Abstract
Emerging all-optical methods provide unique possibilities for noninvasive studies of physiological processes at the cellular and subcellular scale. On the one hand, superresolution microscopy enables observation of living samples with nanometer resolution. On the other hand, light can be used to stimulate cells due to the advent of optogenetics and photolyzable neurotransmitters. To exploit the full potential of optical stimulation, light must be delivered to specific cells or even parts of cells such as dendritic spines. This can be achieved with computer generated holography (CGH), which shapes light to arbitrary patterns by phase-only modulation. We demonstrate here in detail how CGH can be incorporated into a stimulated emission depletion (STED) microscope for photostimulation of neurons and monitoring of nanoscale morphological changes. We implement an original optical system to allow simultaneous holographic photostimulation and superresolution STED imaging. We present how synapses can be clearly visualized in live cells using membrane stains either with lipophilic organic dyes or with fluorescent proteins. We demonstrate the capabilities of this microscope to precisely monitor morphological changes of dendritic spines after stimulation. These all-optical methods for cell stimulation and monitoring are expected to spread to various fields of biological research in neuroscience and beyond.
Highlights
We demonstrate the capabilities of this microscope to precisely monitor morphological changes of dendritic spines after holographic uncaging of glutamate at individually targeted spines
Aberrations introduced in the computer generated holography (CGH) beam path are less critical as they can be corrected by adding compensating phase profiles to the holograms on the phase modulator
We have presented an optical system for noninvasive control and observation of neuronal processes like synaptic plasticity
Summary
Optical developments of the last years provide unique possibilities for the observation and active control of physiological processes at the cellular and subcellular scale.[1]Recordings with high spatial resolution at physiological conditions can be provided by optical superresolution methods.[2,3] stimulated emission depletion (STED) microscopy[2,4] achieves spatial resolution in the nanometer range[5,6] and temporal resolution up to the millisecond range.[7,8,9,10] Due to this high time resolution and compatibility with living thick tissues, it is a valuable tool for functional imaging in neuroscience.The ability to observe neurons at the subcellular scale of dendrites, axons, and synapses is critical for understanding neuronal function. Optical developments of the last years provide unique possibilities for the observation and active control of physiological processes at the cellular and subcellular scale.[1]. The postsynaptic site of most excitatory synapses is formed by spines, which are protrusions from dendrites with dimensions varying from below 100 nm for their neck diameter up to a few microns for total length. The morphology of these spines plays an essential role for the synaptic function,[11,12] for synaptic plasticity,[13,14] and, more generally, for brain development.[15] It was found that the size of spines
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