Biologists are increasingly interested in single-molecule approaches. In this issue, a Focus provides a biologist's guide to this relatively new field, and two papers present advances in nanoscale visualization.

Researchers generated a high-resolution snapshot of the epigenome of Arabidopsis thaliana by constructing and integrating the methylome, transcriptome and small RNAome using next-generation sequencing.

A 3D laser scanning microscopy method requiring no moving parts promises to expand the in vivo study of fast neuronal signaling at cellular and subcellular levels.

By adding new connections between unrelated genes to a gene network, researchers can investigate network robustness and evolvability.

Virtual composite images generate a map of gene expression in Drosophila embryos.

Synthetic peptides that can enter the cell and localize to mitochondria are promising tools for delivering cargo to the organelle.

Technological developments are pushing the emerging super-resolution fluorescence microscopy techniques into the world of three-dimensional imaging.

Screening for photostability in addition to color and brightness creates better fluorescent proteins.

The ability to image thick volumes with invariant high axial and lateral resolution is a challenge for existing super-resolution fluorescence microscopy techniques. The combination of a double-plane detection scheme with fluorescence photoactivation microscopy (FPALM) allows three-dimensional sub-diffraction resolution imaging of samples as thick as whole cells.

Caenorhabditis elegans is an ideal model organism for studying nerve regrowth and functional recovery after in vivo axotomy, but its high mobility makes such experiments challenging. A microfluidic device capable of transient immobilization of individual worms for high-resolution imaging and laser-based nanoaxotomy is described.

To date, the only way to array proteins with high density and high content has been to print purified proteins on a microarray surface. The next generation of nucleic acid programmable protein arrays (NAPPA) now allows thousands of proteins to be produced in situ on a microarray.

A fluorescence microscope relying entirely on focused light allows the generation of spherical focal fluorescence spots much smaller than the wavelength of light. This development, termed isoSTED, overcomes the resolution limitation imposed by the diffraction of light and permits three-dimensional nanoscale imaging inside cells with common fluorophores.

Improved photostability of fluorescent proteins would benefit many applications but is usually an afterthought in selection screens. Setting photostability as the primary selection criterion in screens for improved fluorescent proteins yielded highly photostable variants of existing orange and red fluorescent proteins without compromising other beneficial characteristics.

Analysis of intracellular redox-based processes is constrained by the limited choice of appropriate biosensors. Fusion of human glutaredoxin-1 to an existing redox-sensitive GFP results in a ratiometric biosensor that allows rapid and sensitive dynamic imaging of glutathione redox potential in living cells.

Many extracellular receptors are organized into complexes that may have functional implications. A combination of snap-tag protein labeling technology with time-resolved fluorescence resonance energy transfer (FRET) provides a method for the systematic analysis of higher-order protein-protein interactions on the surface of living cells.

In less than five years the Nano/Bio Interface Center at the University of Pennsylvania has gone from an idea to a nationally funded nanotechnology center. A look inside reveals how they have taken a collaborative approach to technology development.