Structural basis of astrocytic Ca2+ signals at tripartite synapses.

Astrocytic Ca2+ signals can be fast and local, supporting the idea that astrocytes have the ability to regulate single synapses. However, the anatomical basis of such specific signaling remains unclear, owing to difficulties in resolving the spongiform domain of astrocytes where most tripartite synapses are located. Using 3D-STED microscopy in living organotypic brain slices, we imaged the spongiform domain of astrocytes and observed a reticular meshwork of nodes and shafts that often formed loop-like structures. These anatomical features were also observed in acute hippocampal slices and in barrel cortex in vivo. The majority of dendritic spines were contacted by nodes and their sizes were correlated. FRAP experiments and Ca2+ imaging showed that nodes were biochemical compartments and Ca2+ microdomains. Mapping astrocytic Ca2+ signals onto STED images of nodes and dendritic spines showed they were associated with individual synapses. Here, we report on the nanoscale organization of astrocytes, identifying nodes as a functional astrocytic component of tripartite synapses that may enable synapse-specific communication between neurons and astrocytes.

Two-Photon STED Microscopy for Nanoscale Imaging of Neural Morphology In Vivo.

The advent of super-resolution microscopy offers to bridge the gap between electron and light microscopy. It has opened up the possibility of visualizing cellular structures and dynamic signaling events on the "mesoscale" well below the classic diffraction barrier of light microscopy (10-200 nm), while essentially retaining the advantages of fluorescence microscopy concerning multicolor labeling, detection sensitivity, signal contrast, live-cell imaging, and temporal resolution.From among the new super-resolution techniques, STED microscopy stands out as a laser-scanning imaging modality, which enables nanoscale volume-metric imaging of cellular morphology. In combination with two-photon (2P) excitation, STED microscopy facilitates the visualization of the highly complex and dynamic morphology of neurons and glia cells deep inside living brain slices and in the intact brain in vivo.Here, we present an overview of the principles and implementation of 2P-STED microscopy in vivo, providing the neurobiological context and motivation for this technique, and illustrating its capacity by showing images of dendritic spines and microglial processes obtained from living brain tissue.

Excited state proton transfer in strongly enhanced GFP (sGFP2).

Proton transfer is an elementary process in biology. Green fluorescent protein (GFP) has served as an important model system to elucidate the mechanistic details of this reaction, because in GFP proton transfer can be induced by light absorption. We have used pump-dump-probe spectroscopy to study how proton transfer through the 'proton-wire' around the chromophore is affected by a combination of mutations in a modern GFP variety (sGFP2). The results indicate that in H(2)O, after absorption of a photon, a proton is transferred (A* → I*) in 5 ps, and back-transferred from a ground state intermediate (I → A) in 0.3 ns, similar to time constants found with GFPuv, although sGFP2 shows less heterogeneous proton transfer. This suggests that the mutations left the proton-transfer largely unchanged, indicating the robustness of the proton-wire. We used pump-dump-probe spectroscopy in combination with target analysis to probe suitability of the sGFP2 fluorophore for super-resolution microscopy.