Label-free near-infrared reflectance microscopy as a complimentary tool for two-photon fluorescence brain imaging

Year: 2015

Authors: Allegra Mascaro A.L., Costantini I., Margoni E., Iannello G., Bria A., Sacconi L., Pavone F.S.

Autors Affiliation: Univ Florence, European Lab Nonlinear Spect, I-50019 Sesto Fiorentino, Italy; CNR, Natl Inst Opt, I-50125 Florence, Italy; Univ Campus Biomed Roma, Dept Engn, I-00128 Rome, Italy; Univ Cassino & LM, Dept Elect & Informat Engn, Cassino, Italy; Univ Florence, Dept Phys & Astron, I-50019 Sesto Fiorentino, Italy.

Abstract: In vivo two-photon imaging combined with targeted fluorescent indicators is currently extensively used for attaining critical insights into brain functionality and structural plasticity. Additional information might be gained from back-scattered photons from the near-infrared (NIR) laser without introducing any exogenous labelling. Here, we describe a complimentary and versatile approach that, by collecting the reflected NIR light, provides structural details on axons and blood vessels in the brain, both in fixed samples and in live animals under a cranial window. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from a Thy1-GFPm mouse, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Interestingly, NIR reflectance microscopy allowed the label-free detection of axonal elongations over the superficial layers of mouse cortex under a cranial window in vivo. Finally, blood flow can be measured in live preparations, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated. (C) 2015 Optical Society of America

Journal/Review: BIOMEDICAL OPTICS EXPRESS

Volume: 6 (11)      Pages from: 4483  to: 4492

KeyWords: Backscattering; Blood vessels; Brain; Brain mapping; Electromagnetic wave scattering; Fluorescence; Fluorescence microscopy; Hemodynamics; Imaging systems; Light scattering; Mammals; Medical imaging; Neuroimaging; Neurons; Photons; Reflection; Two photon processes, Fluorescent indicators; Label-free detection; Label-free techniques; Medical and biological imaging; Near infra-red reflectances; Near-infrared lasers; Two photon fluorescence; Two-photon fluorescence microscopy, Infrared devices, adult; animal tissue; Article; controlled study; dentate gyrus; dichroic mirror; entorhinal cortex; fluorescence microscopy; hippocampal CA1 region; hippocampal CA2 region; hippocampal CA3 region; image processing; laser; medical device; mouse; near infrared imaging system; neuroimaging; nonhuman; polarizing beam splitter; protein expression; telescope
DOI: 10.1364/BOE.6.004483

Citations: 13
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