Open AccessLabel-free near-infrared reflectance microscopy as a complimentary tool for two-photon fluorescence brain imaging
Author(s) -
Anna Letizia Allegra Mascaro,
Irene Costantini,
Emilia Margoni,
Giulio Iannello,
Alessandro Bria,
Leonardo Sacconi,
Francesco S. Pavone
Publication year - 2015
Publication title -
biomedical optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.362
H-Index - 86
ISSN - 2156-7085
DOI - 10.1364/boe.6.004483
Subject(s) - fluorescence , two photon excitation microscopy , microscopy , fluorescence lifetime imaging microscopy , fluorescence microscope , hippocampal formation , microscope , near infrared spectroscopy , preclinical imaging , materials science , optics , biomedical engineering , in vivo , neuroscience , biology , medicine , physics , microbiology and biotechnology
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.