Stimulated Raman scattering microscopy with phase-controlled light focusing and aberration correction for rapid and label-free, volumetric deep tissue imaging

Abstract

We report a novel stimulated Raman scattering (SRS) microscopy technique featuring phase-controlled light focusing and aberration corrections for rapid, deep tissue 3D chemical imaging with subcellular resolution. To accomplish phasecontrolled SRS (PC-SRS), we utilize a single spatial light modulator to electronically tune the axial positioning of both the shortened-length Bessel pump and the focused Gaussian Stokes beams, enabling z-scanning-free optical sectioning in the sample. By incorporating Zernike polynomials into the phase patterns, we simultaneously correct the system aberrations at two separate wavelengths (~240 nm difference), achieving a ~3-fold enhancement in signal-to-noise ratio over the uncorrected imaging system. PC-SRS provides >2-fold improvement in imaging depth in various samples (e.g., polystyrene bead phantoms, porcine brain tissue) as well as achieves SRS 3D imaging speed of ~13 Hz per volume for real-time monitoring of Brownian motion of polymer beads in water, superior to conventional point-scanning SRS 3D imaging. We further utilize PC-SRS to observe the metabolic activities of the entire tumor liver in living zebrafish in cellsilent region, unraveling the upregulated metabolism in liver tumor compared to normal liver. This work shows that PC-SRS provides unprecedented insights into morpho-chemistry, metabolic and dynamic functioning of live cells and tissue in real-time at the subcellular level.