In:
Journal of Raman Spectroscopy, Wiley, Vol. 51, No. 10 ( 2020-10), p. 1942-1950
Abstract:
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.
Type of Medium:
Online Resource
ISSN:
0377-0486
,
1097-4555
Language:
English
Publisher:
Wiley
Publication Date:
2020
detail.hit.zdb_id:
1481008-6
SSG:
11
