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Single‐beam optogenetic multimodal χ (3) / χ (5) nonlinear microscopy and brain imaging
Author(s) -
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.
Publication year - 2020
Publication title -
journal of raman spectroscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.748
H-Index - 110
eISSN - 1097-4555
pISSN - 0377-0486
DOI - 10.1002/jrs.5933
Subject(s) - microscopy , two photon excitation microscopy , optogenetics , laser , fluorescence lifetime imaging microscopy , optics , fluorescence microscope , biophysics , fluorescence , materials science , chemistry , physics , biology , neuroscience
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, χ (3) and χ (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, cell‐specific 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.