Ex-dSTORM and automated quantitative image analysis of expanded filamentous structures.

This chapter provides a step-by-step protocol how to prepare expansion microcoscopy (ExM) treated biological samples for imaging with single-molecule localization microscopy (SMLM). For this purpose, the protocol describes the stabilization of expanded hydrogels that enables addition of photoswitching buffer without shrinkage of the sample. In addition, a guide for automated image analysis and expansion factor determination of expanded fiber-like structures is provided at the end of the chapter.

Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM).

Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy.

The synaptonemal complex (SC) is a meiosis-specific nuclear multiprotein complex that is essential for proper synapsis, recombination and segregation of homologous chromosomes. We combined structured illumination microscopy (SIM) with different expansion microscopy (ExM) protocols including U-ExM, proExM, and magnified analysis of the proteome (MAP) to investigate the molecular organization of the SC. Comparison with structural data obtained by single-molecule localization microscopy of unexpanded SCs allowed us to investigate ultrastructure preservation of expanded SCs. For image analysis, we developed an automatic image processing software that enabled unbiased comparison of structural properties pre- and post-expansion. Here, MAP-SIM provided the best results and enabled reliable three-color super-resolution microscopy of the SCs of a whole set of chromosomes in a spermatocyte with 20-30 nm spatial resolution. Our data demonstrate that post-expansion labeling by MAP-SIM improves immunolabeling efficiency and allowed us thus to unravel previously hidden details of the molecular organization of SCs.

Imaging cellular ultrastructures using expansion microscopy (U-ExM).

Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.

The N-terminal domains of spider silk proteins assemble ultrafast and protected from charge screening.

Web spiders assemble spidroin monomers into silk fibres of unrivalled tensile strength at remarkably high spinning speeds of up to 1 m s(-1). The spidroin N-terminal domain contains a charge-driven, pH-sensitive relay that controls self-association by an elusive mechanism. The underlying kinetics have not yet been reported. Here we engineer a fluorescence switch into the isolated N-terminal domain from spidroin 1 of the major ampullate gland of the nursery web spider E. australis that monitors dimerization. We observe ultrafast association that is surprisingly insensitive to salt, contrasting the classical screening effects in accelerated, charged protein interfaces. To gain deeper mechanistic insight, we mutate each of the protonatable residue side chains and probe their contributions. Two vicinal aspartic acids are critically involved in an unusual process of accelerated protein association that is protected from screening by electrolytes, potentially facilitating the rapid synthesis of silk fibres by web spiders.