Biosystem function is the result of complex interactions among participating organisms and their local environments; understanding the mechanisms underlying this functionality requires an ability to correlate spatial, temporal, and chemical information with the corresponding morphological conditions at relevant scales. Therefore, the development of bioimaging techniques to visualize biosystem organization and the spatial distribution of molecular species is essential in linking molecular scale processes to macroscopic biological phenomena. Total internal reflection (TIR) based optical microscopy offers a convenient approach to image planar samples and specimens such as plant-microbe systems, biofilms, and other complex interfaces. In this presentation, we will discuss our recently developed TIR enabled multimodal nonlinear optical microscopic platform which combines coherent anti-stokes Raman scattering (CARS), two-photon fluorescence (TPF), second harmonic generation (SHG), and sum frequency generation (SFG) modalities on the same platform. The integration of these different imaging modalities in a single platform offers the advantage of obtaining spatially co-registered images reporting on different chemical and structural aspects of biosystems ranging from pollen grains to living bacterial cells. Specifically, the spatial distribution of different molecular groups in pollen grains is visualized via the vibrational contrast afforded by CARS microscopy, whereas co-registered TPF imaging allows the identification of spatially localized intrinsic fluorophores. We demonstrate the feasibility of our microscope for wide-field CARS imaging on live cells through independent characterization of cell viability using spatially co-registered TPF imaging. Our results highlight the importance of integrating complimentary widefield imaging modalities in a single microscope platform to differentiate spatially and temporally evolving processes in complex biological systems.

Spatially co-registered widefield nonlinear optical imaging of living and complex biosystems in a total internal reflection geometry

ACS Fall 2021

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