Abstract
Wide-field temporal focusing is a novel technique that provides optical sectioning for imaging without the need for beam scanning. However, illuminating over large areas greatly reduces the photon density which limits the technique applicability to small regions, precluding functional imaging of cellular networks. Here we present a strategy that combines beam shaping and temporal focusing of amplified pulses (>1 µJ/pulse) for fast imaging of cells from the central nervous system in acute slices. Multiphoton video-rate imaging over total areas as wide as 4800 µm2 with an optical sectioning under 10 µm at 800 nm is achieved with our setup, leading to imaging of calcium dynamics of multiple cells simultaneously in thick tissue.
Highlights
A major challenge toward understanding brain function is to succeed in monitoring the activity of individual cells, such as neurons, astrocytes, oligodendrocytes and microglia, which constitute the basic functional elements of the nervous system
Optical sectioning characterization is done by scanning a thin layer (∼1 μm) of fluorescein isothiocyanate (FITC) through the focal plane
The fluorescence intensity is fitted by a Lorentzian function and the system optical sectioning is defined as the full-width at half-maximum of that function
Summary
A major challenge toward understanding brain function is to succeed in monitoring the activity of individual cells, such as neurons, astrocytes, oligodendrocytes and microglia, which constitute the basic functional elements of the nervous system. As these cells operate in complex networks, it is necessary to monitor their activity simultaneously over large areas. In the case of neurons, their complex dendritic arbor is thought to integrate numerous excitatory or inhibitory synaptic inputs in order to propagate information throughout the network. Understanding how individual neurons communicate and integrate information is important because such processes are believed to be misregulated in several brain disorders, making their study critical for developing therapeutic strategies
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