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Three‐photon‐resonance‐enhanced third‐harmonic generation for label‐free deep‐brain imaging: In search of a chemical contrast
Author(s) -
Lanin Aleksandr A.,
Chebotarev Artem S.,
Pochechuev Matvey S.,
Kelmanson Ilya V.,
Fedotov Andrei B.,
Belousov Vsevolod V.,
Zheltikov Aleksei M.
Publication year - 2019
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.5566
Subject(s) - microscopy , raman scattering , second harmonic generation , raman spectroscopy , laser , optics , high harmonic generation , two photon excitation microscopy , second harmonic imaging microscopy , nonlinear optics , materials science , nanotechnology , chemistry , optoelectronics , physics , fluorescence
Within the past decade, nonlinear Raman microscopy has earned a well‐deserved status of a gold‐standard technology for chemically selective imaging. Even though second‐ and third‐harmonic microscopy is much less demanding on a laser source and multifrequency beam arrangement, it is increasingly falling behind nonlinear Raman scattering as a method of bioimaging because it offers no mechanism whereby imaging could be made chemically specific. Here, we show, however, that such a mechanism does exist, helping harmonic‐generation microscopy overcome its no‐chemical‐specificity handicap. We demonstrate that, with the laser wavelength tuned to a three‐photon resonance with the Soret band of hemoglobin, third‐harmonic generation provides a chemically specific method for a high‐contrast imaging of red blood cells in a broad class of biological systems, including live brain. Moreover, third‐harmonic generation imaging can be conveniently combined with second‐harmonic microscopy on a compact laser platform, providing, as our experiments on rat brain show, a powerful resource for three‐dimensional, cell‐specific label‐free deep‐brain imaging.