Advances in label-free multimodal multiphoton imaging of living tissues

Many current biomedical applications require the development of advanced non-linear optical microscopy methods to provide functional and non-invasive imaging of live tissues with sub-cellular resolution. In the first part of the talk I will present recent advances in label-free metabolic imaging of living tissues by two-photon fluorescence lifetime microscopy (2P-FLIM) of endogenous biomarkers. We implement simultaneous two-photon excitation of NAD(P)H and FAD by wavelength mixing to acquire 2P-FLIM data of the two biomarkers at the same time and perform efficient multiparametric metabolic imaging in dynamic biological system. We demonstrate how 2P-FLIM of the two metabolic coenzymes reveals the richness and complexity of several metabolic processes in intact tissues with minimal phototoxicity and can be implemented non-invasively for longitudinal studies in vitro and in vivo such as stem cell differentiation, T cell activation and embryo development. In the second part of the talk I will present recent advances in polarization resolved second harmonic generation (pSHG) and modelling from protein molecular structure. We establish a general multi-scale numerical framework relating the micrometer-scale SHG measurements at the optical wavelength to the atomic-scale and molecular structure of the proteins under study and their supramolecular arrangement.