CN107421931A

CN107421931A - A kind of super-resolution imaging method based on fluorescent bleach

Info

Publication number: CN107421931A
Application number: CN201710585804.6A
Authority: CN
Inventors: 樊春海; 邓素辉; 程亚; 陈建芳; 侯尚国
Original assignee: Shanghai Institute of Applied Physics of CAS
Current assignee: Shanghai Institute of Applied Physics of CAS
Priority date: 2017-07-18
Filing date: 2017-07-18
Publication date: 2017-12-01

Abstract

The present invention provides a kind of super-resolution imaging method based on fluorescent bleach, comprises the following steps：1) using the fluorochrome label sample with photobleaching characteristic；2) scanning type laser microscope is excited to focus the laser beam on the testing sample by mark using point, testing sample sends fluorescence through the excitation laser beam；3) fluorescence that the testing sample is sent is collected, obtains fluorescence intensity information；And 4) point by point scanning is carried out to sample directly to bleach sample in scanning process using the exciting light and certain sweep parameter of certain power, so that the valid pixel number collected is relatively reduced, so as to obtain less fluorescence spot, the ultrahigh resolution imaging for breaking through optical diffraction limit is directly obtained.The invention provides it is a kind of it is being realized on common laser flying-spot microscope, suitable for it is all with bleaching characteristic it is fluorescent dye, using the non-linear phenomena of fluorescent bleach come the super-resolution imaging method realized.

Description

A kind of super-resolution imaging method based on fluorescent bleach

Technical field

The present invention relates to the super-resolution far-field optics imaging field for breaking through optical diffraction limit, relate more specifically to a kind of base In the super-resolution imaging method of fluorescent bleach.

Background technology

Light microscope can carry out real-time monitored imaging with lossless to biological sample, be essential in biological study Key tool.And the development of light microscope also promotes the progress of life science.Intracellular organelle and albumen etc. Nanoscale is at, this requires microscopical resolution ratio to reach nanoscale and can be offered a clear explanation.Yet with The presence of optical diffraction causes the lateral resolution of light microscope to be limited in 200nm or so, and this just hinders people's utilization Light microscope carry out deeper into research.

Most in the late two decades, then broken using a variety of nonlinear optical effects, a series of microscopical appearance of super-resolution Limitation of the optical diffraction limit to resolution ratio.Such as by excite illumination light to be modulated realize super-resolution imaging by Swash launch loss microscopy (stimulated emission depletion microscopy, STED) and Structured Illumination shows Micro- art (Structured Illumination Microscopy, SIM)；By to unimolecule be accurately positioned realizing oversubscription Distinguish imaging random optical rebuild microscopy (Stochastic optical reconstruction microscopy, STORM) and photoactivation positions microscopy (Photoactivated localization microscopy, PALM) etc..

Although above super-resolution fluorescence microscope can reach the resolution ratio of tens nanometer, intracellular superelevation point is met The requirement of resolution imaging, but they are required for transforming instrument light path, are realized by complex system, to sample Prepared by product, fluorescence probe and user it is also proposed higher requirement, so as to limit widely using for super resolution technology.If Super-resolution imaging can be carried out directly on commercialized simple microscope can will greatly reduce the threshold of super-resolution imaging.

The content of the invention

It is an object of the invention to provide a kind of super-resolution imaging method based on fluorescent bleach, so as to solve in the prior art The resolution ratio of ordinary optical microscope is inadequate, and requirement of the existing most super-resolution fluorescence microscopes to equipment and technology The problem of universality caused by again too high is relatively low.

In order to solve the above-mentioned technical problem, the present invention uses following technical scheme：

A kind of super-resolution imaging method based on fluorescent bleach is provided, comprised the following steps：1) using has photobleaching The fluorochrome label testing sample of characteristic；2) scanning type laser microscope is excited to focus the laser beam to by glimmering using point On the testing sample of signal, testing sample sends fluorescence through the excitation laser beam；3) testing sample is collected to send Fluorescence, obtain fluorescence intensity information；And 4) sample is entered using the exciting light and certain sweep parameter of certain power Row point by point scanning with scanning process directly bleach sample so that the valid pixel number collected is relatively reduced, so as to obtain Less fluorescence spot is obtained, directly obtains the ultrahigh resolution imaging for breaking through optical diffraction limit.

