CN110501319A - Raman super-resolution microscopy imaging method with multi-channel structured light illumination - Google Patents

Abstract

The multi-channel structured light illumination Raman super-resolution microscopy imaging method relates to the technical field of microscopic particle super-resolution imaging. In the imaging light path of the existing structured light super-resolution microscopy system, a Raman spectroscopic imaging module is added, and a Raman spectroscopic imaging module is added to the imaging light path of the existing structured light super-resolution microscopy system, and through a variety of imaging methods The selection of Mann probes enables specific imaging of different objects in complex systems. It includes an excitation light module, a structured light illumination module and a Raman spectroscopic imaging module; select a specific Raman probe molecule, combine the Raman probe molecule with an antibody, and specifically label the sample to be tested; turn on the excitation light module , using an inverted fluorescence microscope as a platform, the light stripes emitted by the structured light illumination module excite the tested sample; the signals of the excited probes are specifically sorted by the Raman spectroscopic imaging module, and then imaged at the CCD. The invention can be used in research directions such as endocytosis/transport of single molecules and particle systems, the mechanism of virus entry into cells, and the dynamic process of molecules in living cells.

Description

Raman super-resolution microscopy imaging method with multi-channel structured light illumination

technical field

The invention relates to the technical field of microscopic particle super-resolution imaging, in particular to a multi-channel structured light illumination Raman super-resolution microscopic imaging method.

Background technique

Structured light illumination super-resolution fluorescence microscopy (SIM) is a new type of super-resolution wide-field imaging technology developed in recent years. SIM super-resolution technology takes an ordinary inverted fluorescence microscope as a platform, uses a specific excitation light path to generate sinusoidal fringes with a known spatial frequency, excites the fluorescence signal of the sample, and separates and reconstructs the collected signal data. Super-resolution imaging with twice the diffraction limit. SIM super-resolution technology has a fast imaging speed, does not require a long time and a large amount of sample information collection, and is suitable for imaging analysis of living cells.

Optical probes are effective tools for studying specific targets in complex biological systems. Raman probes based on Raman scattering have very sharp spectral lines, high resolution and strong specificity. In addition, the Raman signal of water is very weak, and Raman probes can be directly detected in water environmental samples. But the Raman probe also has its disadvantage, that is, its signal intensity is low (two orders of magnitude lower than the fluorescent signal). The Raman signal intensity can be increased by 10 ² -10 ⁶ through the method of surface-enhanced Raman (SERS) probe and resonance Raman (PR) probe labeling, which can solve the problem of low Raman signal intensity. To sum up, for biological samples, especially the observation of living cells, using Raman probes for specific labeling imaging is an ideal analysis method.

Fluorescence microscope images of cells can display fluorescently labeled molecules, allowing researchers to observe the movement of special molecules or protein complexes in living cells or fixed samples in real time, but the specificity of fluorescent signals is poor and cannot meet the requirements of observing certain ( Or some kinds of research needs of specific targets, in addition, the volume of fluorescent labeling is relatively large, which will change its biological activity when labeling small biological molecules, but compared with the Raman signal, the intensity of the fluorescent signal is higher, which is relatively large. Easy to be captured by camera.

SUMMARY OF THE INVENTION

The invention provides a multi-channel structured light illumination Raman super-resolution microscopy imaging method. In the imaging light path of the existing structured light super-resolution microscopy system, a Raman spectroscopic imaging module is added, and a Raman spectroscopic imaging module is added to pass through various Raman probes. The choice of , to achieve specific imaging of different objects in complex systems.

The multi-channel structured light illumination Raman super-resolution microscopy imaging method is characterized by comprising an imaging system including an excitation light module, a structured light illumination module and a Raman spectroscopic imaging module; the method is realized by the following steps:

Step 1: Select a specific Raman probe molecule, combine the Raman probe molecule with an antibody, and specifically label the sample to be tested;

Step 2: Turn on the excitation light module, take the inverted fluorescence microscope as a platform, and the light stripes emitted by the structured light illumination module excite the sample to be tested;

Step 3: The signals of the excited probes are specifically sorted by the Raman spectroscopic imaging module, and then imaged at the CCD, and reconstructed to obtain super-resolution imaging with twice the diffraction limit.

