Abstract
BackgroundAdvanced light microscopy offers sensitive and non-invasive means to image neural activity and to control signaling with photolysable molecules and, recently, light-gated channels. These approaches require precise and yet flexible light excitation patterns. For synchronous stimulation of subsets of cells, they also require large excitation areas with millisecond and micrometric resolution. We have recently developed a new method for such optical control using a phase holographic modulation of optical wave-fronts, which minimizes power loss, enables rapid switching between excitation patterns, and allows a true 3D sculpting of the excitation volumes. In previous studies we have used holographic photololysis to control glutamate uncaging on single neuronal cells. Here, we extend the use of holographic photolysis for the excitation of multiple neurons and of glial cells.Methods/Principal FindingsThe system combines a liquid crystal device for holographic patterned photostimulation, high-resolution optical imaging, the HiLo microscopy, to define the stimulated regions and a conventional Ca2+ imaging system to detect neural activity. By means of electrophysiological recordings and calcium imaging in acute hippocampal slices, we show that the use of excitation patterns precisely tailored to the shape of multiple neuronal somata represents a very efficient way for the simultaneous excitation of a group of neurons. In addition, we demonstrate that fast shaped illumination patterns also induce reliable responses in single glial cells.Conclusions/SignificanceWe show that the main advantage of holographic illumination is that it allows for an efficient excitation of multiple cells with a spatiotemporal resolution unachievable with other existing approaches. Although this paper focuses on the photoactivation of caged molecules, our approach will surely prove very efficient for other probes, such as light-gated channels, genetically encoded photoactivatable proteins, photoactivatable fluorescent proteins, and voltage-sensitive dyes.
Highlights
In recent years, the use of advanced optical techniques has been generating a continuously growing interest in the field of neurobiology for visualizing neuronal structures and signaling processes, and for controlling neuronal and glial cell activity
We have shown that for large excitation areas holographic illumination has a significantly higher axial resolution than a Gaussian beam and further optical confinement can be achieved in two-photon (2P) holographic illumination combined with temporal focusing (TF) [21,22]
By means of electrophysiological recordings and calcium imaging in acute hippocampal slices, we show that the use of excitation patterns precisely tailored to the shape of multiple neuronal somata represents a very efficient method for the simultaneous excitation of a group of neurons
Summary
The use of advanced optical techniques has been generating a continuously growing interest in the field of neurobiology for visualizing neuronal structures and signaling processes, and for controlling neuronal and glial cell activity This has been made possible by a rapidly expanding set of photosensitive neurotransmitters that can be precisely controlled by light excitation (photolysis) [1]. In conjunction with spatiotemporally resolved photo-stimulation techniques, these photosensible tools represent the most promising alternative to electrical stimulation, providing ways to control precisely in space and time the activity of specific types of brain cells These approaches require fast, flexible and precise illumination schemes, permitting a selective activation and imaging of sub-cellular regions or multi-cellular ensembles, with enough power to drive reactions quickly and fast gating. We extend the use of holographic photolysis for the excitation of multiple neurons and of glial cells
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