According to method provided by the present invention, the progressively bleaching characteristic mainly using high power laser light to fluorescence molecule, And the fluorescence molecule nonlinear effect that fluorescence is suddenly quenched when bleaching, in scanning process by control laser power and Sweep parameter cause fluorescence molecule occur progressively bleach so that the valid pixel number collected is relatively reduced, so as to obtain compared with Small fluorescence spot, as shown in figure 1, the fluorescence spot that the method according to the invention obtains is obvious compared to common co-focusing imaging Diminish, therefore the ultrahigh resolution imaging for breaking through optical diffraction limit can be directly obtained.

Preferably, exciting light, less scanning step and longer single pixel of the step 4) including the use of higher-wattage Residence time carries out point by point scanning to sample.

Sample is marked using the fluorescence labeling probe with the fluorescent dye in the step 1).

Preferably, the higher-wattage be 10 times of common laser power and more than.

It is further preferred that the scanning step representative value is 10~20nm.

It is further preferred that single pixel residence time representative value is 10~20 μ s.

Wherein, 10nm scanning step, 10 μ s single pixel residence time are used to most typically.

It should be understood, however, that according to the difference of the fluorescent dye bleaching characteristic on fluorescence labeling probe, it is specific to swash Luminous power, scanning step and single pixel residence time are not limited in above range, are carried out as long as can realize to sample Sample is directly bleached during point by point scanning so that the valid pixel number collected is relatively reduced, less so as to obtain Fluorescence spot, directly obtain the ultrahigh resolution imaging for breaking through optical diffraction limit.

Heretofore described fluorescence labeling probe is the conventional probe in this area, wherein the probe is preferably comprised core One or more in acid, albumen, antibody, inorganic molecule, organic molecule.

The fluorescent dye marked on heretofore described probe is fluorescent dye commonly used in the art, including but is not limited to In：FITC、Cy3、Cy5、Atto 425、Atto 465、Atto488、Atto514、Atto550、Atto594、Atto633、 One or more in Atto647, Atto740, Alexa405, Alexa488, Alexa546, Alexa555 and Alexa633.

The described method with fluorescence probe mark measuring samples is this area routine techniques.The labeling method is preferably Comprise the following steps：Laser scanning microscope observation can be carried out after cell is marked with described fluorescence probe.Enter The method of line flag is the ordinary skill in the art, is well known to those skilled in the art and grasps.The labeling method compared with It is to be combined probe with sample by the ImmunohistochemistryMethods Methods in cytobiology technology goodly.Except ImmunohistochemistryMethods Methods it Outside, the combination of probe and sample can be also realized by expressing the mode such as fluorescent protein labeling and the coupling of label protein covalent bond. The ImmunohistochemistryMethods Methods are preferably comprised following steps：Closing, primary antibody is marked, mark secondary antibody, wherein described secondary antibody is mark Note has the secondary antibody of fluorescence molecule.

According to method provided by the invention, be can be achieved by a common laser scanning co-focusing microscope to biology The super-resolution imaging of sample.Laser scanning microscope of the present invention is this area conventional laser scanning confocal microscope system System.The manufacturer of laser scanning co-focusing microscope of the present invention and model are preferably comprised：Zessi LSM700, Leica TCS SP5, Leica TCS SP8, Olypums FV500 or NikonC1.

Although it is to have before this to be used for surmounting diffraction limit imaging using fluorescence photobleaching nonlinear optical effect Report, such as bleach/flash position finding microscope (bleaching/blinking assisted localization Microscopy (Balm)) method etc..But these methods are that the mode being imaged using CCD faces obtains image, utilize fluorescent bleach The difference of the caused single molecule signals in front and rear two field pictures, super resolution image is reconstructed by the algorithm in later stage, it is clear that should Method complex operation, take, add the cost of super-resolution imaging.Super resolution technology proposed by the present invention and the technology are completely not Together, image-forming principle, imaging mode and data processing method differ.

It is as follows compared to prior art, beneficial effects of the present invention：

According to method provided by the present invention, realize first on the common laser flying-spot microscope of no any transformation Super-resolution imaging is carried out to different detection objects, is adapted to all laboratory conventional fluorescent dyestuffs, and the super-resolution imaging side Method have it is simple, easily and fast the advantages of, this method takes full advantage of the prior art device in laboratory, considerably reduces The cost of super-resolution imaging technology.

It is obviously improved using the fluorescent bleach scanning imagery resolution ratio of the present invention, obtains the super-resolution for breaking through diffraction limit Effect (<200nm), average mark resolution is about 80nm, and minimum resolution is about 60nm, and relatively existing common laser scanning is aobvious The resolution ratio of micro mirror improves 3 times.

