A set of homo-oligomeric standards allows accurate protein counting.

Techniques based on fluorescence microscopy are increasingly used to count proteins in cells, but few stoichiometrically well-defined standards are available to test their accuracy. A selection of bacterial homo-oligomers were developed that contain 10-24 subunits and fully assemble when expressed in mammalian cells, and they can be used to easily validate/calibrate molecular counting methods. The utility of these standards was demonstrated by showing that nuclear pores contain 32 copies of the Nup107 complex.

Resolving bundled microtubules using anti-tubulin nanobodies.

Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20-40 nm and will thereby blend neighbouring microtubules into one structure.

Click chemistry facilitates direct labelling and super-resolution imaging of nucleic acids and proteins†Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra01027bClick here for additional data file.

We demonstrate high-density labelling of cellular DNA and RNA using click chemistry and perform confocal and super-resolution microscopy. We visualize the crescent and ring-like structure of densely packed RNA in nucleoli. We further demonstrate click chemistry with unnatural amino acids for super-resolution imaging of outer-membrane proteins of .

Multiscale spatial organization of RNA polymerase in Escherichia coli.

Nucleic acid synthesis is spatially organized in many organisms. In bacteria, however, the spatial distribution of transcription remains obscure, owing largely to the diffraction limit of conventional light microscopy (200-300 nm). Here, we use photoactivated localization microscopy to localize individual molecules of RNA polymerase (RNAP) in Escherichia coli with a spatial resolution of ∼40 nm.

Photoswitchable fluorophores for single-molecule localization microscopy.

Over the past decade, fluorescence microscopy has been revolutionized by the development of novel techniques that allow near-molecular resolution. Many such methods-collectively referred to as "single-molecule localization microscopy" (SMLM)-are based upon the repeated imaging of sparse stochastic subsets of the fluorophores in a sample. Active fluorophores are localized by finding the centers of their point spread functions, and a super-resolution image is constructed.

T7 RNA polymerase functions in vitro without clustering.

Many nucleic acid polymerases function in clusters known as factories. We investigate whether the RNA polymerase (RNAP) of phage T7 also clusters when active. Using 'pulldowns' and fluorescence correlation spectroscopy we find that elongation complexes do not interact in vitro with a K(d)<1 µM.

Super-resolution fluorescence imaging of chromosomal DNA.

Super-resolution microscopy is a powerful tool for understanding cellular function. However one of the most important biomolecules - DNA - remains somewhat inaccessible because it cannot be effectively and appropriately labeled. Here, we demonstrate that robust and detailed super-resolution images of DNA can be produced by combining 5-ethynyl-2'-deoxyuridine (EdU) labeling using the 'click chemistry' approach and direct stochastic optical reconstruction microscopy (dSTORM).

Non-specific (entropic) forces as major determinants of the structure of mammalian chromosomes.

Four specific forces (H-bonds, van der Waals forces, hydrophobic and charge interactions) shape the structure of proteins, and many biologists assume they will determine the shape of all structures in the cell. However, as the mass and contour length of a human chromosome are ~7 orders of magnitude larger than those of a typical protein, additional forces can become significant. We review evidence that additional non-specific (entropic) forces are major determinants of chromosomal shape and position.

The depletion attraction: an underappreciated force driving cellular organization.

Cellular structures are shaped by hydrogen and ionic bonds, plus van der Waals and hydrophobic forces. In cells crowded with macromolecules, a little-known and distinct force-the "depletion attraction"-also acts. We review evidence that this force assists in the assembly of a wide range of cellular structures, ranging from the cytoskeleton to chromatin loops and whole chromosomes.