilizes the prior art equipment of the laboratory, and greatly reduces the cost of the super-resolution imaging technology.
  • Figure 1 is a graph showing the nonlinear response of the probe of FRET prepared in Example 1 to excitation light.
  • Figure 2 is a super-resolution imaging of the Cy3-Cy5FRET probe with excitation light intensity near the saturation threshold.
  • the super-resolution effect is obvious, and the resolution of the existing confocal microscope is improved by 3 times and the resolution is 65 nm.
  • Figure 3 is a super-resolution imaging of the Atto488-Atto540 FRET probe.
  • Figure 4 is a super-resolution imaging of the Atto550-Atto647 NFRET probe.
  • Figure 5 is a super-resolution imaging of the Atto488-Atto647 NFRET probe.
  • Figure 6 is a super-resolution imaging of the Alexa488-Cy3FRET probe.
  • Fig. a is an image of a laser confocal microscope.
  • Figure b is an image of an SFM microscope.
  • Figures c and d are enlarged views of the white frame portion of a and b, respectively.
  • the vertical line indicates the area of comparison.
  • the two curves in Fig. e are the fluorescence intensity distribution curves of the vertical line in c and d, respectively, and the Gaussian fitting of the fluorescence curve in the d image gives the full width at half maximum.
  • the invention is further illustrated by the following examples, but the invention is not limited thereto.
  • the experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer.
  • the "room temperature” as used in the examples means the temperature between the operations in which the test is conducted, and is generally 25 .
  • Hela cells were purchased from the Cell Resource Center of the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, and its commercial serial number is Tch20.
  • Fluorescent dyes Cy3, Cy5, Atto488, Atto540, Atto550, Atto647N and Alexa488 were purchased from Takara.
  • Cy3-Cy5 fluorescent probe Preparation of fluorescent nanospheres by reverse microemulsion method.
  • Experimental material sodium silicate solution (water glass) (the water glass modulus is 3. 1, Baume degree is 40), hydrochloric acid (concentration is 1 mol/L), sodium dodecyl sulfate SDS, toluene, positive Pentanol, deionized water (W), fluorescent dye Cy3-Cy5.
  • Osmotic reaction Take 0. lg surfactant SDS (S), 0. 2g fluorescent dye (donor Cy3 and acceptor Cy5, donor acceptor molar ratio 1: 2), 0.2 ml of toluene and 5ml prepared in advance
  • S surfactant SDS
  • 2g fluorescent dye donor Cy3 and acceptor Cy5, donor acceptor molar ratio 1: 2
  • the water glass solution was placed in a beaker, and the mixture was ultrasonically mixed (ultrasonic power 50 W, mixing time: 1 minute) to obtain a turbid emulsion, and n-pentanol was added dropwise until the system was suddenly transparent, that is, microemulsion A was obtained.
  • a lmol/L HCl solution was used instead of the water glass solution, and a microemulsion was prepared as described above. 5 ml of the microemulsion A obtained by the permeation reaction step was poured into a three-necked flask, and the temperature of the water bath was 30 °C. 1 ml of the microemulsion B was added to the microemulsion A obtained by the permeation step using a peristaltic pump, and the solution was added dropwise after 45 minutes to obtain a solution C. The resulting solution C was subjected to dialysis treatment for 24 hours for use in the next experiment, and the dialysis bag used had a molecular weight of 14,000.
  • the fluorescence intensity of the fluorescent dye Dy3 in the solution of the individual donor fluorescent dye Cy3 (diluent concentration is 100 nmol/L) and the solution C obtained by the above polymerization reaction were respectively detected by a fluorescence spectrophotometer; the fluorescence intensity of the Cy3 alone donor was 2000.
  • the fluorescence intensity of Cy3 after polymerization is 30, and the FRET efficiency is calculated to be above 98% under the excitation of 514 nm.
  • the calculation method is:
  • E FRET ( ⁇ - Icy3(FRET) ) x lOO%
  • Biological modification of nanoparticles Weigh 1ml of the nano-fluorescent particle solution C prepared by the above polymerization, wash it with 1ml of PBS buffer, add 1ml of goat anti-mouse IgG
  • the cloned antibody (antibody manufacturer: Abeam, commercial serial number: ab98711) was reacted at room temperature for 30 min, and after centrifugation and washing, antibody-modified fluorescent nanoparticles were obtained, which were dissolved in PBS buffer and stored at 4 ° C until use.
  • the FRET fluorescent probe was labeled on Hela cell microtubules according to the immunohistochemical staining procedure (block-primary-secondary antibody). Specific steps are as follows:
  • the blocking solution (6 % fetal bovine serum) was added to the fixed Hela cells, and the fixed Hela cells were prepared by treating the cells with 4% (v/v) paraformaldehyde for 20 minutes. Blocking was carried out for 1 hour at room temperature and washed with PBS buffer.
  • the antibody is an anti-tubulin monoclonal antibody, antibody concentration: 10 nM, antibody manufacturer: san ta cruz, SEQ ID NO: sc-5286) was added, and the reaction was carried out for 1 hour at room temperature.
  • the secondary antibody was the secondary antibody obtained by cross-linking the nano fluorescent particles as described above, and the concentration of the polyclonal antibody was 30 nM), and the reaction was carried out for 1 hour at room temperature.
  • Fluorescence intensity curves of donors before saturation of FRET process under different excitation light intensities were detected.
  • a 1 mol/L HCl solution was used instead of the water glass solution, and a microemulsion was prepared as described above. 5 ml of the microemulsion A obtained by the permeation reaction step was poured into a three-necked flask, and the temperature of the water bath was 30 °C. 1 ml of the microemulsion B was added to the microemulsion A obtained by the permeation step using a peristaltic pump, and the dropwise addition was completed in 45 minutes to obtain a solution C. The resulting solution C was subjected to dialysis treatment for 24 hours for use in the next experiment, and the dialysis bag used was of a molecular weight of 14,000.
  • the fluorescence intensity of the fluorescent dye Atto488 in the solution of the individual donor fluorescent dye Atto488 (diluent concentration is 100 nmol/L) and the solution C obtained by the above polymerization reaction were respectively detected by a fluorescence spectrophotometer; the fluorescence intensity of the Atto488 single donor was 2000.
  • the fluorescence intensity of the fluorescent dye donor after the polymerization was 32, and the FRET efficiency was calculated to be 95% or more when the excitation light wavelength was 458 nm.
  • Bio-modification of nanoparticles 1 ml of the nano-fluorescent particle solution C prepared by the above polymerization reaction was weighed, and after washing with 1 ml of PBS buffer, 1 ml of goat anti-mouse IgG polyclonal antibody was added (antibody manufacturer: Abeam, commercial serial number: ab98711 The reaction was carried out for 30 min at room temperature, and after centrifugation and washing, antibody-modified fluorescent nanoparticles were obtained, which were dissolved in PBS buffer and stored at 4 ° C until use.
  • the FRET fluorescent probe was labeled on Hela cell microtubules according to the immunohistochemical staining procedure (block-primary-secondary antibody). Specific steps are as follows:
  • the blocking solution (6% fetal bovine serum) was added to the fixed Hela cells, and the fixed Hela cells were prepared by treating the cells with 4% (v/v) paraformaldehyde for 20 minutes. Blocking was carried out for 1 hour at room temperature and washed with PBS buffer.
  • the antibody was an anti-tubulin monoclonal antibody, antibody concentration: 10 nM, antibody manufacturer: santa cruz, SEQ ID NO: sc-5286), and reacted at room temperature for 1 hour.
  • the secondary antibody was the secondary antibody obtained by cross-linking the nano fluorescent particles as described above, and the concentration of the polyclonal antibody was 30 nM), and the reaction was carried out for 1 hour at room temperature.