Super-resolution imaging method of the present invention based on fluorescent bleach can be widely applied to biological study field. Described application field is preferably comprised：To the super-resolution imaging of the biological samples such as cell, microorganism, analyzing proteins and albumen it Between interaction process；Subcellular fraction morphosis is studied, analysis drug molecule is on organelle influence and drug molecule or receives Interaction process between rice structure and organelle.

In a word, the invention provides it is a kind of it is being realized on common laser flying-spot microscope, suitable for it is all have drift White characteristic it is fluorescent dye, using the non-linear phenomena of fluorescent bleach come the super-resolution imaging method realized.

Brief description of the drawings

Fig. 1 is the principle schematic of the inventive method, wherein, A is that the point of common co-focusing imaging (Confocal) expands Function distribution is dissipated, B is the imaging point spread function distribution based on fluorescent bleach (Bleaching), and compared to A, B is in a dimension Fluorescence spot size be obviously reduced；

Fig. 2 is that the Alexa 405 for having used embodiment 1 marks 40nm fluorescence beads to carry out the result of fluorescent bleach imaging Comparison diagram, wherein A are imaged for common laser Laser Scanning Confocal Microscope, and B is to have used the imaging after fluorescent bleach imaging method, takes figure A bead in B analyzes its center fluorescence intensity distribution, half obtained using Gaussian function fitting fluorescence intensity profile High overall with (resolution ratio), as shown at c；

Fig. 3 is the result pair for having used the Alexa 405 of embodiment 2 to mark Hela cellular microtubules to carry out fluorescent bleach imaging Than figure, wherein A is imaged for common laser Laser Scanning Confocal Microscope, and B is to have used the imaging after fluorescent bleach imaging method；Along figure A The distribution of its fluorescence intensity is analyzed with a micro-pipe of white linear mark in B, is distributed using Gaussian function fitting fluorescence intensity bent The full width at half maximum (resolution ratio) that line obtains, is respectively displayed in figure C and D, compared to the microscopical resolution of plain scan in figure C Rate (282nm), the resolution ratio (figure D, 86nm) of the ultra-resolution method based on fluorescent bleach imaging improve plain scan microscope point Nearly 3.3 times of resolution；

Fig. 4 is the result pair for having used the Atto 488 of embodiment 3 to mark Hela cellular microtubules to carry out fluorescent bleach imaging Than figure, wherein A is imaged for common laser Laser Scanning Confocal Microscope, and B is to have used the imaging after fluorescent bleach imaging method.In micro-pipe Close quarters, the distribution of its fluorescence intensity of white line analysis, is respectively displayed in figure C and D along figure A and B, shows the present invention's Imaging method has clearly distinguished intensive micro-pipe distribution, and resolution capability is higher, as shown in Figure 4 D；

Fig. 5 is the result pair for having used the Alexa 514 of embodiment 4 to mark Hela cellular microtubules to carry out fluorescent bleach imaging Than figure, wherein A is imaged for common laser Laser Scanning Confocal Microscope, and B is to have used the imaging after fluorescent bleach imaging method；Take along The micro-pipe of white linear mark analyzes the distribution of its fluorescence intensity, half obtained using Gaussian function fitting fluorescence intensity profile High overall with (resolution ratio), as shown in C figures, wherein Grey curves are to be distributed in A figures along the micro-pipe fluorescence intensity of white linear mark, Black curve is to be distributed in B figures along the micro-pipe fluorescence intensity of white linear mark；

Fig. 6 is the result pair for having used the Alexa 633 of embodiment 5 to mark Hela cellular microtubules to carry out fluorescent bleach imaging Than figure, wherein A is imaged for common laser Laser Scanning Confocal Microscope, and B is to have used the imaging after fluorescent bleach imaging method；Take figure A The distribution of its fluorescence intensity is analyzed with the micro-pipe marked in white box in B, uses Gaussian function fitting fluorescence intensity profile Obtained full width at half maximum (resolution ratio), is respectively displayed in figure C and D.

Embodiment

Below in conjunction with specific embodiment, the present invention will be further described.It should be understood that following examples are merely to illustrate this Invention is not for limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, according to conventional side Method and condition, or selected according to catalogue.

Agents useful for same and raw material of the present invention are commercially available.

Described room temperature is the conventional room temperature in this area, generally 15~25 DEG C.

