reSOLUTION_Research_09_Neuroscience - Leica Microsystems

suPerresolution
fig. 2: single synapses of different
sizes show considerable structural
rearrangement. sted images of gfP
show dliprin-αgfP as discrete dots
arranged around the synapse core labeled
by brPnc82 (magenta), ranging
from one to two dots at small, freshly
assembled synapses and from four
to five dots at mature ones. these architectural
features weren‘t stipulated
in em pictures nor in confocal images,
demonstrating the importance of sted
analysis for structural characterization
at the drosophila neuromuscular junction.
scale bar: 250 nm.
12 resolutioN
em often involves quite elaborate dehydration and
contrasting procedures. even though very small
structures are nicely displayed, attributing the visualized
structures to the localization of one or more
proteins via immuno-labeling remains tricky. furthermore,
time-consuming difficulties arise when high
resolution images are needed from bigger or thicker
samples, and several em slices need to be merged or
reconstructed.
recent advances in light microscopy, such as the development
of sted microscopy 1 , greatly contribute to
this issue by offering a revolutionary simple method
of fluorescence visualization with image resolution
ranging down to 30 nm, and creating the fully new
concept of nanobiophotonics.
contributions of sted to neurobiology
conventional fluorescent microscopy is perfectly suited
for analysis of a biological specimen, since the localization
of fluorescent dyes is easily assessed in both
fixed and living specimens. sted goes one step further
by enabling the detailed discrimination of even
smaller cellular organelles and sub-compartments. in
neurobiology many considerable achievements have
been made, as described in a few examples below:
synaptic vesicles (50 – 80 nm) are transport units used
by the cell to harvest neurotransmitters, which on
demand, are fused with the presynaptic membrane and
release their content into the synaptic cleft. understanding
the process of how such vesicles are formed,
transported, and docked to the proper release
site and how the endo/exocytosis vesicle recycling
works is hugely important to the scientific community.
recently, video rate sted imaging of live specimens
was used to describe vesicle mobility along
axons 2 . With the help of sted, the transport of vesicles
was described more precisely, detecting even small
changes in speed and direction otherwise unrecognizable
in conventional image acquisition. in other
experiments 3 the localization of a synaptic vesicle’s
associated protein (synaptotagmin) was characterized
upon vesicle fusion. their findings contributed
to an overall understanding of how vesicle-specific
proteins may be retrieved from the plasma membrane
during endocytosis.
temporal aspects of how single components of the
synapse are incorporated into the protein matrix
throughout synapse maturation, e.g. via synaptic precursor
vesicles, are not yet fully understood.
studies on the drosophila nmJ were performed
to analyze the synapse structure and assembly 4,5,6 .
Presynaptic electron dense structures named “tbars”
(owing to their characteristic shape in electron
micrographs) were shown to comprise bruchpilot
(brP). brP is thought to play a role in signal transduction
by acting as a presynaptic scaffolding protein.
through the application of sted technology, in
a synergistic combination with established imaging
techniques, valuable information concerning the architecture
of the roughly 250 nm size t-bar and adjacent
structures was obtained (fig. 1). similar studies
as in drosophila were performed on murine retina
cells, where the composition of presynaptic proteins
associated to precursor vesicles, which are thought
to promote synaptogenesis, was described 7 .
in the examples above, sted microscopy revealed
a very precise distribution of fluorescently-tagged
synaptic proteins, which until then were unrecognizable
via conventional confocal imaging. When compared
to data from electron micrographs, a whole
new set of information was retrieved. but unlike em,
sted, due to its simple methodology, allowed image
acquisition not only on an uncomplicated and quick
fashion, but also in a larger scale, thereby assisting in