阅读说明：本技术 多通路结构光照明的拉曼超分辨显微成像方法 (The Raman super-resolution micro imaging method of multi-path Structured Illumination ) 是由 王宏达 孙佳音 初宏亮 吴强 于 2019-08-29 设计创作，主要内容包括：多通路结构光照明拉曼超分辨显微成像方法,涉及微观粒子超分辨成像技术领域,在现有的结构光超分辨显微系统的成像光路中,加入拉曼分光成像模块,通过多种拉曼探针的选取,实现复杂体系中不同观测物的特异性成像。包括激发光模块、结构光照明模块和拉曼分光成像模块；选取特定的拉曼探针分子,将所述拉曼探针分子与抗体结合,对被测样品进行特异性标记；开启激发光模块,以倒置荧光显微镜为平台,结构光照明模块出射的光条纹激发被测样品；被激发出来探针的信号通过拉曼分光成像模块实现信号的特异性分选,然后在CCD处成像。本发明可用于内吞/转运单个分子及颗粒体系、病毒进入细胞机理、活细胞内分子的动态过程等研究方向。(Multi-path Structured Illumination Raman super-resolution micro imaging method, it is related to microcosmic particle super-resolution imaging technical field, in the imaging optical path of existing structure light super-resolution microscopic system, raman spectroscopy image-forming module is added, by the selection of a variety of Raman microprobes, the specificity imaging of different observation objects in complex system is realized.Including excitation module, Structured Illumination module and raman spectroscopy image-forming module；Specific Raman microprobe molecule is chosen, by the Raman microprobe molecule in conjunction with antibody, specific marker is carried out to sample；Excitation module is opened, using inverted fluorescence microscope as platform, the striations of Structured Illumination module outgoing excites sample；Be excited out probe signal by raman spectroscopy image-forming module realize signal specificity sorting, then CCD at imaging.The present invention can be used for endocytosis/transhipment individual molecule and granular system, cell entry celelular mechanism, the research directions such as dynamic process of molecule in living cells.)

1. multi-path Structured Illumination Raman super-resolution micro imaging method, it is characterized in that；Including imaging system, the imaging system System includes excitation module, Structured Illumination module and raman spectroscopy image-forming module；This method is realized by following steps:

Step 1: choosing specific Raman microprobe molecule, by the Raman microprobe molecule in conjunction with antibody, sample is carried out Specific marker；

Step 2: excitation module is opened, and using inverted fluorescence microscope as platform, the striation of the Structured Illumination module outgoing Line excites sample；

Step 3: the signal for the probe that is excited out realizes the specificity sorting of signal by raman spectroscopy image-forming module, then It is imaged at CCD, reconstruct obtains the super-resolution imaging with two times of diffraction limits.

2. multi-path Structured Illumination Raman super-resolution micro imaging method according to claim 1, it is characterised in that: institute State the laser that excitation module includes coupler (1), single mode optical fiber (2) and different wave length；

The Structured Illumination module includes collimator (3), acousto-optic modulator (4), expands microscope group (5), half-wave plate (6), PBS point Beam device (7), ferroelectric liquid crystals (8), first group of lens (9), Pockers cell (10), reflecting mirror (11), second group of lens (12), first Converging lenses (13), mask plate (14), collimating mirror (15) and the second converging lenses (16)；

The raman spectroscopy image-forming module includes Guan Jing (17), the electronic runner (18) and CCD for being loaded with narrow band filter slice；

The laser of the laser emitting of the different wave length converges at coupler (1) through the reflection of corresponding dichroscope respectively, passes through Coupler (1) is coupled to single mode optical fiber (2)；

The divergent beams of single mode optical fiber (2) the coupling outgoing are collimated by collimator (3)；It is selected again by acousto-optic modulator (4) The laser of different wave length expands beam size by expanding microscope group (5)；Then pass through half-wave plate (6) for light polarization It is adjusted to be reflexed to the light beam in the direction S on ferroelectric liquid crystals (8), by PBS beam splitter (7) by ferroelectric liquid crystals picture behind the direction S Light beam is diffracted into multi beam outgoing by the load of element；Multi-beam is successively through first group of lens (9), Pockers cell (10), reflecting mirror (11) and after second group of lens (12), mask plate (14) is reached by the first converging lenses (13), pass through ± the 1 of mask plate (14) Grade light beam by collimating mirror (15) collimation and the second converging lenses (16) focusing, into inverted fluorescence microscope, finally in object The focal plane of mirror realizes that striations excites sample；

The Raman signal of sample excitation is imaged after filtering by Guan Jing (17) and by narrow band filter in CCD camera.

3. multi-path Structured Illumination Raman super-resolution micro imaging method according to claim 1, which is characterized in that use In specific marker probe be Raman microprobe or fluorescence probe.

4. multi-path Structured Illumination Raman super-resolution micro imaging method according to claim 1, which is characterized in that quilt Sample includes the system of living cells, cell membrane, artificial phospholipid's film, energy endocytosis or transhipment individual molecule and particle.

5. multi-path Structured Illumination Raman super-resolution micro imaging method according to claim 1, which is characterized in that institute Stating antibody is that EGF-R ELISA (EGFR) antibody or glucose turn albumen (GLUT1) antibody.

6. multi-path Structured Illumination Raman super-resolution micro imaging method according to claim 2, which is characterized in that swash The laser of different wave length includes outgoing tetra- excitation wavelengths of 488nm, 532nm, 633nm and 785nm in light emitting module.