The primary antibody of the cellular microtubules of rabbit-anti people is purchased from Abcam companies.

The goat-anti rabbit secondary antibody for indicating fluorescent dye is purchased from Invitrogen companies.

Laser confocal microscope Lecia TCS SP8 are purchased from Leca company.

Hela cells are purchased from Shanghai Inst. of Life Science, CAS cell resource center, its commodity Serial No. Tch20。

Embodiment 1:

The fluorescence beads super-resolution imaging of Alexa405 marks.

1.1 by the single-stranded (sequences of Alexa405DNA：Alexa 405-5'AAAAAACAGGACCAGAAAAAA-3'biotin, TAKARA 100 μM) are diluted to using ultra-pure water.

The polystyrene sphere (Invitrogen) for being marked with biological Avidin is diluted to 100 μM by 1.2 using ultra-pure water.

1.3 toward adding 97 μ L PBS in 100 μ L centrifuge tubes, and adds the single-stranded 2 μ L of DNA just diluted and be marked with life The μ L of polystyrene sphere 1 of thing Avidin.Its 25 DEG C are incubated 1 hour.

1.4 is inner by the solution being incubated addition 100KD super filter tubes (Millipore), adds 300 microlitres of PBS.

1.5 are put it into centrifuge and are centrifuged 2 minutes with 2700g rotating speed.Take out centrifuge tube after outwell filter it is molten Liquid, then toward 400 microlitres of PBS of addition in centrifuge tube, centrifuged with 2700g rotating speed.The liquid after centrifugation is taken out after in triplicate.

1.6 are put into circular cover glass in 12 porocyte culture plates, add 1 milliliter of 0.1M hydrofluoric acid solution.It is incubated Hydrofluoric acid solution is washed out after 30 seconds, and using PBS cover glass three times.

Fluorescence beads solution after centrifugation is diluted to 1 milliliter of volume by 1.7, takes 500 microlitres to be added on cover glass, is incubated 2 Hour.

Slide is cleaned up and dried using lens wiping paper by 1.8 using ultra-pure water, and 10 microlitres of fluorescence adhesive is added dropwise.

1.9 using tweezers by with fluorescence beads cover glass take out, be buckled in a manner of former upper surface is directed downwardly added it is glimmering On the wave carrier piece of light adhesive.

1.10 can be imaged sample lucifuge room temperature for 12 hours under the microscope afterwards.

1.11 choose suitable laser power and scan frequency, wherein, scan frequency is 100Hz/ rows, when single pixel stops Between be 10 μ s, scanning step 10nm.Excitation light power is 50 μ W, fluorescent bleach imaging is carried out to sample, referring to Fig. 2.By Fig. 2 Understand, be imaged (Fig. 2A) compared to common confocal scanning, be obviously improved using fluorescent bleach scanning imagery (Fig. 2 B) resolution ratio, Fig. 2 C center fluorescence intensity curves distribution shows that resolution ratio be 62nm, obtain breakthrough diffraction limit super-resolution effect (< 200nm)。

Embodiment 2:The cellular microtubules fluorescent bleach super-resolution imaging that Alexa 405 is marked

2.1 are put into 12 porocyte culture plates18mm dome slides, about 60,000 Hela cells are spread on slide.

2.2 are cleaned 3 times using 37 DEG C of 1 × phosphate buffer (PBS) 1mL, every time between at intervals of five minutes.

2.3, which add the paraformaldehydes of 1mL 4% (w/v)+4% sucrose (w/v), fixes 15min.

2.4 are cleaned 3 times using 1 × PBS, per minor tick 5min.

Triton X-100 are diluted to 0.25% (w/v) by 2.5 using 6% bovine serum albumin(BSA) (w/v).Add 500 μ L 0.25%Triton X-100 are incubated 45 minutes into 12 orifice plates.

2.6 add 400mL primary antibodies (10nM), are incubated 1h.

2.7 are cleaned three times using 1 × PBS, every minor tick 10 minutes.

2.8 addition 400mL indicate Alexa 405 secondary antibody, are incubated 45min.

2.9 are cleaned three times using 1 × PBS, every minor tick 10 minutes.

2.10 paster.