  • the fluorescence intensity curve of the donor before saturation of the FRET process under different intensity excitation light and the donor fluorescence intensity curve after saturation of the FRET process were detected respectively at the excitation light wavelength of 458 nm, and the changes of the two curves were compared to obtain the probe.
  • the nonlinear optical response is calculated by calculating the ratio of the fluorescence intensity of the individual donor fluorescent molecule to the donor fluorescence intensity of the FRET molecule. When the ratio changes, FRET saturation occurs, and the excitation threshold of FRET saturation is 400 ⁇ .
  • the intensity is 480 laser excitation, and the imaging is started to obtain a super-resolution image.
  • the resolution of the image is 90 nm, as shown in Fig. 3.
  • Experimental material sodium silicate solution (water glass) (the water glass modulus is 3.3, Baume is 40), hydrochloric acid (concentration is 1 mol/L), sodium dodecyl sulfate SDS, toluene, positive Pentanol, deionized water (W), fluorescent dye Atto550-Atto647N.
  • Osmotic reaction Take 0. lg surfactant SDS (S), 0. 2g fluorescent dye (donor Atto550 and acceptor Atto647N, donor acceptor molar ratio 1: 2), 0.2 ml of toluene and 5ml prepared in advance
  • S surfactant SDS
  • 2g fluorescent dye donor Atto550 and acceptor Atto647N, donor acceptor molar ratio 1: 2
  • 0.2 ml of toluene and 5ml prepared in advance
  • the water glass solution is placed in a beaker, and the ultrasonic mixing is uniform (ultrasonic power 50W, mixing time 1 minute) to obtain colostrum, dripping Adding n-pentanol to the system is suddenly transparent, that is, obtaining microemulsion A.
  • a lmol/L HCl solution was used instead of the water glass solution, and a microemulsion was prepared as described above. 5 ml of the microemulsion A obtained by the permeation reaction step was poured into a three-necked flask, and the temperature of the water bath was 30 °C. 1 ml of the microemulsion B was added to the microemulsion A obtained by the permeation step using a peristaltic pump, and the solution was added dropwise after 45 minutes to obtain a solution C. The resulting solution C was subjected to dialysis treatment for 24 hours for use in the next experiment, and the dialysis bag used had a molecular weight of 14,000.
  • the fluorescence intensity of the fluorescent dye Atto550 in the solution of the individual donor fluorescent dye Atto550 (diluent concentration is 100 nmol/L) and the solution C obtained by the above polymerization reaction were respectively detected by a fluorescence spectrophotometer; the fluorescence intensity of the Atto550 individual donor was 3000.
  • the fluorescence intensity of the fluorescent dye Atto550 donor after the polymerization reaction was 50, and the FRET efficiency was calculated to be 95% or more when the excitation light wavelength was 514 nm.
  • Bio-modification of nanoparticles Weigh 1 ml of the nano-fluorescent particle solution C prepared by the above polymerization, and after washing with 1 ml of PBS buffer, add 1 ml of goat anti-mouse IgG polyclonal antibody (antibody manufacturer: Abeam, commodity serial number: ab98711 The reaction was carried out for 30 min at room temperature, and after centrifugation and washing, antibody-modified fluorescent nanoparticles were obtained, which were dissolved in PBS buffer and stored at 4 ° C until use.
  • the blocking solution (6% fetal bovine serum) was added to the fixed Hela cells, and the fixed Hela cells were prepared by treating the cells with 4% (v/v) paraformaldehyde for 20 minutes. Block for 1 hour at room temperature and wash with PBS buffer.
  • the antibody is an anti-tubulin monoclonal antibody, antibody concentration: 10 nM, antibody manufacturer: san ta cruz, SEQ ID NO: sc-5286) was added, and the reaction was carried out for 1 hour at room temperature.
  • the secondary antibody was the secondary antibody obtained by cross-linking the nano fluorescent particles as described above, and the concentration of the polyclonal antibody was 30 nM), and the reaction was carried out for 1 hour at room temperature.
  • the fluorescence intensity curves of the donor before saturation of the FRET process at different excitation light intensities and the donor fluorescence intensity after saturation of the FRET process were measured at 514 nm, and the changes of the two curves were compared to obtain the probe.
  • the nonlinear optical response is calculated by calculating the ratio of the fluorescence intensity of the individual donor fluorescent molecule to the donor fluorescence intensity of the FRET molecule. When the ratio changes, FRET saturation occurs, thereby obtaining a FRET saturated excitation light threshold of 450 ⁇ ⁇ .
  • a laser of intensity 500 is excited to start imaging, and a super-resolution image is obtained with a resolution of 88 nm, as shown in FIG.
  • Atto488-Atto647N fluorescent probe Construction of high FRET efficiency Atto488-Atto647N fluorescent probe: Experimental material: sodium silicate solution (water glass) (the water glass modulus is 3.4, Baume is 40), hydrochloric acid (concentration is 1 mol/L), sodium dodecyl sulfate SDS, toluene, positive Pentanol, deionized water (W), fluorescent dye Atto488-Atto647No
  • Osmotic reaction Take 0. lg surfactant SDS (S), 0. 2g fluorescent dye (donor Atto488 and acceptor Atto647N, donor acceptor molar ratio 1: 2), 0.2 ml of toluene and 5ml prepared in advance The water glass solution was placed in a beaker, and the mixture was ultrasonically mixed (ultrasonic power 50 W, mixing time: 1 minute) to obtain a turbid emulsion, and n-pentanol was added dropwise until the system was suddenly transparent, that is, microemulsion A was obtained.
  • a lmol/L HCl solution was used instead of the water glass solution, and a microemulsion was prepared as described above. 5 ml of the microemulsion A obtained by the permeation reaction step was poured into a three-necked flask, and the temperature of the water bath was 30 °C. 1 ml of the microemulsion B was added to the microemulsion A obtained by the permeation step using a peristaltic pump, and the solution was added dropwise after 45 minutes to obtain a solution C. The resulting solution C was subjected to dialysis treatment for 24 hours for use in the next experiment, and the dialysis bag used had a molecular weight of 14,000.
  • the fluorescence intensity of the fluorescent dye Atto488 in the solution of the individual donor fluorescent dye Atto488 (diluent concentration is 100 nmol/L) and the solution C obtained by the above polymerization reaction were respectively detected by a fluorescence spectrophotometer; the fluorescence intensity of the Atto488 single donor was 2500.
  • the fluorescence intensity of the donor after the FRET polymerization reaction was 50.
  • the FRET efficiency was calculated to be 90% or more when the excitation light wavelength was 458 nm.
  • Bio-modification of nanoparticles Weigh 1 ml of the nano-fluorescent particle solution C prepared by the above polymerization, and after washing with 1 ml of PBS buffer, add 1 ml of goat anti-mouse IgG polyclonal antibody (antibody manufacturer: Abeam, commodity serial number: ab98711 The reaction was carried out for 30 min at room temperature, and after centrifugation and washing, antibody-modified fluorescent nanoparticles were obtained, which were dissolved in PBS buffer and stored at 4 ° C until use.
  • the FRET fluorescent probe was labeled on Hela cell microtubules according to the immunohistochemical staining procedure (block-primary-secondary antibody). Specific steps are as follows:
  • the blocking solution (6% fetal bovine serum) was added to the fixed Hela cells, and the fixed Hela cells were prepared by treating the cells with 4% (v/v) paraformaldehyde for 20 minutes. Blocking was carried out for 1 hour at room temperature and washed with PBS buffer.
  • ⁇ -antibody 200 ⁇ M ⁇ -antibody (the antibody is anti-tubulin monoclonal antibody, antibody concentration: 10 nM, antibody manufacturer: san ta cruz, SEQ ID NO: sc-5286) was added, and the reaction was carried out for 1 hour at room temperature.
