Superresolving dendritic spine morphology with STED microscopy under holographic photostimulation

Abstract

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. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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“…Apart from the challenge of a compact distribution of the labels, the precise timing of light pulses and forming the drought shape are challenges in developing the optical setup. STED is useful for subcellular nanoscopy, e.g., dendritic spines of a nerve cell [89] and organization of cytoskeleton proteins of a HeLa cell (Figure 19j). [90] Structured Illumination Microscopy (SIM): Structured illumination can be utilized for extracting high spatial frequency details of a fluorescent sample surface (which are normally beyond the resolvability of objective lenses).…”