Research On Raman Wide-field Super-resolution Imaging Based On Structured Illumination Microscopy

The optical microscope has been associated with life sciences since its birth,opening the door for humans to understand the microscopic world.Among them,the milestone development is the invention of the fluorescence microscope.However,the existence of the diffraction limit makes traditional optical microscopes unable to distinguish objects with dimensions below 200 nanometers.To surpass this resolution limit,scientists have developed various super-resolution microscopy techniques.However,these super-resolution methods are based on fluorescent probes,and fluorescence has its inherent defects.Therefore,we proposed the use of Raman scattering for super-resolution microscopy.Raman spectroscopy is known as molecular fingerprint spectroscopy because it reflects the structure of material molecules through vibration and rotation energy level.The traditional Raman scattering imaging adopts the form of point scanning and spectrometer,while the newer uses coherent Raman light and point scanning.The wide-field Raman can only be realized by line scanning or fiber bundle.We propose a novel method to achieve wide-field Raman microscopy,which combines super-resolution structured light microscopy with tunable filters to obtain super-resolution microscopy images and Raman spectrum information.Super-resolution structured illumination microscopy comes from the application of Moiré effect.It modulates the high-frequency information in the frequency domain into the OTF of the optical microscope,and then uses the SIM algorithm to demodulate the high-frequency information,and then splice and reconstruct it into super-resolution microscopic image.In the work of this article,we use a 532 nm wavelength single longitudinal mode laser as the light source on the frame of the Olympus IX73 inverted microscope.Based on the fringe structured illumination generated by the DMD,a set of wide-field Raman super-resolution structured illumination microscopy system is built.This system uses DMD as a device to generate fringe structured illumination,because it has the advantages of fast refresh speed and high laser power.For the sample,SERS particles are used.This kind ofSERS particles contains a gap mode structure,which can be used for SERS enhancement more efficiently,and is convenient for Raman scattering imaging.At the same time,because SIM is naturally wide-field imaging,ordinary spectrometers are not wide-field and cannot be connected to the SIM filter position,so we built a tunable narrowband filter and replaced the spectrometer with this filter.The location of traditional Raman spectroscopy imaging.The filter is composed of a group of special filters whose cut-off frequency changes with the angle of incident light.Adjusting the angle of this group of filters can select the appropriate Raman peak band,and can reach 2nm The narrow bandwidth.Because it is not a grating-type dispersive device,this filter can be directly connected to the position of the SIM system filter,and the entire system becomes a wide-field Raman super-resolution structured light microscope system.On this system,we performed imaging experiments on fluorescence and Raman molecules respectively,and proved that the resolution can reach the level of 110 nm,which meets the requirements of our design.