Two-photon imaging combined with targeted fluorescent indicators is extensively used for visualizing critical features of brain functionality and structural plasticity. Back-scattered photons from the NIR laser provide complimentary information without introducing any exogenous labelling. Here, we describe a versatile approach that, by collecting the reflected NIR light, provides structural details on the myelinated axons and blood vessels in the brain, both in fixed samples and in live animals. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from Thy1-GFPm mice, 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. Label-free detection of axonal elongations over the layer 2/3 of mouse cortex under a cranial window was also possible in live brain. Finally, blood flow could be measured in vivo, 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 simultaneously.
Anna Letizia Allegra Mascaro, Irene Costantini, Emilia Margoni, Giulio Iannello, Alessandro Bria, Leonardo Sacconi, and Francesco S. Pavone, "Label-free NIR reflectance imaging as a complimentary tool for two-photon fluorescence microscopy: multimodal investigation of stroke
(Conference Presentation)," Proc. SPIE 9712, Multiphoton Microscopy in the Biomedical Sciences XVI, 97121K (Presented at SPIE BiOS: February 16, 2016; Published: 27 April 2016); https://doi.org/10.1117/12.2208886.4848767256001.
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