Single molecule localization-based superresolution microscopy methods, such as PALM or STORM, have been breakthrough techniques of the last years. Until now however, they require 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, as demonstrated on microtubules, living neurons and yeast cells. 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.

The labeling density in superresolution microscopy based on photoactivatable fluorophores is limited by the fact that a small, but significant fraction is always in the bright state. To overcome this limitation we implemented binding-activated localization microscopy (BALM), which is based on the localization of individual binding events of fluorophores that show a fluorescence enhancement upon binding to their target structures. Using nucleic acid stains on double-stranded DNA we yielded a resolution of ∼14 nm (fwhm) and a spatial sampling of 1/nm in vitro and could visualize the organization of the bacterial chromosome in fixed Escherichia coli cells. In general, the principle of binding-activated localization microscopy can be extended to other dyes and targets such as protein structures.


Wednesday, June 12, 2013, 10:30. Seminar Room

Hosted by Prof. Melike Lakadamyali