CN103575716A - Magnetic field regulated and controlled super-resolution fluorescence imaging method - Google Patents
Magnetic field regulated and controlled super-resolution fluorescence imaging method Download PDFInfo
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- CN103575716A CN103575716A CN201310571202.7A CN201310571202A CN103575716A CN 103575716 A CN103575716 A CN 103575716A CN 201310571202 A CN201310571202 A CN 201310571202A CN 103575716 A CN103575716 A CN 103575716A
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Abstract
The invention relates to a magnetic field regulated and controlled super-resolution fluorescence imaging method, relates to the technical field of optical microscope imaging measurement methods and solves the problems of insufficient switching times and insufficient emitted photons of an existing fluorescence probe applied to a super-resolution imaging method. According to the super-resolution fluorescence imaging method in which a magnetic field is additionally arranged to influence light emission of a probe, the outer magnetic field is applied near a sample to be measured, which is marked with the fluorescence probe, and simultaneously, data acquisition of super-resolution imaging is carried out; acquired data is analyzed by utilizing software. By the magnetic field regulated and controlled super-resolution fluorescence imaging method disclosed by the invention, the number of the emitted photons of the fluorescence probes such as dye is increased, resolution of super-resolution imaging is improved and the super-resolution imaging method is further developed.
Description
Technical field
The present invention relates to optical microscope imaging measurement method and technology field, be specifically related under a kind of externally-applied magnetic field condition, the super-resolution fluorescence formation method of magnetic field regulation and control.
Background technology
The invention of super-resolution fluorescence microtechnic has broken through diffraction limit, makes resolution can reach tens nanometers, thereby can on single molecules level, observe well and research biological process.(G.Patterson,M.Davidson,S.Manley,and?J.L.Schwartz,Superresolution?Imaging?using?Single-Molecule?Localization,Annual?Review?of?Physical?Chemistry,2010(61):345–367)。
Super-resolution imaging method based on unimolecule location in prior art, as random optics is rebuild microscope (STORM), photoactivation position finding microscope (PALM) etc., with its remarkable resolution characteristic, the superiority such as simple device is being brought into play more and more important effect in biological study relatively.The super-resolution imaging method of STORM and PALM has following step: first using can be at fluorescent dye or the fluorescent protein labeling sample of fluorescence state/dark state conversion, at any one time, in imaging process, fluorescence probe in most of samples remains on dark state, only has random sub-fraction to be converted to fluorescence state; Luminous fluorescence probe is changed by light or photobleaching is dark state afterwards; Another fraction fluorescence probe is fluorescence state by random transition subsequently; And so forth, make the independent imaging of molecule that is labeled; Finally, by all imaging accumulations, finally reconstitute super-resolution imaging.
The resolution of above-mentioned STORM and PALM super-resolution imaging method and the photochemistry/physical property of fluorescence probe (as the photon number of conversion cycle number of times, each switching emission, contrast etc.) are in close relations.Desirable fluorescence probe should have each conversion can send a large amount of photon number, the very high character of contrast of fluorescence state/dark state.But be widely used at present fluorescence probe (as spent cyanine type dye etc.) the circulation conversion times of the super-resolution imaging methods such as STORM and change the photon number of sending at every turn limited, thereby limited imaging resolution, and then limited further developing of super-resolution imaging method.
Summary of the invention
In order to overcome existing fluorescence probe conversion times and the few problem of utilizing emitted light subnumber that is applied to super-resolution imaging method, the invention provides that a kind of by externally-applied magnetic field, to regulate and control fluorescence probe luminous, the super-resolution fluorescence formation method of magnetic field regulation and control.
The present invention is that the technical scheme that adopts of technical solution problem is as follows:
A super-resolution fluorescence formation method for regulation and control, the method has following steps:
Step 1, use fluorescence probe mark testing sample, be layered on them on microslide, adds imaging damping fluid, covered mounting;
Step 2, near testing sample, apply an external magnetic field, control the size and Orientation of external magnetic field;
Step 3, to having modified the testing sample of fluorescence probe, carry out the data acquisition of super-resolution imaging;
Step 4, by the data analysis collecting.
In technique scheme, described fluorescence probe is: organic molecule fluorescent dye or fluorescin.
In technique scheme, described organic molecule fluorescent dye is: flower cyanines class or rhodamine or piperazine class.
In technique scheme, described organic molecule fluorescent dye is: Cy3, Cy5, Alexa Fluor 647, Alexa Fluor 532 or ATTO 655.
In technique scheme, fluorescin is: mEOS2 or GFP.
In technique scheme, imaging damping fluid contains mercaptoethanol, hydrogen peroxidase and glucose oxidase.
In technique scheme, testing sample is: albumen, DNA or living cells.
In technique scheme, the external magnetic field in step 2 is produced by hot-wire coil, or is produced by magnet.
In technique scheme, described step 3 specifically comprises:
The Ear Mucosa Treated by He Ne Laser Irradiation fluorescence probe that utilizes laser system to produce, make its by ground state transition to excited state, send fluorescence; The signal that fluorescent dye flicker produces is gathered and is sent to computer by CCD.