  • the secondary antibody was the secondary antibody obtained by cross-linking the nano fluorescent particles as described above, and the concentration of the polyclonal antibody was 30 nM), and the reaction was carried out for 1 hour at room temperature.
  • the fluorescence intensity curves of the donor before saturation of the FRET process at different excitation light intensities and the donor fluorescence intensity after saturation of the FRET process were measured separately at the excitation light wavelength of 458 nm, and the curves of the two curves were compared. Change, obtain the nonlinear optical response of the probe, calculate the ratio of the fluorescence intensity of the individual donor fluorescent molecule to the donor fluorescence intensity of the FRET molecule, and FRET saturation occurs when the ratio changes, thereby obtaining FRET saturated excitation light.
  • the threshold is 350 ⁇ .
  • a laser with a intensity of 400 is selected for excitation, and imaging is started to obtain a super-resolution image with a resolution of 69 nm, as shown in FIG.
  • Osmotic reaction Take 0. lg surfactant SDS (S), 0. 2g fluorescent dye (donor Alexa488 and acceptor Cy3, donor acceptor molar ratio 1: 2), 0.2 ml of toluene and 5ml prepared in advance
  • S surfactant SDS
  • 2g fluorescent dye donor Alexa488 and acceptor Cy3, donor acceptor molar ratio 1: 2
  • n-pentanol was added dropwise until the system was suddenly transparent, that is, microemulsion A was obtained.
  • a lmol/L HCl solution was used instead of the water glass solution, and a microemulsion was prepared as described above. 5 ml of the microemulsion A obtained by the permeation reaction step was poured into a three-necked flask, and the temperature of the water bath was 30 °C. 1 ml of the microemulsion B was added to the microemulsion A obtained by the permeation step using a peristaltic pump, and the solution was added dropwise after 45 minutes to obtain a solution C. The resulting solution C was subjected to dialysis treatment for 24 hours for use in the next experiment, and the dialysis bag used was of a molecular weight of 13,000.
  • the fluorescence intensity of the fluorescent dye Alexa488 in the solution of the individual donor fluorescent dye Alexa488 (diluent concentration is 100 nmol/L) and the solution C obtained by the above polymerization reaction were respectively detected by a fluorescence spectrophotometer; the fluorescence intensity of the Alexa488 alone donor was 2500.
  • the fluorescence intensity of the donor is 50, and the FRET efficiency is calculated to be 90% or more when the excitation light wavelength is 458 nm.
  • Bio-modification of nanoparticles Weigh 1 ml of the nano-fluorescent particle solution C prepared by the above polymerization, and after washing with 1 ml of PBS buffer, add 1 ml of goat anti-mouse IgG polyclonal antibody (antibody manufacturer: Abeam, commodity serial number: ab98711 The reaction was carried out for 30 min at room temperature, and after centrifugation and washing, antibody-modified fluorescent nanoparticles were obtained, which were dissolved in PBS buffer and stored at 4 ° C until use.
  • the FRET fluorescent probe was labeled on Hela cell microtubules according to the immunohistochemical staining procedure (block-primary-secondary antibody). Specific steps are as follows:
  • the blocking solution (6% fetal bovine serum) was added to the fixed HeLa cells.
  • the fixed HeLa cells were prepared by treating the cells with 4% (v/v) paraformaldehyde for 20 minutes. Blocking was carried out for 1 hour at room temperature and washed with PBS buffer.
