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.
Highlights
In vivo two-photon fluorescence (TPF) imaging is extensively used for attaining critical insights into brain plasticity
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
Summary
In vivo two-photon fluorescence (TPF) imaging is extensively used for attaining critical insights into brain plasticity. We show that axons can be imaged longitudinally under a cranial window in vivo This complimentary technique allows monitoring the blood flow velocity in real time without the need of introducing exogenous dyes. This imaging modality could be used in many pathologies involving the degeneration of neurons and oligodendrocytes, the myelin-producing cells, and of blood flow interruption and angiogenesis. We applied it to a mouse model of cortical stroke to study demyelination and neuronal degeneration dynamics in vivo in parallel
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