High-sensitivity High-speed Stimulated Raman Scattering Microscopy

Stimulated Raman scattering(SRS)microscopy is a new imaging technique based on stimulated radiation.As a label-free imaging tool with high chemical specificity,SRS has been widely used in biomedical imaging.However,the sensitivity and imaging speed limit the further development and application of SRS imaging.In this work,conjugated polydiacetylene derivatives signals are developed for high sensitivity SRS biological imaging;the ultrafast SRS microscopic imaging system with2000 frames per second has been developed to visualize the radical polymerization process.The thesis mainly includes three parts to describe SRS imaging system in detail:hyperspectral SRS system combined with conjugated polydiacetylene derivatives achieve organelle imaging in living cells;collinear-multiple-beams ultrafast SRS microscope based on acousto-optic deflectors is demonstrated;free radical polymerization imaging by the ultrafast SRS microscope.(1)Spectra-focusing hyperspectral SRS microscopic imaging system was developed based on SF57 glass rod.By high-frequency amplitude modulation of Stokes laser via electro-optic modulator,the system achieved shot-noise-limit detection by a narrow-band RF amplifier.In combination with the conjugated polymer PDDA,an ultrastrong biorthogonal Raman tag,SRS imaging of lysosomes,mitochondria and nucleus was achieved in live cells.(2)To increase the imaging speed of SRS,collinear multiple beam-based stimulated Raman scattering(COMB-SRS)was designed and developed.Two AODs were independently fed with 46 RF driving frequencies,which simultaneously generated 46 Gaussian laser beams with evenly separated diffraction angles.Furthermore,the46-channel SRS signals were extracted by 46-channel lock-in free circuits,which enabled parallel SRS imaging at speed of 2000 frames per second.(3)The Raman spectra of acrylamide monomer and polymer at 3043 cm-1 and 2930cm-1 were identified.Polymerization initiated by 396 nm UV laser was observed by SRS.Furthermore,the COMB-SRS system was used to visualize the ultrafast dynamics of the polymerization process at 2000 frames per second.COMB-SRS has been proved to be a universal method for the study of polymerization.The thesis pushed the limits of the SRS sensitivity and imaging speed.The shot-noise-limit detection hyperspectral SRS microscope and 2000 frames per second COMB-SRS were developed for imaging of chemical reaction.