Single-Beam Optogenetic Multimodal Chi((3))/Chi((5)) Nonlinear Microscopy And Brain Imaging

We demonstrate single-beam optogenetic multimodal nonlinear-optical microscopy that combines third-harmonic generation (THG) and three-photon-excited fluorescence (3PEF) - two nonlinear-optical processes related to the third- and fifth-order susceptibilities, chi((3)) and chi((5)). A carefully tailored unamplified short-pulse output of mode-locked solid-state lasers is shown to provide an ample parameter space for the optimization of such a single-beam multimodal microscopy, enabling subcellular-resolution, cellspecific imaging using genetically encoded fluorescent-protein-based reporters in a vast variety of biological systems and settings, ranging from HeLa to brain cells. Experiments on brain slices and cell cultures presented in this paper demonstrate the potential of short-pulse 3PEF/THG microscopy for a subcellular-resolution, high-contrast imaging of HeLa-line cancer-cell derivatives, as well as mitochondrial and somatic intracellular structures within deep-brain neurons and astrocytes. As a step beyond the state of the art in optical brain imaging, single-beam subcellular-resolution, cell-specific optogenetic 3PEF/THG imaging of fundamental functional brain units is experimentally demonstrated. One fundamental question that this work brings up for the future nonlinear absorption and Raman/hyper-Raman studies is whether the Herzberg-Teller corrections, needed for an accurate description of two-photon absorption in fluorescent proteins (FPs), would be also sufficient for an adequate treatment of higher-n n-photon absorption in FP-based systems or models that include other quantum pathways would be necessary for the analysis of FP agents for higher-n nonlinear microscopy.