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Allegra Mascaro AL, Costantini I, Margoni E, et al. Label-free near-infrared reflectance microscopy as a complimentary tool for two-photon fluorescence brain imaging. Biomed Opt Express. 2015;6(11):4483-92doi: 10.1364/BOE.6.004483.
Allegra Mascaro, A. L., Costantini, I., Margoni, E., Iannello, G., Bria, A., Sacconi, L., & Pavone, F. S. (2015). Label-free near-infrared reflectance microscopy as a complimentary tool for two-photon fluorescence brain imaging. Biomedical optics express, 6(11), 4483-92. https://doi.org/10.1364/BOE.6.004483
Allegra Mascaro, Anna Letizia, et al. "Label-free near-infrared reflectance microscopy as a complimentary tool for two-photon fluorescence brain imaging." Biomedical optics express vol. 6,11 (2015): 4483-92. doi: https://doi.org/10.1364/BOE.6.004483
Allegra Mascaro AL, Costantini I, Margoni E, Iannello G, Bria A, Sacconi L, Pavone FS. Label-free near-infrared reflectance microscopy as a complimentary tool for two-photon fluorescence brain imaging. Biomed Opt Express. 2015 Oct 21;6(11):4483-92. doi: 10.1364/BOE.6.004483. eCollection 2015 Nov 01. PMID: 26601011; PMCID: PMC4646555.
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Biomed Opt Express. 2015 Oct 21;6(11):4483-92. doi: 10.1364/BOE.6.004483. eCollection 2015 Nov 01.

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

Biomedical optics express

Anna Letizia Allegra Mascaro, Irene Costantini, Emilia Margoni, Giulio Iannello, Alessandro Bria, Leonardo Sacconi, Francesco S Pavone

Affiliations

  1. 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.
  2. European Laboratory for Non-Linear Spectroscopy, University of Florence, Via Nello Carrara 1, Sesto Fiorentino 50019, Italy.
  3. Department of Engineering, Università Campus Bio-Medico di Roma, 00128 Rome, Italy.
  4. 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.
  5. 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.

PMID: 26601011 PMCID: PMC4646555 DOI: 10.1364/BOE.6.004483

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.

Keywords: (170.0110) Imaging systems; (170.3880) Medical and biological imaging

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