Near-resonance Enhanced High-resolution Stimulated Raman Scattering Microscopy And Applications

Label-free optical microscopy plays essential role in biomedical research.However,high resolution imaging of cells or tissues is still a huge challenge due to optical diffraction limit.Fluorescent labeling based super-resolution microscopes have been widely applied to image specific proteins and nucleic acids.However,the large-size exogenous fluorescent labels will potentially interfere with cell functions.Therefore,the high resolution label-free microscopy based on endogenous biomolecules is in urgent need of development.At present,spontaneous Raman spectroscopy has been widely used for biochemical analysis of cells,but its slow acquisition speed,poor resolution and low sensitivity hinder the further development of this technology.With the development of science and technology,stimulated Raman scattering(SRS)microscopy has gradually matured.This technology has faster imaging speed,higher resolution and sensitivity.However,as a labeled-free imaging technology based on detecting molecular vibrations,the resolution of SRS has been limited to-300 nanometers.Therefore,an optical imaging tools that can break the traditional spatial resolution limit of 180 nm are urgently needed.Herein,we report near resonance enhanced label-free stimulated Raman scattering(SRS)microscopy with spatial resolution near 130 nanometers.The contrast of high resolution images is directly derived from endogenous biological molecules with low concentration.By introducing BBO crystals into the imaging system,a visible-light-based SRS microscope is realized for the first time.In contrast to near-infrared SRS system,the molecular virtual state in the visible SRS process is closer to the electronic excited state.The Raman scattering cross section becomes larger and the imaging sensitivity is increased by about 23 times to that of traditional NIR SRS microscopy.In addition,we have implemented the hyperspectral SRS microscope based on spectral focusing technology using one 0.3 m-long single-mode polarization-maintaining optical fiber,which enables chemical analysis in high resolution image.Moreover,the optical fiber provides absolute collinearity,stability and excellent Gauss mode of the pump and Stokes lasers,which further ensures high resolution imaging.Finally,based on the improvement of spatial resolution and imaging sensitivity,we demonstrated high-resolution three-dimensional(3D)imaging of single cells and high-resolution imaging of large-scale unprocessed mouse brain tissue.In addition,we chemically observed the distribution of myelin in brain tissue.Our concept further advances the development of SRS imaging technology and provides a new platform for high resolution biomedical imaging with spatial resolution?130 nm.