Stimulated Raman scattering tomography for rapid three-dimensional chemical imaging of cells and tissue

Three-dimensional (3D) imaging is essential for understanding intricate biological and biomedical systems, yet live cell and tissue imaging applications still face challenges due to constrained imaging speed and strong scattering in turbid media. Here, we present a unique phase-modulated stimulated Raman scattering tomography (PM-SRST) technique to achieve rapid label-free 3D chemical imaging in cells and tissue. To accomplish PM-SRST, we utilize a spatial light modulator to electronically manipulate the focused Stokes beam along the needle Bessel pump beam for SRS tomography without the need for mechanical z scanning. We demonstrate the rapid 3D imaging capability of PM-SRST by real-time monitoring of 3D Brownian motion of polystyrene beads in water with 8.5 Hz volume rate, as well as the instant biochemical responses to acetic acid stimulants in MCF-7 cells. Further, combining the Bessel pump beam with a longer wavelength Stokes beam (NIR-II window) provides a superior scattering resilient ability in PM-SRST, enabling rapid tomography in deeper tissue areas. The PM-SRST technique provides ∼twofold enhancement in imaging depth in highly scattering media (e.g., polymer beads phantom and biotissue like porcine skin and brain tissue) compared with conventional point-scan SRS. We also demonstrate the rapid 3D imaging ability of PM-SRST by observing the dynamic diffusion and uptake processes of deuterium oxide molecules into plant roots. The rapid PM-SRST developed can be used to facilitate label-free 3D chemical imaging of metabolic activities and functional dynamic processes of drug delivery and therapeutics in live cells and tissue.