2.11 choose suitable laser power and scan frequency, wherein, scan frequency is 100Hz/ rows, when single pixel stops Between be 10 μ s, scanning step 10nm, excitation light power is 50 μ W, to sample carry out fluorescent bleach imaging, referring to Fig. 3.By Fig. 3 Understand, be imaged (Fig. 3 A) compared to common confocal scanning, be obviously improved using fluorescent bleach scanning imagery (Fig. 3 B) resolution ratio, Single micro-pipe fluorescence intensity distribution shows that resolution ratio be 86nm (Fig. 3 D), obtain breakthrough diffraction limit super-resolution effect (< 200nm).Compared to the microscopical resolution ratio of plain scan (282nm) in Fig. 3 C, the super-resolution side based on fluorescent bleach imaging The resolution ratio (Fig. 3 D, 86nm) of method improves nearly 3.3 times of the plain scan resolution of microscope.

Embodiment 3:The cellular microtubules fluorescent bleach super-resolution imaging that Atto 488 is marked

3.1 are put into φ 18mm dome slides in 12 porocyte culture plates, and about 60,000 Hela cells are spread on slide.

3.2 are cleaned 3 times using 37 DEG C of 1 × phosphate buffer (PBS) 1mL, every time between at intervals of five minutes.

3.3, which add the paraformaldehydes of 1mL 4% (w/v)+4% sucrose (w/v), fixes 15min.

3.4 are cleaned 3 times using 1 × PBS, per minor tick 5min.

Triton X-100 are diluted to 0.25% (w/v) by 3.5 using 6% bovine serum albumin(BSA) (w/v).Add 500 μ L 0.25%Triton X-100 are incubated 45 minutes into 12 orifice plates.

3.6 add 400mL primary antibodies (10nM), are incubated 1h.

3.7 are cleaned three times using 1 × PBS, every minor tick 10 minutes.

3.8 addition 400mL indicate Atto 488 secondary antibody, are incubated 45min.

3.9 are cleaned three times using 1 × PBS, every minor tick 10 minutes.

3.10 paster.

3.11 choose suitable laser power and scan frequency, wherein, scan frequency is 100Hz/ rows, when single pixel stops Between be 10 μ s, scanning step is arranged to 20nm, and it is 400 μ W to use excitation light power, to sample carry out fluorescent bleach imaging, ginseng See Fig. 4.As shown in Figure 4, (Fig. 4 A) is imaged compared to common confocal scanning, is differentiated using fluorescent bleach scanning imagery (Fig. 4 B) Rate is obviously improved, and has been told two micro-pipes (Fig. 4 D) that distance is 144nm, has been obtained the super-resolution effect for breaking through diffraction limit (<200nm)。

Embodiment 4:The cellular microtubules fluorescent bleach super-resolution imaging that Alexa 514 is marked

4.1 are put into φ 18mm dome slides in 12 porocyte culture plates, and about 60,000 Hela cells are spread on slide.

4.2 are cleaned 3 times using 37 DEG C of 1 × phosphate buffer (PBS) 1mL, every time between at intervals of five minutes.

4.3, which add the paraformaldehydes of 1mL 4% (w/v)+4% sucrose (w/v), fixes 15min.

4.4 cleaned 3 times using 1 × PBS, per minor tick 5min.

Triton X-100 are diluted to 0.25% (w/v) by 4.5 using 6% bovine serum albumin(BSA) (w/v).Add 500 μ L 0.25%Triton X-100 are incubated 45 minutes into 12 orifice plates.

4.6 add 400mL primary antibodies (10nM), are incubated 1h.

4.7 are cleaned three times using 1 × PBS, every minor tick 10 minutes.

4.8 addition 400mL indicate Alexa 405 secondary antibody, are incubated 45min.

4.9 are cleaned three times using 1 × PBS, every minor tick 10 minutes.

4.10 paster.

4.11 choose suitable laser power and scan frequency, wherein.Scan frequency is 100Hz/ rows, when single pixel stops Between be 12 μ s, scanning step is arranged to 10nm, and it is 400 μ W to use excitation light power, to sample carry out fluorescent bleach oversubscription Imaging is distinguished, referring to Fig. 5.As shown in Figure 5, (Fig. 5 A) is imaged compared to common confocal scanning, uses fluorescent bleach scanning imagery (Fig. 5 B) resolution ratio is obviously improved, and is taken the fluorescence intensity of single micro-pipe to be distributed and is shown that resolution ratio is 137nm (Fig. 5 C), obtains prominent Broken diffraction limit super-resolution effect (<200nm).