7. multi-path Structured Illumination Raman super-resolution micro imaging method according to claim 2, which is characterized in that In In raman spectroscopy image-forming module, using the narrow band filter of electronic runner and different central wavelengths with multiple hole locations to difference The peak signal of Raman microprobe carries out sorting imaging.

8. multi-path Structured Illumination Raman super-resolution micro imaging method according to claim 2, which is characterized in that institute It states and is loaded with the hole location that the electronic runner (18) of narrow band filter slice has been provided with, the hole location of the sky is under fluorescence probe label Traditional structure light super-resolution imaging.

Technical field

The present invention relates to microcosmic particle super-resolution imaging technical fields, and in particular to a kind of multi-path Structured Illumination Raman Super-resolution micro imaging method.

Background technique

Structured Illumination super-resolution fluorescence microtechnic (SIM) is a kind of novel super-resolution wide field that development in recent years comes Imaging technique.SIM super resolution technology generates space using common inverted fluorescence microscope as platform, using specific excitation light path Sine streak known to frequency, to sample carry out fluorescence signal excitation, to collected signal carry out data separation with again Structure can get the super-resolution imaging with two times of diffraction limits.SIM super resolution technology image taking speed is very fast, do not need for a long time, A large amount of acquisition sample message, the imaging analysis suitable for active somatic cell.

Optical probe is the effective tool for studying specific objective in complex biological system.Raman based on Raman scattering is visited Needle, spectral line is very sharp keen, high resolution, high specificity.In addition, the Raman signal of water is very weak, Raman microprobe can be in water environment sample It is directly detected in product.But Raman microprobe also has its shortcoming, is exactly that its signal strength lower (compares fluorescence signal Low two orders of magnitude).The method marked by surface-enhanced Raman (SERS) probe and resonance raman (PR) probe can incite somebody to action The intensity of Raman signal improves 10²-10⁶, can solve the problems, such as that Raman signal intensity is low.To sum up, for biological sample, especially It is the observation of active somatic cell, carries out specific marker imaging with Raman microprobe, is a kind of ideal analysis means.

The fluorescence microscopy images of cell can show the molecule of fluorescent marker, and researcher is allowed to observe living cells or solid in real time The movement of particular molecule or albumen composition in random sample product, but the specificity of fluorescence signal is poor, is unable to satisfy under complex system The Research Requirements of certain (or certain is several) specific objective are observed, in addition, the volume of fluorescent marker is relatively large, it is small in label biology Its bioactivity can be changed when molecule, but for Raman signal, the intensity of fluorescence signal is higher, is easier to be adopted by camera Collection.

Summary of the invention

The present invention provides a kind of multi-path Structured Illumination Raman super-resolution micro imaging methods, in existing structure light In the imaging optical path of super-resolution microscopic system, raman spectroscopy image-forming module is added, by the selection of a variety of Raman microprobes, realizes multiple The specificity imaging of different observation objects in miscellaneous system.

Multi-path Structured Illumination Raman super-resolution micro imaging method, it is characterized in that；Including imaging system, the imaging System includes excitation module, Structured Illumination module and raman spectroscopy image-forming module；This method is realized by following steps:

Step 1: specific Raman microprobe molecule is chosen, by the Raman microprobe molecule in conjunction with antibody, to sample Carry out specific marker；

Step 2: excitation module is opened, using inverted fluorescence microscope as platform, the Structured Illumination module outgoing Striations excites sample；

Step 3: the signal for the probe that is excited out realizes the specificity sorting of signal by raman spectroscopy image-forming module, Then it is imaged at CCD, reconstruct obtains the super-resolution imaging with two times of diffraction limits.

Beneficial effects of the present invention:

The present invention provides multi-path Structured Illumination Raman super-resolution micro imaging method, have chosen 488,532,633, The laser of tetra- wavelength of 785nm, can meet the needs of different probe is to excitation wavelength；

The present invention provide multi-path Structured Illumination Raman super-resolution micro imaging method, by Raman microprobe labelling technique with Structure light super-resolution imaging technology combines, and can be realized specific super-resolution imaging, improves the resolution of wide field Raman image Rate；

The present invention provides multi-path Structured Illumination Raman super-resolution micro imaging method, using raman spectroscopy image-forming module The characteristic peaks signal of different probe is gated, system stability is high, and image taking speed is fast；

The present invention provides multi-path Structured Illumination Raman super-resolution micro imaging method, requirement to sample preparation compared with It is low, relatively it is suitable for the observational study of active somatic cell, endocytosis/transhipment individual molecule and granular system, cell entry cell can be used for The research directions such as dynamic process of molecule in mechanism, living cells.

Detailed description of the invention

Fig. 1 is Single-Mode Fiber Coupling in multi-path Structured Illumination Raman super-resolution micro imaging method of the present invention Multi-path excitation module schematic diagram；

Fig. 2 is Structured Illumination mould in multi-path Structured Illumination Raman super-resolution micro imaging method of the present invention Block schematic diagram；

Fig. 3 is raman spectroscopy imaging in multi-path Structured Illumination Raman super-resolution micro imaging method of the present invention Module diagram；

Fig. 4 is the whole of imaging system in multi-path Structured Illumination Raman super-resolution micro imaging method of the present invention Body light channel structure figure；

Fig. 5 is the flow chart in multi-path Structured Illumination Raman super-resolution micro imaging method of the present invention.

Specific embodiment