  • 200 ⁇ M ⁇ -antibody was added (the antibody was an anti-tubulin monoclonal antibody, antibody concentration: 10 nM, antibody manufacturer: san ta cruz, SEQ ID NO: sc-5286), and reacted at room temperature for 1 hour.
  • the secondary antibody was the secondary antibody obtained by cross-linking the nano fluorescent particles as described above, and the concentration of the polyclonal antibody was 30 nM), and the reaction was carried out for 1 hour at room temperature.
  • the fluorescence intensity curves of the donor before saturation of the FRET process at different intensity and the donor fluorescence intensity curve after saturation of the FRET process were measured at 458 nm, and the changes of the two curves were compared to obtain the nonlinearity of the probe.
  • the optical response is mainly calculated by calculating the ratio of the fluorescence intensity of the individual donor fluorescent molecules to the donor fluorescence intensity of the FRET molecule pair. When the ratio changes, FRET saturation occurs, thereby obtaining the FRET saturated excitation light threshold of 400 ⁇ Wo.
  • the 450 ⁇ W laser was excited and the imaging was started to obtain a super-resolution image with a resolution of 125 nm, as shown in Fig. 6.

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Abstract

一种基于荧光共振能量转移的超分辨成像方法，所述超分辨成像方法包括以下步骤：1）用高FRET效率的荧光探针标记待检样品，所述的高FRET效率的荧光探针标记有FRET分子对，所述的FRET分子对包括第一荧光基团（供体）和第二荧光基团（受体），第一荧光基团能够向第二荧光基团发生荧光共振能量转移（FRET）；2）采用激发光强度能够使步骤1）所述的FRET分子对发生荧光共振能量转移的激发光阈值，进行激光扫描共聚焦显微镜成像。上述基于饱和荧光共振能量转移的超分辨技术能够在一台普通的激光共聚焦显微镜上实现对生物样品的超分辨成像，该方法分辨率高。

Description

一种基于荧光共振能量转移的超分辨成像方法
技术领域
本发明属于超分辨成像技术领域， 特别涉及一种基于荧光共振能量转移 (FRET, Fluorescence Resonance Energy Transfer)的超分辨成像方法。 背景技术
现代生命科学中的许多重大研究成果都依赖于显微成像技术的进步， 然而很 长时间以来人们一直认为光学显微镜能达到的极限分辨率大约为光波长的一半 200纳米左右。 直到 20世纪 90年代初， 随着光学领域中一系列新技术的出现才打 破光学衍射极限， 将光学显微镜的分辨率提高到数十纳米。
超分辨成像技术的基础主要借助于各种非线性光学效应。 1991年康乃尔大学 的一个研究组实现了多光子荧光成像技术，成为利用非线性光学效应进行显微成像 的一个先驱性的工作， 多光子荧光显微镜实现了光学衍射极限的超越，但是由于多 光子荧光激发过程必须采取更长的激发光波长，因此最后所能达到的实际分辨率与 单光子激发技术相比反而有所下降。在原理上彻底突破光学衍射极限的远场光学成 像技术是 1994年由 Stefan Hell提出的 STED (stimulated emission deplet ion microscopy)受激发射损耗显微术。
超分辨成像技术的三维分辨率取决于该聚焦激光焦斑内所激发的荧光分子所 占据的体积。 因此， 为了减小荧光激发体积， STED 采用了两束组合激光， 即一束 光被聚焦成正常的衍射极限焦斑，其将焦斑内的荧光分子到激发态； 而第二束光则 选取在荧光分子的发射波长范围，并被聚焦成一个中心与第一束光的焦斑中心完全 重合但却是中空的环状焦斑。由于利用第二束光可以将被第一束激发光激发到激发 态上去的荧光分子从激发态淬灭到基态， 因此它也被称作淬灭光束。 由于淬灭光束 的光强分布在焦点处为零， 因此从原理上讲， 只要淬灭光足够强， 则由第一束光激 发的荧光分子所占据的体积可以被压缩到极小的范围内，因为在焦点以外这些荧光 分子都会被淬灭。
另一类超分辨成像技术则基于单分子的精确定位。 光学衍射极限的概念对于 单个发光点并不适用， 这是因为艾里斑的中心可以被非常精确地定位。 2006 年华 裔科学家庄小威提出了随机光学重建显微术 (STORM) , 通过将相互位置过于靠近而 无法被传统光学显微镜同时分辨的荧光标记分子逐个予以分别激发，使各个荧光分 子所成的艾里斑像之间不再相互干扰，从而能够对每个独立的荧光分子逐个进行定 位。这样，在全部荧光标记分子的定位完成后，一幅超越衍射极限的图像即已形成， 随后还有 PALM、 FPALM等单分子超分辨成像技术。
然而现有的超分辨成像技术都需要对现有的荧光显微镜进行大量的改造， 成 本非常昂贵： 商品化的 Leica STED 市场价高达 700万人民币， 成像软件也非常复 杂； STORM需要快速的对图片中每个单分子形成的艾里斑进行定位， 而完成一张完 整的生物样品图片往往要拍成千上万张观测图片。这些在技术及资金上的存在的问 题极大地限制了超分辨成像技术的普及和应用。 发明内容
因此， 本发明要解决的技术问题就是针对现有的超分辨成像技术都需要对荧 光显微镜进行大量的改造， 成本非常昂贵， 成像软件也非常复杂的缺陷， 提供一种 基于荧光共振能量转移 (FRET)的超分辨成像方法。
为解决上述技术问题， 本发明采取的技术方案之一是： 一种基于荧光共振能 量转移的超分辨成像方法， 其中包括以下步骤：
1) 用高 FRET效率的荧光探针标记待检样品， 所述的高 FRET效率的荧光探针 标记有 FRET分子对，所述的 FRET分子对包括第一荧光基团 (供体)和第二荧光基团 (受体）， 第一荧光基团能够向第二荧光基团发生荧光共振能量转移 (FRET)；
2) 采用激发光强度能够使步骤 1)所述的 FRET分子对发生荧光共振能量转移 的激发光阈值， 进行激光扫描共聚焦显微镜成像。