Embodiment 5：The cellular microtubules fluorescent bleach super-resolution imaging that Alexa 633 is marked

5.1 are put into φ 18mm dome slides in 12 porocyte culture plates, and about 60,000 Hela cells are spread on slide.

5.2 are cleaned 3 times using 37 DEG C of 1 × phosphate buffer (PBS) 1mL, every time between at intervals of five minutes.

5.3, which add the paraformaldehydes of 1mL 4% (w/v)+4% sucrose (w/v), fixes 15min.

5.4 are cleaned 3 times using 1 × PBS, per minor tick 5min.

Triton X-100 are diluted to 0.25% (w/v) by 5.5 using 6% bovine serum albumin(BSA) (w/v).Add 500 μ L 0.25%Triton X-100 are incubated 45 minutes into 12 orifice plates.

5.6 add 400mL primary antibodies (10nM), are incubated 1h.

5.7 are cleaned three times using 1 × PBS, every minor tick 10 minutes.

5.8 addition 400mL indicate Alexa 633 secondary antibody, are incubated 45min.

5.9 are cleaned three times using 1 × PBS, every minor tick 10 minutes.

5.10 paster.

5.11 choose suitable laser power and scan frequency, wherein, scan frequency is 100Hz/ rows, when single pixel stops Between be 10 μ s, scanning step is arranged to 10nm, and it is 400 μ W to use excitation light power, to sample carry out fluorescent bleach imaging, ginseng See Fig. 6.It will be appreciated from fig. 6 that being imaged (Fig. 6 A) compared to common confocal scanning, the imaging method scanned based on fluorescent bleach is used Super-resolution imaging (Fig. 6 B) is realized, the fluorescence intensity distribution of the single micro-pipe of extracting waste collimation mark note shows that resolution ratio substantially changes Kind, resolution ratio be 130nm (Fig. 6 D), obtain breakthrough diffraction limit super-resolution effect (<200nm).

Above-described, only presently preferred embodiments of the present invention is not limited to the scope of the present invention, of the invention is upper Stating embodiment can also make a variety of changes.What i.e. every claims and description according to the present patent application were made Simply, equivalent changes and modifications, the claims of patent of the present invention are fallen within.The not detailed description of the present invention is Routine techniques content.

Claims

1. a kind of super-resolution imaging method based on fluorescent bleach, it is characterised in that comprise the following steps：

1) using the fluorochrome label testing sample with photobleaching characteristic；

2) scanning type laser microscope is excited to focus the laser beam on the testing sample by fluorescence labeling using point, it is to be measured Sample sends fluorescence through the excitation laser beam；

3) fluorescence that the testing sample is sent is collected, obtains fluorescence intensity information；And

4) point by point scanning is carried out with scanning process to sample using the exciting light and certain sweep parameter of certain power Directly bleach sample so that the valid pixel number collected is relatively reduced, so as to obtain less fluorescence spot, directly obtains Break through the ultrahigh resolution imaging of optical diffraction limit.

2. super-resolution imaging method according to claim 1, it is characterised in that the step 4) is including the use of higher-wattage Exciting light, less scanning step and longer single pixel residence time to sample carry out point by point scanning.

3. super-resolution imaging method according to claim 2, it is characterised in that the higher-wattage is common laser power 10 times and more than.

4. super-resolution imaging method according to claim 2, it is characterised in that the scanning step is 10~20nm.

5. super-resolution imaging method according to claim 2, it is characterised in that the single pixel residence time is 10~20 μs。

6. super-resolution imaging method according to claim 1, it is characterised in that described glimmering using having in the step 1) Sample is marked the probe of photoinitiator dye.

7. super-resolution imaging method according to claim 6, it is characterised in that the probe includes nucleic acid, albumen, resisted One or more in body, inorganic molecule, organic molecule.

8. super-resolution imaging method according to claim 1, it is characterised in that the fluorescent dye include FITC, Cy3, Cy5、Atto 425、Atto 465、Atto488、Atto514、Atto550、Atto594、Atto633、Atto647、 One or more in Atto740, Alexa405, Alexa488, Alexa546, Alexa555 and Alexa633.

9. super-resolution imaging method according to claim 1, it is characterised in that the point excites scanning type laser microscope Scanning confocal microscope system including doing light source using laser.

10. super-resolution imaging method according to claim 1, it is characterised in that described to be swept using laser light source Retouch Laser Scanning Confocal Microscope include Zessi LSM700, Leica TCS SP5, Leica TCS SP8, Olypums FV500 or NikonC1。