Single-molecule localization based superresolution microscopy (localization microscopy) nowadays reaches a resolution sufficient to determine structures of protein assemblies in the cellular context. It is therefore a technique complementary to classical structural techniques such as x-ray crystallography or electron microscopy to investigate, how molecular machines are organized.
Here we present two alternative labeling schemes for localization microscopy and report on our progress towards resolving a fundamental multi-protein machinery on the nanometer scale, namely the endocytotic machinery in S. cerevisiae.
Localization microscopy requires a high degree of labeling with bright and switchable dyes. Until now however, this required special fluorescent proteins to be cloned or high-affinity antibodies to be generated for specific labeling. On the other hand, many laboratories will have most of their constructs in GFP form and entire genomes are available as functional GFP-fusion proteins. Here, we report a method that makes all these constructs available for superresolution microscopy by targeting GFP with tiny, high-affinity antibodies coupled to blinking dyes . It thus combines the molecular specificity of genetic tagging with the high photon yield of organic dyes and minimal linkage error. We show that in combination with GFP-libraries, virtually any known protein can immediately be used in superresolution microscopy and that high-throughput superresolution imaging using simplified labeling schemes is possible.
As an alternative to using photo-switchable fluorophores, we introduce binding-activated localization microscopy (BALM), which employs fluorescence enhancement of fluorogenic dyes upon binding to target structures for superresolution microscopy. We used this approach to study DNA structures  and a-synuclein amyloids  and could demonstrate a superb labeling density combined with a very high resolution.
Endocytosis is a highly intricate cellular process, which in yeast involves the ordered recruitment and disassembly of around 60 proteins. Our current efforts focus on understanding the intermediate and late coat assembly preceding scission. Here, we were able to reveal subdiffraction features regarding shape and structure of endocytic coat proteins that were previously inaccessible. By visualizing many proteins pairs with dual-color superresolution microscopy, we are pursuing to obtain a comprehensive structural picture of the endocytic proteome.
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