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 phase-controlled 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 cell-silent 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.

Keywords

• srs 3d imaging
• phase-controlled light focusing
• image aberration corrections
• deep tissue imaging