Recent applications of superresolution microscopy in neurobiology

Abstract

Chemical synapses in brain are structural differentiations where excitatory or inhibitory signals are vectorially transmitted between two neurons. Excitatory synapses occur mostly on dendritic spines, submicron sized protrusions of the neuronal dendritic arborizations. Axons establish contacts with these tiny specializations purported to be the smallest functional processing units in the central nervous system. The minute size of synapses and their macromolecular constituents creates an inherent difficulty for imaging but makes them an ideal object for superresolution microscopy. Here we discuss some representative examples of nanoscopy studies, ranging from quantification of receptors and scaffolding proteins in postsynaptic densities and their dynamic behavior, to imaging of synaptic vesicle proteins and dendritic spines in living neurons or even live animals.

A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy

“…[1][2][3][4][5][6][7][8][9] In STED imaging,afluorescent dye is exposed to irradiation from two different laser sources,namely an excitation laser and aSTED beam, where the STED beam is adonut-shaped beam for the depletion of excited molecules.T he spatial resolution of STED imaging increases with the I STED /I sat ratio,where I STED is the STED beam intensity and I sat represents the saturation intensity at 50 %depletion. We herein developed ap hotoresistant fluorescent dye C-Naphoxa sapractical tool for STED imaging.Withexcitation using either a l = 405 or 488 nm laser in protic solvents,C -Naphox exhibited an intense red/orange fluorescence (quantum yield F F > 0.7) with alarge Stokes shift (circa 5900 cm À1 ).…”

“…[1][2][3][4][5][6][7][8][9] In STED imaging,afluorescent dye is exposed to irradiation from two different laser sources,namely an excitation laser and aSTED beam, where the STED beam is adonut-shaped beam for the depletion of excited molecules.T he spatial resolution of STED imaging increases with the I STED /I sat ratio,where I STED is the STED beam intensity and I sat represents the saturation intensity at 50 %depletion. [1][2][3][4][5][6][7][8][9] In STED imaging,afluorescent dye is exposed to irradiation from two different laser sources,namely an excitation laser and aSTED beam, where the STED beam is adonut-shaped beam for the depletion of excited molecules.T he spatial resolution of STED imaging increases with the I STED /I sat ratio,where I STED is the STED beam intensity and I sat represents the saturation intensity at 50 %depletion.…”

Nanoclustering as a dominant feature of plasma membrane organization

“…Whereas examples of nanoclustering in neurobiology can be found in a recent review (Willig and Barrantes, 2014), we will focus here on some other salient examples of receptor nanoclustering, including those involved in pathogen binding, cell adhesion and the immune response.…”

Cell-surface translational dynamics of nicotinic acetylcholine receptors

“…Fluorescence microscopy has recently experienced a series of interesting developments that have opened new avenues to study the fine structure and dynamics of synaptic constituents with unprecedented resolution (see recent reviews in Eggeling et al, 2013; Willig and Barrantes, 2014). Localization-based superresolution imaging techniques based on stochastic activation of photo-convertible (switchable) fluorescent probes have in theory inherently low temporal resolution because of the need to collect a considerable number of photons per molecule to accurately localize a given particle.…”