本发明中， 当两个荧光基团——第一荧光基团（供体)和第二荧光基团（受体) 之间足够靠近，并且如果第一荧光基团的发射光谱与第二荧光基团的吸收光谱之间 有所重叠， 贝 Ij根据 FRET (Fluorescence Resonance Energy Transfer: 焚光共振能 量转移)原理， 将允许处于激发态的第一荧光基团将能量交给第二荧光基团， 然后 无辐射直接跃迁回到基态。 根据 FRET原理， FRET过程的效率与两个荧光基团之间 的间距的 6次方成倒数， 因此距离越小， FRET过程的效率越高。 当第一荧光基团 处于激发状态，会将其能量传递给相邻的第二荧光基团，引起第二荧光基团的激发。 当第一荧光基团的激发光强度较低时， 被辐照的第一荧光基团基本上发生 FRET效 应而不自发荧光。当激发光的激发光强度被提高时，会导致激发光焦斑中心附近的 第一荧光基团发生 FRET饱和， 第一荧光基团就被迫发射荧光。 如果选择合适的激 光值刚好稍大于引起 FRET饱和的激发光强度阈值， 就能控制激光焦斑中心很小区 域内第一荧光基团发光。于是就得到了一个小于衍射极限尺度的发光点，从而获得 超越光学衍射极限的分辨能力。
本发明中所述的探针是本领域常规的探针， 其中所述探针较佳地包括但不限 于核酸、 抗体、 蛋白（如酶、 受体)、 有机小分子 (如核苷酸)、 无机分子 (如钙离子) 中的一种或几种，只要这些探针上能够标记第一荧光基团和第二荧光基团即可， 并 且这些探针能够和目标物质 (如待检样本）结合即可。探针和和目标物质的结合是特 异性的。本发明所述的探针优选地包括： 脱氧核糖核苷酸寡聚体， 核糖核酸寡聚体 和多肽寡聚体中的一种或几种。
本发明中所述探针上标记的 FRET分子对包括第一荧光基团 (供体)和第二荧光 基团 (受体），第一荧光基团能够向第二荧光基团发生荧光共振能量转移 (FRET)。本 发明所述的 FRET分子对优选具有较高的 FRET效率 (优选 >85%)，供体与受体的荧 光信号串扰较小， 较高的供体荧光量子产率和较小的荧光光漂白问题的 FRET分子 对。其中所述的荧光基团为本领域常规的荧光基团。所述荧光基团是指： 在吸收可 见光和紫外光后， 能把紫外光转变为波长较长的可见光波而反射出来，呈闪亮的鲜 艳色彩。 例如， 酸性曙红、 荧光黄、 红汞以及某些分散染料等。 所述荧光基团大多 是含有苯环或杂环并带有共轭双键的化合物。
其中所述 FRET分子对为本领域常规的 FRET分子对。本发明所述 FRET分子对 较佳地包括： CFP-YFP (青色荧光蛋白 -黄色荧光蛋白）， Cy3_Cy5 (菁染料 3-5)， Atto488-Atto540, FITORhodamine (异硫氰酸荧光素 -罗丹明）， BFP-GFP (蓝色荧光 蛋白-绿色荧光蛋白）， Atto488-Atto647N, Atto550- Atto647N， Atto488- Atto590， Atto550- Atto655， Atto590- Atto655， CFP- dsRED (青色荧光蛋白-红色荧光蛋白）， Alexa488-Alexa555 (Alexaf luor488— Alexaf luor555)； Alexa488— Cy3 (Alexaf luor4 88-菁染料 3)， YFP-TRITC (黄色荧光蛋白-四甲基罗丹明）和 YFP_Cy3 (黄色荧光蛋白 -菁染料 3)中的一种或几种。 本发明 FRET 分子对优选地包括： Cy3-Cy5， Atto488- Atto540， Atto550- Atto647N， Atto488- Atto647N和 Alexa488- Cy3。
本发明中所述的 FRET分子对之间的距离较佳地小于 10nm，所述的分子对距离 更佳地为 2〜5nm， 距离优选地为小于 2. 5nm。
其中所述的高 FRET效率的荧光探针的制备方法为本领域常规制备方法。本发 明所述的荧光探针制备方法较佳地包括以下步骤：首先利用反相微乳液法制备荧光 纳米小球，再将该小球与抗体结合即得。其中所述反相微乳液法较佳地包括以下步 骤： 渗透反应： 表面活性剂的添加量较佳地为 2〜5 %， 所述表面活性剂的添加量 优选地为 2 %， 所述表面活性剂为本领域常规的表面活性剂， 优选地为十二垸基硫 酸钠。 荧光染料供体和受体的添加量较佳地为 4〜10 %， 其添加量优选地为 4%， 甲苯的添加量较佳地为 4〜10 %，其添加量优选地为 4%，和事先配制好的 5〜10ml 水玻璃溶液放入烧杯中，所述水玻璃模数较佳地为 3. 1〜3. 4，波美度优选地是 40， 超声混合均匀（超声波功率 50W， 混合时间 1〜5分钟）， 得到初乳浊液， 滴加正戊 醇至体系突然透明， 即获得微乳液 A， 其中所述百分比均为质量百分比。
聚合反应： 用 l〜5mol/L的 HC1溶液代替水玻璃溶液， 按上述方法制得微乳 液^ 将微乳液 A倒入三口烧瓶中， 水浴温度较佳地为 30〜35°C， 优选地为 30°C， 用蠕动泵将 l〜5ml微乳液 B加入渗透步骤所得的微乳液 A中， 滴加时间较佳地为 40〜60min， 滴加完毕即得荧光纳米小球溶液 C。 将所得溶液 C进行透析处理， 以 备下一步实验所用， 透析时间较佳地为 8〜24小时， 优选地为 24小时， 所用透析 袋规格较佳地为 10000〜 14000分子量， 其规格优选地为 14000分子量。
本发明所述荧光纳米小球与抗体结合的方法为本领域常规方法。 所述荧光纳 米小球与抗体的结合方法较佳地包括以下步骤：称取一定体积上述制备的荧光纳米 小球溶液 C， 经 PBS缓冲液洗涤后， 加入等体积的抗体溶液， 在室温下反应， 反应 时间较佳地为 30〜60min， 经离心、 洗涤后得到抗体修饰的荧光纳米粒子， 将其溶 于 PBS缓冲液中 4°C保存备用。
其中所述的用高 FRET 效率的荧光探针标记待检样品的方法是本领域常规技 术。 所述标记方法较佳地包括以下步骤： 将细胞用所述的高 FRET效率的荧光探针 进行标记， 洗涤充分之后， 即可进行激光扫描共聚焦显微镜观察。进行标记的方法 是本领域的常规技术， 为本领域的技术人员所熟知和掌握。所述标记方法较佳地为 通过细胞生物学技术中的免疫组化方法将探针与样品结合，所述免疫组化方法较佳 地包括以下步骤： 封闭， 标记一抗， 标记二抗， 其中所述的二抗是与前述荧光纳米 小球相结合的二抗。
本发明中步骤 2)为： 采用激发光强度能够使步骤 1)所述的 FRET分子对发生 荧光共振能量转移的激发光阈值，进行激光扫描共聚焦显微镜成像。其中采用的激 发光强度是能够使步骤 1)所述的 FRET 分子对发生荧光共振能量转移的激发光阈 值。其中所述的能够使步骤 1)所述的 FRET分子对发生荧光共振能量转移的激发光 阈值是指使 FRET分子对发生荧光共振能量转移达到 FRET饱和的激发光强度，称之 为饱和激发光阈值。 本发明通过本领域常规方法检测所述 FRET分子对的饱和激发光阈值。其中所 述饱和激发光阈值检测方法较佳地包括以下步骤：将单独的供体荧光分子和标记着 FRET 分子对的荧光探针分别置于激光扫描共聚焦显微镜下检测供体荧光分子的荧 光随激发光强变化的曲线， 比较两个曲线的变化， 可以发现在一定的激发光强时 FRET 探针的供体荧光发生了非线性的光学响应， 即在一定激发光阈值附近供体荧 光发生了明显的增加，达到了 FRET饱和， 该激发光阈值即称之为饱和激发光阈值。 通过优选的多次试验， 获得该 FRET 分子对的饱和激发光阈值。 本发明所述 FRET 分子对的饱和激发光阈值优选地为 350〜450 μ Ι所述检测方法参考文献为： Chen, J. F. &Cheng， Y. Far-field superresolution imaging with dual-dye-doped nanoparticles. Optics Letters34, 1831-1833 (2009)。
其中所述的激光扫描共聚焦显微镜成像的方法是本领域常规技术。 在本领域 常规的激光扫描共聚焦显微镜下采用常规的操作方法即可实现超分辨成像。其中所 述的超分辨成像方法较佳地为：利用现有的普通激光扫描共聚焦显微镜的激光器使 本发明的荧光探针实现饱和荧光共振能量转移。
本发明所述超分辨成像方法较佳地为选择稍高于饱和阈值的激发光强度下进 行扫描成像， 实现系统的超分辨成像能力。所述激发光强度较佳地为高于饱和阈值 1. 1倍到 2倍， 所述的激发光强度范围优选地为 400 μ ϊ〜500 μ Ι在本发明中， 采 用的激发光强度通常可以是饱和激发光阈值的 1. 05-5倍， 较佳地为 1. 1-2倍。
