AbstractWe demonstrate singlebeam optogenetic multimodal nonlinearoptical microscopy that combines thirdharmonic generation (THG) and threephotonexcited fluorescence (3PEF) two nonlinearoptical processes related to the third and fifthorder susceptibilities, (3) and (5). A carefully tailored unamplified shortpulse output of modelocked solidstate lasers is shown to provide an ample parameter space for the optimization of such a singlebeam multimodal microscopy, enabling subcellularresolution, cellspecific imaging using genetically encoded fluorescentproteinbased 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 shortpulse 3PEF/THG microscopy for a subcellularresolution, highcontrast imaging of HeLaline cancercell derivatives, as well as mitochondrial and somatic intracellular structures within deepbrain neurons and astrocytes. As a step beyond the state of the art in optical brain imaging, singlebeam subcellularresolution, cellspecific 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/hyperRaman studies is whether the HerzbergTeller corrections, needed for an accurate description of twophoton absorption in fluorescent proteins (FPs), would be also sufficient for an adequate treatment of highern nphoton absorption in FPbased systems or models that include other quantum pathways would be necessary for the analysis of FP agents for highern nonlinear microscopy.
Lanin, A. A., Chebotarev, A. S., Pochechuev, M. S., Kelmanson, I. V., Kotova, D. A., Bilan, D. S., ... Zheltikov, A. M.
citation count
3
complete list of authors
Lanin, Aleksandr A||Chebotarev, Artem S||Pochechuev, Matvey S||Kelmanson, Ilya V||Kotova, Daria A||Bilan, Dmitry S||Ivanov, Anatoly A||Panova, Anastasiya S||Tarabykin, Viktor S||Fedotov, Andrei B||Belousov, Vsevolod V||Zheltikov, Aleksei M
