Showing papers by "Helge Ewers published in 2012"

A simple, versatile method for GFP-based super-resolution microscopy via nanobodies

Abstract: We developed a method to use any GFP-tagged construct in single-molecule super-resolution microscopy By targeting GFP with small, high-affinity antibodies coupled to organic dyes, we achieved nanometer spatial resolution and minimal linkage error when analyzing microtubules, living neurons and yeast cells We show that in combination with libraries encoding GFP-tagged proteins, virtually any known protein can immediately be used in super-resolution microscopy and that simplified labeling schemes allow high-throughput super-resolution imaging

mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities

Abstract: Recent advances in fluorescence microscopy have extended the spatial resolution to the nanometer scale. Here, we report an engineered photoconvertible fluorescent protein (pcFP) variant, designated as mMaple, that is suited for use in multiple conventional and super-resolution imaging modalities, specifically, widefield and confocal microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy. We demonstrate the versatility of mMaple by obtaining super-resolution images of protein organization in Escherichia coli and conventional fluorescence images of mammalian cells. Beneficial features of mMaple include high photostability of the green state when expressed in mammalian cells and high steady state intracellular protein concentration of functional protein when expressed in E. coli. mMaple thus enables both fast live-cell ensemble imaging and high precision single molecule localization for a single pcFP-containing construct.

Analysis of Virus Entry and Cellular Membrane Dynamics by Single Particle Tracking

Abstract: Viruses have evolved to mimic cellular ligands in order to gain access to their host cells for replication. Since viruses are simple in structure, they rely on host cells for all their transportation needs. Following single virus particles during the initial phase of infection, that is, virus entry into target cells, can reveal crucial information on the mechanism of pathogen infections and likewise cellular transport and membrane dynamics. Here, we give an overview on how to fluorescently label virus particles for live cell microscopy, and on how virus entry can be analyzed by single particle tracking experiments. Highlighted are strategies, on how to chemically introduce fluorophores into virions, and on how to extract quantitative information from live cell data.

Nano Resolution Optical Imaging Through Localization Microscopy

Abstract: The recent development of superresolution microscopy methods has found great resonance in the neurosciences. One approach is based on the stochastic fluorescence-activation and subsequent localization of many molecules one by one over time to generate an outline from the cumulative localizations. Not only does this technique yield an outline of the labeled structure, it also outlines the position of the many molecules that constitute the structure of interest, and is especially appealing for the study of the dense, complex, and dynamic multiprotein structures found at synapses. Here, thousands of molecules are spatially organized within an area only slightly larger than a single diffraction-limited spot and it is likely that their spatial organization will reflect an aspect of their function. A detailed investigation of the relative position of the receptor, scaffold, signaling, and structural molecules within the postsynaptic density; the vesicle fusion; and recycling machinery at the presynapse and the perisynaptic trafficking and metabolic structures seems within reach with the novel single molecule localization techniques. This will undoubtedly lead to important insights into synaptic cell biology. In this chapter the different technical approaches, available probes, and labeling techniques and their potential for the investigation of neuronal and synaptic cell biology is discussed.