title: Label-free near-infrared reflectance microscopy as a complimentary tool for two-photon fluorescence brain imaging.
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.
authors.affiliation: European Laboratory for Non-Linear Spectroscopy, University of Florence, Via Nello Carrara 1, Sesto Fiorentino 50019, Italy ; National Research Council, National Institute of Optics, Largo Fermi 6, Florence 50125, Italy.
authors.forename: Anna Letizia
authors.initials: AL
authors.lastname: Allegra Mascaro
authors.affiliation: European Laboratory for Non-Linear Spectroscopy, University of Florence, Via Nello Carrara 1, Sesto Fiorentino 50019, Italy.
authors.forename: Irene
authors.initials: I
authors.lastname: Costantini
authors.affiliation: European Laboratory for Non-Linear Spectroscopy, University of Florence, Via Nello Carrara 1, Sesto Fiorentino 50019, Italy.
authors.forename: Emilia
authors.initials: E
authors.lastname: Margoni
authors.affiliation: Department of Engineering, Università Campus Bio-Medico di Roma, 00128 Rome, Italy.
authors.forename: Giulio
authors.initials: G
authors.lastname: Iannello
authors.affiliation: Department of Engineering, Università Campus Bio-Medico di Roma, 00128 Rome, Italy ; Department of Electrical and Information Engineering, University of Cassino and L. M. Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bria
authors.affiliation: European Laboratory for Non-Linear Spectroscopy, University of Florence, Via Nello Carrara 1, Sesto Fiorentino 50019, Italy ; National Research Council, National Institute of Optics, Largo Fermi 6, Florence 50125, Italy.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Sacconi
authors.affiliation: European Laboratory for Non-Linear Spectroscopy, University of Florence, Via Nello Carrara 1, Sesto Fiorentino 50019, Italy ; National Research Council, National Institute of Optics, Largo Fermi 6, Florence 50125, Italy ; University of Florence, Department of Physics and Astronomy, Via Sansone 1, Sesto Fiorentino 50019, Italy.
authors.forename: Francesco S
authors.initials: FS
authors.lastname: Pavone
keywords: (170.0110) Imaging systems; (170.3880) Medical and biological imaging