Abstract
We perform high-resolution, non-invasive, in vivo deep-tissue imaging of the mouse neocortex using multiphoton microscopy with a high repetition rate optical parametric amplifier laser source tunable between λ=1,100 and 1,400 nm. By combining the high repetition rate (511 kHz) and high pulse energy (400 nJ) of our amplifier laser system, we demonstrate imaging of vasculature labeled with Texas Red and Indocyanine Green, and neurons expressing tdTomato and yellow fluorescent protein. We measure the blood flow speed of a single capillary at a depth of 1.2 mm, and image vasculature to a depth of 1.53 mm with fine axial steps (5 μm) and reasonable acquisition times. The high image quality enabled analysis of vascular morphology at depths to 1.45 mm.
Highlights
The ability to visualize deep structures in vivo with high spatial resolution is of rising interest to investigate neuronal physiology and cerebral vasculature
We demonstrate deep-tissue imaging of vasculature labeled with Texas Red and Indocyanine green (ICG), and neurons expressing tdTomato and yellow fluorescent protein (YFP)
Two-photon microscopy images of vasculature were separately acquired in different mice using two fluorescent dyes – Texas Red and ICG
Summary
The ability to visualize deep structures in vivo with high spatial resolution is of rising interest to investigate neuronal physiology and cerebral vasculature. Optical imaging offers non-invasive, high-resolution in vivo microscopy techniques to observe brain tissue and its surrounding environment. Confocal fluorescence microscopy using one-photon excitation is limited to imaging the superficial tissue surface due to the scattering of short excitation wavelength light in heterogeneous brain tissue. Two-photon fluorescence laser-scanning microscopy (2PM), developed in the early 1990’s [1], uses an ultrafast laser to cause two-photon excitation of a fluorophore in a confined excitation volume. Ti:S oscillators provide a tunable excitation wavelength between 700 and 1,000 nm, which covers the peak two-photon absorption of a myriad of common fluorophores. Ti:S oscillators provide a tunable excitation wavelength between 700 and 1,000 nm, which covers the peak two-photon absorption of a myriad of common fluorophores. 2PM performed with Ti:S oscillators has been shown to reach a maximum imaging depth of 800 μm in the neocortex [2]
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