In technique scheme, the external magnetic field in described step 2 is for being less than 0.1T.
The invention has the beneficial effects as follows: by applying external magnetic field around at sample, can carry out modulation to the photophysical properties such as utilizing emitted light subnumber of the conversion times of the fluorescence probes such as dyestuff, each conversion, and then affect the number of super-resolution reconstruct map point.When applying a very little external magnetic field (magnetic field intensity is less than 0.1T), the number showed increased of super-resolution reconstruct map point.The resolution that this can improve super-resolution imaging, makes further developing of super-resolution imaging method.
The super-resolution fluorescence formation method of magnetic field of the present invention regulation and control can be for improving the super-resolution imaging resolution of albumen, DNA and the living cells etc. of fluorescent dye/fluorescent protein labeling.
Accompanying drawing explanation
Fig. 1 is the STORM imaging schematic diagram of prior art.
Fig. 2 is the device schematic diagram of the super-resolution fluorescence formation method of magnetic field of the present invention regulation and control.
Fig. 3 is the molecular structure of Alexa Fluor 647.
Fig. 4 is the molecular structure of Alexa Fluor 532.
Fig. 5 is 120G (12mT), the STORM super-resolution imaging figure under the externally-applied magnetic field that the right-hand rule makes progress (the super-resolution imaging restructuring graph of monodispersed Alexa Fluor 647).
Fig. 6 is the histogram that the anchor point number corresponding with Fig. 5 distributes.
Fig. 7 is under the same terms corresponding with Fig. 5, the STORM super-resolution imaging figure while there is no magnetic field (the super-resolution imaging restructuring graph of monodispersed Alexa Fluor 647).
Fig. 8 is the histogram that the anchor point number corresponding with Fig. 7 distributes.
Fig. 9 is 900G (90mT), the STORM super-resolution imaging figure under the externally-applied magnetic field that the right-hand rule makes progress (the super-resolution imaging restructuring graph of monodispersed Alexa Fluor 647).
Figure 10 is the histogram that the anchor point number corresponding with Fig. 9 distributes.
Figure 11 is under the same terms corresponding with Fig. 9, the STORM super-resolution imaging figure while there is no magnetic field (the super-resolution imaging restructuring graph of monodispersed Alexa Fluor 647).
Figure 12 is the histogram that the anchor point number corresponding with Figure 11 distributes.
Figure 13 is 100G (10mT), the STORM super-resolution imaging figure under the externally-applied magnetic field that the right-hand rule makes progress (the super-resolution imaging restructuring graph of monodispersed Alexa Fluor 532).
Figure 14 is the histogram that the anchor point number corresponding with Figure 13 distributes.
Figure 15 is under the same terms corresponding with Figure 13, the STORM super-resolution imaging figure while there is no magnetic field (the super-resolution imaging restructuring graph of monodispersed Alexa Fluor 532).
Figure 16 is the histogram that the anchor point number corresponding with Figure 15 distributes.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail.
As shown in Figure 2, the super-resolution fluorescence formation method of magnetic field regulation and control, the method has following steps:
Step 1, first, with fluorescence probe (as fluorescent dye or fluorescin) mark testing sample, is layered on them on microslide, adds imaging damping fluid, covered mounting;
Step 2, near testing sample, apply an external magnetic field, for example, can twine some turn coil, coil two ends connect respectively positive pole and the negative pole of direct supply, connect direct supply, at this moment at sample, just have an external magnetic field around.By changing the number of turn of coil or the size of electric current, control the size of external magnetic field, by changing coil winding mode, control the direction of external magnetic field;
Step 3, experiment parameter is set, to having modified the testing sample of fluorescence probe, carries out the data acquisition of super-resolution imaging;
Step 4, the data that collect are utilized to related software analysis.
In above-mentioned step 1, imaging damping fluid mainly contains mercaptoethanol, hydrogen peroxidase and glucose oxidase, and their effect is respectively extend the time of fluorescent dye in fluorescence state and remove the oxygen in system.
In above-mentioned step 1, test substance comprises the unimolecule of the available super-resolution fluorescence microscopic examination such as albumen, DNA or cell, fluorescence probe comprises and can be used for the microscopical organic molecule dyestuff of super-resolution fluorescence and fluorescin etc., as Cy3, Cy5, Alexa Fluor 647, Alexa Fluor 532 and GFP etc.The molecular structure of Alexa Fluor 647 and Alexa Fluor 532 as shown in Figures 3 and 4.
In above-mentioned step 2, external magnetic field both can be produced by near the conductive coil being wrapped in sample, also can be produced by near permanent magnet sample.
In above-mentioned step 3, super-resolution imaging system is mainly comprised of laser, microscope, CCD etc., Ear Mucosa Treated by He Ne Laser Irradiation fluorescence probe, make its by ground state transition to excited state, send fluorescence.The signal that fluorescent dye flicker produces is gathered and is sent to computer by CCD.
In above-mentioned step 4, related software analysis refers to and utilizes software analysis method to obtain super-resolution imaging restructuring graph, heavier structure map point number, determine imaging resolution.