Beneficial effects of the present invention:

The invention provides a multi-channel structured light illumination Raman super-resolution microscopic imaging method, and four wavelengths of 488, 532, 633, and 785 nm lasers are selected, which can meet the excitation wavelength requirements of different probes;

The invention provides a multi-channel structured light illumination Raman super-resolution microscopic imaging method, which combines the Raman probe labeling technology with the structured light super-resolution imaging technology, can realize specific super-resolution imaging, and improves the performance of wide-field Raman imaging. resolution;

The invention provides a multi-channel structured light illumination Raman super-resolution microscopic imaging method, which adopts a Raman spectroscopic imaging module to gate the characteristic peak signals of different probes, and has high system stability and fast imaging speed;

The invention provides a multi-channel structured light illumination Raman super-resolution microscopic imaging method, which has lower requirements for sample preparation, is more suitable for observation and research of living cells, and can be used for endocytosis/transport of single molecules and particle systems, and virus entry into cells Mechanism, dynamic process of molecules in living cells and other research directions.

Description of drawings

1 is a schematic diagram of a multi-channel excitation light module coupled with a single-mode fiber in the multi-channel structured light illumination Raman super-resolution microscopy imaging method according to the present invention;

2 is a schematic diagram of a structured light illumination module in the multi-channel structured light illumination Raman super-resolution microscopy imaging method according to the present invention;

3 is a schematic diagram of a Raman spectroscopic imaging module in the multi-channel structured light illumination Raman super-resolution microscopy imaging method according to the present invention;

4 is an overall optical path structure diagram of an imaging system in the multi-channel structured light illumination Raman super-resolution microscopy imaging method according to the present invention;

FIG. 5 is a flowchart of the multi-channel structured light illumination Raman super-resolution microscopy imaging method according to the present invention.

Detailed ways

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS First, the present embodiment will be described with reference to FIG. 1 to FIG. 5 , a multi-channel structured light illumination Raman super-resolution microscopy imaging method, the imaging method includes an imaging system, and the imaging system includes an excitation light module, a structured light illumination module and Raman spectroscopic imaging module; the excitation light module includes lasers with wavelengths of 488 nm, 532 nm, 633 nm and 785 nm, a coupler 1 and a single-mode fiber 2; the structured light illumination module includes a collimator 3, an acousto-optic modulator 4. Beam expander group 5, half-wave plate 6, PBS beam splitter 7, ferroelectric liquid crystal 8, first group lens 9, Pockels cell 10, reflector 11, second group lens 12, first converging mirror 13. The mask plate 14, the collimating mirror 15 and the second converging mirror 16; the Raman spectroscopic imaging module includes a tube mirror 17, a motorized runner 18 carrying a filter, and a CCD;

The lasers with wavelengths of 488 nm, 532 nm, 633 nm, and 785 nm are reflected (transmitted) at the coupler 1 through the corresponding dichroic mirrors, and then coupled into the single-mode fiber 2 by the coupler.

The divergent beam coupled out of the single-mode fiber is firstly collimated by the collimator 3; then selected from the above four excitation wavelengths by the acousto-optic modulator 4; then the beam diameter is expanded by the beam expander group 5; The wave plate 6 adjusts the polarization state of the beam to the S direction; further, through the PBS beam splitter 7, the beam in the S direction is reflected to the ferroelectric liquid crystal 8. By loading the ferroelectric liquid crystal pixel, a beam of light can be diffracted into multiple The beam exits; in order to fit the clear aperture of the Pockels cell 10, the first group of lenses 9 and the second group of lenses 12 are placed before and after it, so that the light beam is unobstructed in the Pockels box, and the function of the Pockels box is is to modulate the polarization direction of the outgoing beam to ensure that the interference fringes have the best contrast; the function of the mirror 11 in the system is to turn the beam to compress the length of the entire system; the modulated polarization state of the beam passes through the first convergence The mirror 13 reaches the mask plate 14. There are 6 holes on the mask plate 14. These holes correspond to the spatial positions of the ±1-level beams in three different illumination directions in the plane, which can filter out the 0-level beams and other high-level beams. Diffracted light; the light beam further propagates forward, successively passes through 15) the collimation of the collimating lens and the focusing of the second converging mirror 16, and then enters the inverted fluorescence microscope, and finally realizes structured light illumination at the focal plane of the objective lens.

