Abstract
Super-resolution (SR) microscopy techniques have been advancing the understanding of neuronal protein networks and interactions. Unraveling the arrangement of proteins with molecular resolution provided novel insights into neuron cytoskeleton structure and actin polymerization dynamics in synaptic spines. Recent improvements in quantitative SR imaging have been applied to synaptic protein clusters and with improved multiplexing technology, the interplay of multiple protein partners in synaptic active zones has been elucidated. While all SR techniques come with benefits and drawbacks, true molecular quantification is a major challenge with the most complex requirements for labeling reagents and careful experimental design. In this perspective, we provide an overview of quantitative SR multiplexing and discuss in greater detail the quantification and multiplexing capabilities of the SR technique DNA-PAINT. Using predictable binding kinetics of short oligonucleotides, DNA-PAINT provides two unique approaches to address multiplexed molecular quantification: qPAINT and Exchange-PAINT. With precise and accurate quantification and spectrally unlimited multiplexing, DNA-PAINT offers an attractive route to unravel complex protein interaction networks in neurons. Finally, while the SR community has been pushing technological advances from an imaging technique perspective, the development of universally available, small, efficient, and quantitative labels remains a major challenge in the field.
Highlights
In recent years, SR microscopy has been a rising technique to investigate complex biological systems and molecular mechanisms
If researchers only aim to address a small number of targets, several different approaches can be implemented
Among the most sophisticated labeling approaches are the use of primary nanobodies, which combine all the advantages of stochiometric labeling and small linkage error, but they are only available for very few targets
Summary
SR microscopy has been a rising technique to investigate complex biological systems and molecular mechanisms. This approach is called Qpaint (Jungmann et al, 2016) and uses the predictable second-order association kinetics of imager strands to their docking strands to obtain quantitative molecule numbers (Figure 1C).
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