本发明能够在一台普通的激光扫描共聚焦显微镜上实现对生物样品的超分辨 成像。 本发明所述激光扫描共聚焦显微镜为本领域常规激光扫描共聚焦显微镜系 统。所述激光扫描共聚焦显微镜系统是指采用激光为光源，在传统荧光显微镜成像 的基础上， 附加了激光扫描装置和共轭聚焦装置，通过计算机控制来进行数字化图 像采集和处理的光学成像系统。其中所述的激光光源较佳地包括单激光和多激光系 统， 其中所述激光光源使用的激光器较佳地包括： 半导体激光器、 氩离子激光器、 氦氖激光器和 UV激光器 (紫外激光器）。 本发明所述激光扫描共聚焦显微镜生产商 和型号较佳地包括： ZessiLSM700, Leica TCSSP5II , Olypums FV500, Nikon CI , 本发明激光共聚焦显微镜生产厂商和型号优选地为： 德国徕卡公司， Lecia TCS SP5II。
本发明提供的基于饱和荧光共振能量转移的超分辨成像方法可以广泛应用于 生物学研究领域。所述的应用较佳地包括： 对细胞、微生物等生物样品的三维超分 辨成像， 如检测天然和致癌的融合蛋白、在实体瘤中检测出现的致癌融合蛋白、研 究蛋白复合物之间的组分以及各个组分之间的距离和状态；通过研究转录复合物来 研究基因调控机理，研究蛋白质复合物引起的转录活化，研究活体细胞及细胞间相 互作用。所述应用实例较佳地包括： 检测细胞内融合蛋白、分子复合物或细胞器的 状态。 由于本发明提供的超分辨成像技术不需要破坏细胞完整性， 可以在保留细胞 形态完整状态下，在单细胞水平上通过流式细胞技术或荧光显微镜对细胞或亚细胞 结构进行分析。其中所述亚细胞结构为本领域常规的亚细胞结构。本发明所述亚细 胞结构优选地为 Hela细胞的微管系统。
本发明所用的原料或试剂除特别说明之外， 均市售可得。
相比于现有技术， 本发明的有益效果如下： 本发明所述基于荧光共振能量转 移的超分辨成像方法利用非线性光学效应，能够在没有任何改造的激光共聚焦显微 镜上对不同检测对象进行超分辨成像检测分析，所述超分辨成像方法具有快速、简 便的优点，该方法充分利用了实验室的现有技术设备，大幅度降低了超分辨成像技 术的成本。 附图说明
以下结合附图说明本发明的特征和有益效果。
图 1是实施例 1制备 FRET的探针对激发光的非线性响应图。
图 2是 Cy3-Cy5FRET探针， 激发光强度在饱和阈值附近时的超分辨成像图。 超分辨效果明显， 改善现有的共聚焦显微镜分辨率 3倍， 分辨率达 65nm。
图 3是 Atto488-Atto540FRET探针超分辨成像图。
图 4是 Atto550-Atto647NFRET探针超分辨成像图。
图 5是 Atto488-Atto647NFRET探针超分辨成像图。
图 6是 Alexa488-Cy3FRET探针超分辨成像图。
其中图 2〜图 6中， 图 a是激光共聚焦显微镜成像图。 图 b是 SFM显微镜成像 图。 图 c、 d分别是 a、 b中白色框部分的放大图。 其中竖线表示比较的区域。 其中 图 e的两条曲线分别是 c、 d图中竖线部分的荧光强度分布曲线， 并且对 d图中的 荧光曲线进行了高斯拟合得到半高宽即为分辨率。 具体实施方式
下面用实施例来进一步说明本发明， 但本发明并不受其限制。 下列实施例中 未注明具体条件的实验方法， 通常按照常规条件， 或按照制造厂商所建议的条件。 实施例中所述的 "室温"是指进行试验的操作间的温度， 一般为 25 。
激光共聚焦显微镜 Lecia TCS SP5, 德国徕卡公司。
Hela细胞购自中国科学院上海生命科学研究院细胞资源中心， 其商品序列号 为 Tch20。
荧光染料 Cy3、 Cy5、 Atto488、 Atto540、 Atto550、 Atto647N和 Alexa488购 自 Takara公司。
实施例 1 Cy3-Cy5探针标记的 Hela细胞微管的超分辨成像
构建高 FRET效率的 Cy3-Cy5荧光探针:采用反相微乳液法制备荧光纳米小球。 实验材料： 硅酸钠溶液 (水玻璃）（所述水玻璃模数是 3. 1， 波美度是 40)， 盐 酸 (浓度为 lmol/L)， 十二垸基硫酸钠 SDS， 甲苯， 正戊醇， 去离子水 (W)， 荧光染 料 Cy3-Cy5。
该技术具体步骤包括：
渗透反应： 取 0. lg表面活性剂 SDS (S)、 0. 2g荧光染料 (供体 Cy3和受体 Cy5， 供体受体摩尔比 1 : 2)、 0. 2ml甲苯和事先配制好的 5ml水玻璃溶液放入烧杯中， 超声混合均匀（超声波功率 50W， 混合时间 1分钟）， 得到初乳浊液， 滴加正戊醇至 体系突然透明， 即获得微乳液 A。
聚合反应： 用 lmol/L的 HC1溶液代替水玻璃溶液， 按上述方法制得微乳液^ 将 5ml渗透反应步骤所得的微乳液 A倒入三口烧瓶中， 水浴控温为 30°C。 用蠕动 泵将 lml微乳液 B加入渗透步骤所得的微乳液 A中， 45min滴加完毕， 得溶液 C。 将所得溶液 C 进行 24 小时透析处理， 以备下一步实验所用， 所用透析袋规格为 14000分子量。
用荧光分光光度计分别检测单独供体荧光染料 Cy3 稀释液（稀释液浓度是 lOOnmol/L)和前述聚合反应所得的溶液 C中的荧光染料 Cy3的荧光强度； Cy3单独 供体的荧光强度是 2000， 聚合反应后的 Cy3荧光强度是 30， 计算得出在 514nm的 波长激发下， FRET效率在 98%以上， 计算方法为：
EFRET = (\ - Icy3(FRET) ) x lOO% 纳米颗粒的生物修饰： 称取 lml上述聚合反应制备的纳米荧光粒子溶液 C， 经 lml的 PBS缓冲液洗涤后， 加入 lml羊抗鼠 IgG多克隆抗体 (抗体生产商： Abeam, 商品序列号： ab98711)， 在室温下反应 30min， 经离心、 洗涤后得到抗体修饰的荧 光纳米粒子， 将其溶于 PBS缓冲液中 4°C保存备用。 按照免疫组化染色步骤操作 (封闭- 一抗- 二抗）， 将 FRET 荧光探针标记在 Hela细胞微管上。 具体步骤如下：
1： 将封闭液 (6 %胎牛血清）加入固定的 Hela细胞， 固定的 Hela细胞的制备 方法为： 用 4% (V/V)的多聚甲醛处理细胞 20分钟。 在室温下进行封闭 1小时， 用 PBS缓冲液洗涤。
2：加入 200 μ 1 —抗 (所述抗体为抗微管蛋白单克隆抗体，抗体浓度为： 10ηΜ， 抗体的生产商： santa cruz , 序列号： sc-5286)， 室温下反应 1小时。
3： 加入 100 μ ΐ二抗 (所述二抗是上述所得交联有纳米荧光颗粒的二抗， 多克 隆抗体浓度为 30ηΜ)， 室温下反应 1小时。
分别检测不同激发光强度下 FRET 过程饱和前供体的荧光强度变化曲线和
FRET过程饱和后的供体荧光强度变化曲线。 结果见图 1。 比较这两个曲线的变化， 计算单独供体荧光分子在不同强度的 514nm激光照射下的荧光强度与 FRET分子对 中供体荧光强度的比值， 当比值发生变化时则发生 FRET饱和现象， 获得该探针的 非线性光学响应， 得 FRET饱和的激发光阈值为 400 μ ϊ。 所述检测方法参考文献： Chen, J. F. &Cheng， Y. Far- fieldsuperresolution imaging with dual- dye- doped nanoparticles. Optics Letters 34， 1831-1833 (2009)。 选取强度为 500 的激 光进行激发开始成像， 得到超分辨图像， 其分辨率为 65nm， 如图 2所示。
实施例 2 Atto488-Atto540探针标记的 Hela细胞微管的超分辨成像
构建高 FRET效率的 Atto488-Atto540荧光探针
实验材料： 硅酸钠溶液 (水玻璃）（所述水玻璃模数是 3. 2， 波美度是 40)， 盐 酸 (浓度为 lmol/L)， 十二垸基硫酸钠 SDS， 甲苯， 正戊醇， 去离子水 (W)， 荧光染 料 Atto488- Atto540。
取 0. lg表面活性剂 SDS (S)、 0. 2g荧光染料（供体 Atto488和受体 Atto540， 供体受体摩尔比 1 : 2)、 0. 2ml甲苯和事先配制好的 5ml水玻璃溶液放入烧杯中， 超声混合均匀（超声波功率 50W， 混合时间 1分钟）， 得到初乳浊液， 滴加正戊醇至 体系突然透明， 即获得微乳液 A。