The super-resolution fluorescence formation method of magnetic field of the present invention regulation and control for all kinds of unimolecule materials of fluorescence probe mark, the laser of each wavelength is all applicable.
Embodiment 1120G (12mT), the distribution situation research of monodispersed Alexa Fluor647 on slide under the external magnetic field condition that the right-hand rule makes progress.
Print is fixed on objective table, opens laser it is irradiated on sample, bring into focus, time shutter and photograph number.A selected region, carries out image acquisition.The data that collect are utilized to related software analysis.
As shown in Fig. 5-8, experimental result shows that advantage of the present invention is: under (1) the same terms, apply after 120G external magnetic field, dyestuff Alexa Fluor 647 flicker increases, thereby makes the super-resolution reconstruct map point that obtains more.(2) can improve the resolution of super-resolution imaging, super-resolution imaging method is further developed.
Embodiment 2900G (90mT), the distribution situation research of monodispersed Alexa Fluor 647 on slide under the external magnetic field condition that the right-hand rule makes progress.
Print is fixed on objective table, opens laser it is irradiated on sample, bring into focus, time shutter and photograph number.A selected region, carries out image acquisition.The data that collect are utilized to related software analysis.
As shown in Fig. 9-12, experimental result shows that advantage of the present invention is: under (1) the same terms, apply after 900G external magnetic field, dyestuff Alexa Fluor 647 flicker increases, thereby makes the super-resolution reconstruct map point that obtains more.(2) can improve the resolution of super-resolution imaging, super-resolution imaging method is further developed.
Embodiment 3100G (10mT), the distribution situation research of monodispersed Alexa Fluor 532 on slide under the external magnetic field condition that the right-hand rule makes progress.
Print is fixed on objective table, opens laser it is irradiated on sample, bring into focus, time shutter and photograph number.A selected region, carries out image acquisition.The data that collect are utilized to related software analysis.
As shown in Figure 13-16, experimental result shows that advantage of the present invention is: under (1) the same terms, apply after 100G external magnetic field, dyestuff Alexa Fluor 532 flicker increases, thereby makes the super-resolution reconstruct map point that obtains more.(2) can improve the resolution of super-resolution imaging, super-resolution imaging method is further developed.
Obviously, above-described embodiment is only for example is clearly described, and the not restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without also giving all embodiments.And the apparent variation of being extended out thus or change are still among the protection domain in the invention.
Claims (10)
1. a super-resolution fluorescence formation method for magnetic field regulation and control, is characterized in that, the method has following steps:
Step 1, use fluorescence probe mark testing sample, be layered on them on microslide, adds imaging damping fluid, covered mounting;
Step 2, near testing sample, apply an external magnetic field, control the size and Orientation of external magnetic field;
Step 3, to having modified the testing sample of fluorescence probe, carry out the data acquisition of super-resolution imaging;
Step 4, by the data analysis collecting.
2. the super-resolution fluorescence formation method of magnetic field according to claim 1 regulation and control, is characterized in that, described fluorescence probe is: organic molecule fluorescent dye or fluorescin.
3. the super-resolution fluorescence formation method of magnetic field according to claim 2 regulation and control, is characterized in that, described organic molecule fluorescent dye is: flower cyanines class or rhodamine or piperazine class.
4. the super-resolution fluorescence formation method of magnetic field according to claim 3 regulation and control, is characterized in that, described organic molecule fluorescent dye is: Cy3, Cy5, Alexa Fluor 647, Alexa Fluor 532 or ATTO655.
5. the super-resolution fluorescence formation method of magnetic field according to claim 2 regulation and control, is characterized in that, fluorescin is: mEOS2 or GFP.
6. the super-resolution fluorescence formation method of magnetic field according to claim 1 regulation and control, is characterized in that, imaging damping fluid contains mercaptoethanol, hydrogen peroxidase and glucose oxidase.
7. the super-resolution fluorescence formation method of magnetic field according to claim 1 regulation and control, is characterized in that, testing sample is: albumen, DNA or living cells.
8. according to the super-resolution fluorescence formation method of the magnetic field regulation and control described in any one in claim 1 to 7, it is characterized in that, the external magnetic field in step 2 is produced by hot-wire coil, or is produced by magnet.
9. according to the super-resolution fluorescence formation method of the magnetic field regulation and control described in any one in claim 1 to 7, it is characterized in that, described step 3 specifically comprises:
The Ear Mucosa Treated by He Ne Laser Irradiation fluorescence probe that utilizes laser system to produce, make its by ground state transition to excited state, send fluorescence; The signal that fluorescent dye flicker produces is gathered and is sent to computer by CCD.
10. according to the super-resolution fluorescence formation method of the magnetic field regulation and control described in any one in claim 1 to 7, it is characterized in that, the external magnetic field in described step 2 is for being less than 0.1T.
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Cited By (2)
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| CN104515760A (en) * | 2014-12-17 | 2015-04-15 | 香港纳观生物有限公司 | Two-color fluorescence localization super-resolution biological microscopy method and system |
| CN104777137A (en) * | 2015-03-25 | 2015-07-15 | 深圳市前海安测信息技术有限公司 | Spectral position adjustment device for biomarker detection |
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Application publication date: 20140212 |