FIG. 3 is a schematic diagram of a Raman spectroscopic imaging module. The Raman (fluorescence) signal excited by the sample first passes through the tube lens 17, and then further forwards to narrow-band filters with different center wavelengths (eg, Semrock FF01-575/5-25, FF01-680/13-25 type filter, etc.) of the motorized wheel 18. The central wavelength of the narrow-band filter corresponds to the Raman characteristic peak of the Raman probe, and its function is to filter out other background signals and only pass the desired specific Raman signal. The Raman signal further propagates forward to the CCD camera and participates in data acquisition. When the system is performing fluorescence imaging, the wheel can be turned to the empty position, allowing the fluorescence signal to pass through. The use of the filter wheel improves the imaging speed of the system, and the non-scanning one-time imaging method also greatly reduces the instability of the system. Through the characteristic signals excited by different observation targets labeled with different probes, specific and fast super-resolution imaging can be performed on multiple observation targets in a complex system.

In the Raman spectroscopic imaging module, it also has the function of fluorescence imaging, so that the Raman probe and the fluorescent probe can be used to complement each other. Since the probe is wavelength-selective, the system uses a multi-channel excitation light module as the excitation light source for Raman signals.

In this embodiment, combined with the imaging system structure diagram in FIG. 4 , the multi-channel structured light illumination super-resolution Raman microscopy imaging method based on each module can realize the super-resolution microscopy imaging analysis of various target components in a complex system, The imaging speed is fast and the reliability is strong. In addition, the imaging method can also meet the needs of different application fields by changing or optimizing one of the modules, and has strong flexibility and wide applicability.

This embodiment is described with reference to FIG. 5 , and the imaging method of this embodiment is specifically implemented by the following steps:

Step 1: Select lasers with wavelengths of 488nm, 532nm, 633nm and 785nm, use single-mode fiber as the coupling medium, and combine to form a multi-channel excitation light module;

Step 2: Select a specific Raman probe molecule (fluorescent probe molecule), combine it with an antibody, and specifically label the sample;

Step 3: Turn on the excitation light module, place the sample to be tested with the inverted fluorescence microscope as a platform, and use the structured light stripes generated by the excitation light path to excite the probe signal of the sample; it is worth noting that in order to achieve isotropic super-resolution For imaging, three sinusoidal fringes in different directions are used to excite and illuminate the sample. Since the excitation light source module contains four different excitation wavelengths, an acousto-optic modulator capable of sorting wavelengths is added to the optical path, and the incident wavelength is gated according to the specific probe used;

Step 4. The signal of the excited probe will reach the Raman spectroscopic imaging module, and the specific sorting of the signal will be realized by the filter wheel in the imaging module, and then imaged on the CCD;

Step 5: Perform data processing on the collected signals, and reconstruct to obtain super-resolution imaging with twice the diffraction limit.

In this embodiment, the probes used for specific labeling are Raman probes or fluorescent probes. Test samples include living cells, cell membranes, artificial phospholipid membranes, and systems capable of endocytosing or transporting single molecules and particles. The antibody can be epidermal growth factor receptor (EGFR) antibody or glucose transfectin (GLUT1) antibody, and the like.

In this embodiment, the multi-channel structured light illumination super-resolution microscopy imaging method is combined with the specific Raman imaging technology, which can obtain the specific super-resolution imaging of multiple target observations marked with different Raman probes in a complex system. Resolved imaging information can be used in research directions such as endocytosis/transport of single molecules and particle systems, the mechanism of virus entry into cells, and the dynamic process of molecules in living cells.

Claims (8)

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.