聚合反应： 用 lmol/L的 HC1溶液代替水玻璃溶液， 按上述方法制得微乳液^ 将 5ml渗透反应步骤所得的微乳液 A倒入三口烧瓶中， 水浴控温为 30°C。 用蠕动 泵将 lml微乳液 B加入渗透步骤所得的微乳液 A中， 45min滴加完毕， 得溶液 C。 将所得溶液 C 进行 24 小时透析处理， 以备下一步实验所用， 所用透析袋规格为 14000分子量。 用荧光分光光度计分别检测单独供体荧光染料 Atto488稀释液 (稀释液浓度是 lOOnmol/L)和前述聚合反应所得的溶液 C 中的荧光染料 Atto488 的荧光强度； Atto488 单独供体的荧光强度是 2000， 聚合反应后的荧光染料供体的荧光强度是 32， 计算出出激发光波长是 458nm时 FRET效率 95%以上。
纳米颗粒的生物修饰： 称取 1ml上述聚合反应制备的纳米荧光粒子溶液 C， 经 lml的 PBS缓冲液洗涤后， 加入 1ml羊抗鼠 IgG多克隆抗体 (抗体生产商： Abeam, 商品序列号： ab98711)， 在室温下反应 30min， 经离心、 洗涤后得到抗体修饰的荧 光纳米粒子， 将其溶于 PBS缓冲液中 4°C保存备用。
按照免疫组化染色步骤操作 (封闭- 一抗- 二抗）， 将 FRET 荧光探针标记在 Hela细胞微管上。 具体步骤如下：
1： 将封闭液 (6%胎牛血清）加入固定的 Hela细胞， 固定的 Hela细胞的制备 方法为： 用 4% (V/V)的多聚甲醛处理细胞 20分钟。 在室温下进行封闭 1小时， 用 PBS缓冲液洗涤。
2： 加入 200 μ ΐ—抗 (所述抗体为抗微管蛋白单克隆抗体，抗体浓度为： 10ηΜ， 抗体的生产商： santa cruz， 序列号： sc-5286)， 室温下反应 1小时。
3： 加入 100 μ ΐ二抗 (所述二抗是上述所得交联有纳米荧光颗粒的二抗， 多克 隆抗体浓度为 30ηΜ)， 室温下反应 1小时。
分别检测激发光波长为 458nm时不同强度激发光下 FRET过程饱和前供体的荧 光强度变化曲线和 FRET过程饱和后的供体荧光强度变化曲线， 比较这两个曲线的 变化， 获得该探针的非线性光学响应， 计算单独供体荧光分子的荧光强度与 FRET 分子对中供体荧光强度的比值，当比值发生变化时则发生 FRET饱和，从而获得 FRET 饱和的激发光阈值为 400 μ ϊ。 选取强度为 480 激光进行激发， 开始成像， 得到 超分辨图像， 该图像的分辨率是 90nm， 如图 3所示。
实施例 3 Atto550-Atto647N探针标记的 Hela细胞微管的超分辨成像 构建高 FRET效率的 Atto550-Atto647N荧光探针：
实验材料： 硅酸钠溶液 (水玻璃）（所述水玻璃模数是 3. 3， 波美度是 40)， 盐酸 (浓度为 lmol/L)， 十二垸基硫酸钠 SDS， 甲苯， 正戊醇， 去离子水 (W)， 荧光染料 Atto550-Atto647N。
渗透反应： 取 0. lg表面活性剂 SDS (S)、 0. 2g荧光染料 (供体 Atto550和受体 Atto647N, 供体受体摩尔比 1 : 2)、 0. 2ml甲苯和事先配制好的 5ml水玻璃溶液放 入烧杯中， 超声混合均匀（超声波功率 50W， 混合时间 1分钟）， 得到初乳浊液， 滴 加正戊醇至体系突然透明， 即获得微乳液 A。
聚合反应： 用 lmol/L的 HC1溶液代替水玻璃溶液， 按上述方法制得微乳液^ 将 5ml渗透反应步骤所得的微乳液 A倒入三口烧瓶中， 水浴控温为 30°C。 用蠕动 泵将 lml微乳液 B加入渗透步骤所得的微乳液 A中， 45min滴加完毕， 得溶液 C。 将所得溶液 C 进行 24 小时透析处理， 以备下一步实验所用， 所用透析袋规格为 14000分子量。
用荧光分光光度计分别检测单独供体荧光染料 Atto550稀释液 (稀释液浓度是 lOOnmol/L)和前述聚合反应所得的溶液 C 中的荧光染料 Atto550 的荧光强度； Atto550单独供体的荧光强度是 3000，聚合反应后的荧光染料 Atto550供体的荧光 强度是 50， 计算出激发光波长是 514nm时 FRET效率 95%以上。
纳米颗粒的生物修饰： 称取 lml上述聚合反应制备的纳米荧光粒子溶液 C， 经 lml的 PBS缓冲液洗涤后， 加入 lml羊抗鼠 IgG多克隆抗体 (抗体生产商： Abeam, 商品序列号： ab98711)， 在室温下反应 30min， 经离心、 洗涤后得到抗体修饰的荧 光纳米粒子， 将其溶于 PBS缓冲液中 4°C保存备用。
按照免疫组化染色步骤操作 (封闭- 一抗- 二抗）， 将 FRET 荧光探针标记在
Hela细胞微管上。 具体步骤如下：
1： 将封闭液 (6%胎牛血清）加入固定的 Hela细胞， 固定的 Hela细胞的制备 方法为： 用 4% (V/V)的多聚甲醛处理细胞 20分钟。 在室温下封闭 1小时， 用 PBS 缓冲液洗涤。
2： 加入 200 μ 1—抗 (所述抗体为抗微管蛋白单克隆抗体，抗体浓度为： 10ηΜ， 抗体的生产商： santa cruz, 序列号： sc-5286)， 室温下反应 1小时。
3： 加入 100 μ ΐ二抗 (所述二抗是上述所得交联有纳米荧光颗粒的二抗， 多克 隆抗体浓度为 30ηΜ)， 室温下反应 1小时。
分别检测激发光波长是 514nm时不同激发光强度下 FRET过程饱和前供体的荧 光强度变化曲线和 FRET过程饱和后的供体荧光强度变化曲线， 比较这两个曲线的 变化， 获得该探针的非线性光学响应， 计算单独供体荧光分子的荧光强度与 FRET 分子对中供体荧光强度的比值，当比值发生变化时则发生 FRET饱和，从而获得 FRET 饱和的激发光阈值 450 μ ΐ 选取激发光强度 500 的激光进行激发， 开始成像， 得到超分辨图像， 其分辨率是 88nm， 如图 4所示。
实施例 4 Atto488-Atto647N探针标记的 Hela细胞微管的超分辨成像 构建高 FRET效率 Atto488-Atto647N荧光探针： 实验材料： 硅酸钠溶液 (水玻璃）（所述水玻璃模数是 3. 4， 波美度是 40)， 盐 酸 (浓度为 lmol/L)， 十二垸基硫酸钠 SDS， 甲苯， 正戊醇， 去离子水 (W)， 荧光染 料 Atto488-Atto647No
渗透反应： 取 0. lg表面活性剂 SDS (S)、 0. 2g荧光染料 (供体 Atto488和受体 Atto647N, 供体受体摩尔比 1 : 2)、 0. 2ml甲苯和事先配制好的 5ml水玻璃溶液放 入烧杯中， 超声混合均匀（超声波功率 50W， 混合时间 1分钟）， 得到初乳浊液， 滴 加正戊醇至体系突然透明， 即获得微乳液 A。
聚合反应： 用 lmol/L的 HC1溶液代替水玻璃溶液， 按上述方法制得微乳液^ 将 5ml渗透反应步骤所得的微乳液 A倒入三口烧瓶中， 水浴控温为 30°C。 用蠕动 泵将 lml微乳液 B加入渗透步骤所得的微乳液 A中， 45min滴加完毕， 得溶液 C。 将所得溶液 C 进行 24 小时透析处理， 以备下一步实验所用， 所用透析袋规格为 14000分子量。
用荧光分光光度计分别检测单独供体荧光染料 Atto488 稀释液 (稀释液浓度 是 lOOnmol/L)和前述聚合反应所得的溶液 C中的荧光染料 Atto488 的荧光强度； Atto488单独供体的荧光强度是 2500， 发生 FRET聚合反应后的供体的荧光强度是 50。 计算出激发光波长为 458nm时 FRET效率 90%以上。
纳米颗粒的生物修饰： 称取 lml上述聚合反应制备的纳米荧光粒子溶液 C， 经 lml的 PBS缓冲液洗涤后， 加入 lml羊抗鼠 IgG多克隆抗体 (抗体生产商： Abeam, 商品序列号： ab98711)， 在室温下反应 30min， 经离心、 洗涤后得到抗体修饰的荧 光纳米粒子， 将其溶于 PBS缓冲液中 4°C保存备用。
按照免疫组化染色步骤操作 (封闭- 一抗- 二抗）， 将 FRET 荧光探针标记在 Hela细胞微管上。 具体步骤如下：
1： 将封闭液 (6%胎牛血清）加入固定的 Hela细胞， 固定的 Hela细胞的制备 方法为： 用 4% (V/V)的多聚甲醛处理细胞 20分钟。 在室温下进行封闭 1小时， 用 PBS缓冲液洗涤。
2： 加入 200 μ ΐ—抗 (所述抗体为抗微管蛋白单克隆抗体，抗体浓度为： 10ηΜ， 抗体的生产商： santa cruz, 序列号： sc-5286)， 室温下反应 1 小时。
