PALMsiever: a tool to turn raw data into results for single-molecule localization microscopy

During the past decade, localization microscopy (LM) has transformed into an accessible, commercially available technique for life sciences. However, data processing can be challenging to the non-specialist and care is still needed to produce meaningful results. PALMsiever has been developed to provide a user-friendly means of visualizing, filtering and analyzing LM data. It includes drift correction, clustering, intelligent line profiles, many rendering algorithms and 3D data visualization. It incorporates the main analysis and data processing modalities used by experts in the field, as well as several new features we developed, and makes them broadly accessible. It can easily be extended via plugins and is provided as free of charge open-source software. Contact: [email protected]

Live-Cell dSTORM of Cellular DNA Based on Direct DNA Labeling

We have implemented the super-resolution method of direct stochastic optical reconstruction microscopy (dSTORM) to image nuclear and mitochondrial DNA in living cells. We also demonstrate time-lapse imaging, all using a dye that associates directly with cellular DNA: the commercially available dye Picogree

Making giant unilamellar vesicles via hydration of a lipid film

This unit describes protocols for making giant unilamellar vesicles (GUVs) based on rehydration of dried lipid films. These model membranes are useful for determining the impact of membrane and membrane-binding components on lipid bilayer stiffness and phase behavior. Due to their large size, they are especially amenable to studies using fluorescence and light microscopy, and may also be manipulated for mechanical measurements with optical traps or micropipets. In addition to their use in encapsulation, GUVs have proven to be useful model systems for studying many cellular processes, including tubulation, budding, and fusion, as well as peptide insertion. The introduction of enzymes or proteins can result in reorganization, leading to such diverse behavior as vesicle aggregation, fusion, and fission

Quantitative super-resolution imaging reveals protein stoichiometry and nanoscale morphology of assembling HIV-Gag virions

The HIV structural protein Gag assembles to form spherical particles of radius ∼70 nm. During the assembly process, the number of Gag proteins increases over several orders of magnitude from a few at nucleation to thousands at completion. The challenge in studying protein assembly lies in the fact that current methods such as standard fluorescence or electron microscopy techniques cannot access all stages of the assembly process in a cellular context. Here, we demonstrate an approach using super-resolution fluorescence imaging that permits quantitative morphological and molecular counting analysis over a wide range of protein cluster sizes. We applied this technique to the analysis of hundreds of HIV-Gag clusters at the cellular plasma membrane, thus elucidating how different fluorescent labels can change the assembly of virions

Resolution Doubling in 3D-STORM Imaging through Improved Buffers

Super-resolution imaging methods have revolutionized fluorescence microscopy by revealing the nanoscale organization of labeled proteins. In particular, single-molecule methods such as Stochastic Optical Reconstruction Microscopy (STORM) provide resolutions down to a few tens of nanometers by exploiting the cycling of dyes between fluorescent and non-fluorescent states to obtain a sparse population of emitters and precisely localizing them individually. This cycling of dyes is commonly induced by adding different chemicals, which are combined to create a STORM buffer. Despite their importance, the composition of these buffers has scarcely evolved since they were first introduced, fundamentally limiting what can be resolved with STORM. By identifying a new chemical suitable for STORM and optimizing the buffer composition for Alexa-647, we significantly increased the number of photons emitted per cycle by each dye, providing a simple means to enhance the resolution of STORM independently of the optical setup used. Using this buffer to perform 3D-STORM on biological samples, we obtained images with better than 10 nanometer lateral and 30 nanometer axial resolution

Multicolor Single Molecule Tracking of Stochastically Active Synthetic Dyes

Single particle tracking can reveal dynamic information at the scale of single molecules in living cells but thus far has been limited either in the range of potential protein targets or in the quality and number of tracks attainable. We demonstrate a new approach to single molecule tracking by using the blinking properties of synthetic dyes targeted to proteins of interest with genetically encoded tags to generate high-density tracks while maintaining flexibility in protein labeling. We track membrane proteins using different combinations of dyes and show that the concept can be extended to three-color imaging. Moreover, we show that this technique is not limited to the membrane by performing live tracking of proteins in intracellular compartments