3： 加入 100 μ ΐ二抗 (所述的二抗是上述所得交联有纳米荧光颗粒的二抗， 多 克隆抗体浓度为 30ηΜ)， 室温下反应 1小时。
分别检测激发光波长为 458nm时不同激发光强度下 FRET过程饱和前供体的荧 光强度变化曲线和 FRET过程饱和后的供体荧光强度变化曲线， 比较这两个曲线的 变化， 获得该探针的非线性光学响应， 计算单独供体荧光分子的荧光强度与 FRET 分子对中供体荧光强度的比值，当比值发生变化时则发生 FRET饱和，从而获得 FRET 饱和的激发光阈值为 350 μ ϊ。 选取强度为 400 的激光进行激发， 开始成像， 得 到超分辨图像， 其分辨率是 69nm， 如图 5所示。
实施例 5 Alexa488-Cy3探针标记的 Hela细胞微管的超分辨成像
构建高 FRET效率的 Alexa488-Cy3荧光探针：
实验材料： 硅酸钠溶液 (水玻璃）（所述水玻璃模数是 3. 4， 波美度是 40)， 盐 酸 (浓度为 lmol/L)， 十二垸基硫酸钠 SDS， 甲苯， 正戊醇， 去离子水 (W)， 荧光染 料 Alexa488- Cy3。
渗透反应： 取 0. lg表面活性剂 SDS (S)、 0. 2g 荧光染料 (供体 Alexa488和受 体 Cy3， 供体受体摩尔比 1 : 2)、 0. 2ml 甲苯和事先配制好的 5ml水玻璃溶液放入 烧杯中， 超声混合均匀（超声波功率 50W， 混合时间 1分钟）， 得到初乳浊液， 滴加 正戊醇至体系突然透明， 即获得微乳液 A。
聚合反应： 用 lmol/L的 HC1溶液代替水玻璃溶液， 按上述方法制得微乳液^ 将 5ml渗透反应步骤所得的微乳液 A倒入三口烧瓶中， 水浴控温为 30°C。 用蠕动 泵将 lml微乳液 B加入渗透步骤所得的微乳液 A中， 45min滴加完毕， 得溶液 C。 将所得溶液 C 进行 24 小时透析处理， 以备下一步实验所用， 所用透析袋规格为 13000分子量。
用荧光分光光度计分别检测单独供体荧光染料 Alexa488稀释液 (稀释液浓度 是 lOOnmol/L)和前述聚合反应所得的溶液 C中的荧光染料 Alexa488的荧光强度； Alexa488单独供体的荧光强度是 2500， 发生 FRET时供体的荧光强度是 50， 计算 出激发光波长为 458nm时 FRET效率 90%以上。
纳米颗粒的生物修饰： 称取 lml上述聚合反应制备的纳米荧光粒子溶液 C， 经 lml的 PBS缓冲液洗涤后， 加入 lml羊抗鼠 IgG多克隆抗体 (抗体生产商： Abeam, 商品序列号： ab98711)， 在室温下反应 30min， 经离心、 洗涤后得到抗体修饰的荧 光纳米粒子， 将其溶于 PBS缓冲液中 4°C保存备用。
按照免疫组化染色步骤操作 (封闭- 一抗- 二抗）， 将 FRET 荧光探针标记在 Hela细胞微管上。 具体步骤如下：
1： 将封闭液 (6%胎牛血清）加入固定的 Hela细胞， 固定的 Hela细胞的制备 方法为： 用 4% (V/V)的多聚甲醛处理细胞 20分钟。 在室温下进行封闭 1小时， 用 PBS缓冲液洗涤。 2： 加入 200 μ ΐ—抗 (所述抗体为抗微管蛋白单克隆抗体，抗体浓度为： 10ηΜ， 抗体的生产商： santa cruz , 序列号： sc-5286)， 室温下反应 1小时。
3： 加入 100 μ ΐ二抗 (所述二抗是上述所得交联有纳米荧光颗粒的二抗， 多克 隆抗体浓度为 30ηΜ)， 室温下反应 1小时。
分别检测激发光波长为 458nm时不同强度下 FRET过程饱和前供体的荧光强度 变化曲线和 FRET过程饱和后的供体荧光强度变化曲线， 比较这两个曲线的变化， 获得该探针的非线性光学响应， 主要是通过计算单独供体荧光分子的荧光强度与 FRET分子对中供体荧光强度的比值， 当比值发生变化时则发生 FRET饱和， 从而获 得 FRET饱和的激发光阈值 400 μ Wo 选取强度为 450 μ W的激光进行激发， 开始成 像， 得到超分辨图像， 其分辨率为 125nm， 如图 6所示。 在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单 独引用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领域 技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利 要求书所限定的范围。

Claims

权 利 要 求
1. 一种基于荧光共振能量转移的超分辨成像方法， 其特征在于， 包括以下步 骤：
1) 用高 FRET效率的荧光探针标记待检样品， 所述的高 FRET效率的荧光探针 标记有 FRET分子对，所述的 FRET分子对包括第一荧光基团 (供体)和第二荧光基团 (受体）， 第一荧光基团能够向第二荧光基团发生荧光共振能量转移 (FRET)；
2) 采用激发光强度能够使步骤 1) 所述的 FRET分子对发生荧光共振能量转 移的激发光阈值， 进行激光扫描共聚焦显微镜成像。
2. 如权利要求 1所述的超分辨成像方法，其特征在于，所述的探针选自核酸、 抗体、 蛋白质分子、 有机小分子和无机分子中的一种或几种。
3. 如权利要求 1所述的超分辨成像方法， 其特征在于， 所述的 FRET分子对 具有 > 85% FRET效率。
4. 如权利要求 1所述的超分辨成像方法， 其特征在于， 所述的 FRET分子对 包括： CFP-YFP, Cy3-Cy5, Atto488- Atto540， FITC- Rhodamine, BFP- GFP， FITC- EITC，
Atto488- Atto647N， Atto550- Atto647N， Atto488- Atto590， Atto550- Atto655， Atto590- Atto655， CFP- dsRED , Alexa488- Alexa555， Alexa488- Cy3， YFP- TRITC 和 YFP-Cy3中的一种或几种。
5. 如权利要求 1所述的超分辨成像方法， 其特征在于， 所述的 FRET分子对 之间的距离小于 10nm。
6. 如权利要求 1所述的超分辨成像方法， 其特征在于， 所述的能够使步骤 2) 所述的 FRET分子对发生荧光共振能量转移的激发光阈值是指使 FRET分子对发生荧 光共振能量转移达到 FRET饱和的激发光强度， 称之为饱和激发光阈值。
7. 如权利要求 1所述的超分辨成像方法， 其特征在于， 通过以下方法获得所 述 FRET分子对的饱和激发光阈值：将单独的供体荧光分子和标记着 FRET分子对的 荧光探针分别置于激光扫描共聚焦显微镜下检测供体荧光分子的荧光随激发光强 变化的曲线， 比较两个曲线的变化， 发现在一定的激发光强时 FRET探针的供体荧 光发生了非线性的光学响应， 即在一定激发光阈值附近供体荧光发生了明显的增 力口， 达到了 FRET饱和， 该激发光阈值即为饱和激发光阈值。
8. 如权利要求 1所述的超分辨成像方法， 其特征在于， 步骤 2)所述的激发光 强度稍高于饱和激发光阈值， 激发光强度是 400 μ W〜500 μ W o
9. 如权利要求 1所述的超分辨成像方法， 其特征在于， 所述的激光扫描共聚 焦显微镜包括采用激光做光源的显微镜系统。
10. 如权利要求 9所述的超分辨成像方法， 其特征在于， 所述的激光扫描共 聚焦显微镜包括 Zessi LSM700, Leica TCS SP5II , Olypums FV500 或 NikonCl。
11. 如权利要求 6所述的超分辨成像方法， 其特征在于， 所述饱和激发光阈 值为 350〜450 μ W o
12. 如权利要求 6所述的超分辨成像方法， 其特征在于， 步骤 2)所述的激发 光强度高于饱和激发光阈值， 为饱和激发光阈值的 1. 1倍到 2倍。
13. 如权利要求 1 所述的超分辨成像方法， 其特征在于， 所述待检样品为生 物样品。
14. 如权利要求 1 所述的超分辨成像方法， 其特征在于， 所述超分辨成像包 括三维超分辨成像。
15. 如权利要求 1 所述的超分辨成像方法， 其特征在于， 所述超分辨成像方 法用于检测细胞内融合蛋白、 分子复合物或细胞器的状态。
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