Microscopy

Super-resolution imaging goes multicolor

Recently several different techniques for super-resolution imaging of fluorescently tagged biological molecules have been described. Until now, only up to two colors could be imaged at one time. By finding new pairs of photoswitchable dyes for use with the previously described single-molecule photoswitching method, stochastic optical reconstruction microscopy (STORM), Bates et al. now demonstrate multicolor super-resolution imaging with three colors.

Bates, M. et al. Science, published online 16 August 2007.

Genomics

Saturating the mouse genome

Multi-lab efforts are underway to create a knockout mouse for each of its ∼25,000 genes. Gragerov et al. enrich the available toolbox with a retroviral insertion strategy that leads to saturating random mutagenesis in 90% of the genome. With a targeted selection for the mutated embyonic stem cells, they can efficiently and rapidly identify knockout clones for almost any gene of interest.

Gragerov, A. et al. Proc. Nat. Acad. Sci. USA 104, 14406–14411 (2007).

Proteomics

Assessing ETD

Electron transfer dissociation (ETD) is a relatively new method for fragmenting peptides and proteins within a mass spectrometer to obtain more information about their molecular structures. With a systematic large-scale analysis, Good et al. demonstrate that ETD is highly complementary to the more traditional fragmentation method known as collision-activated dissociation (CAD), by showing that there was relatively little overlap in peptides identified by each of the methods.

Good, D.M. et al. Mol. Cell. Proteomics, published online 1 August 2007.

Protein biochemistry

Genetic selection with tunable stringency

Selection schemes are often based on complementation of a metabolic defect in a model microorganism. Kleeb et al. now design flexible stringency selection by adopting strategies similar to those that control metabolic flux in vivo. By providing variable amounts of an enzyme that shunts a given metabolite down an alternative pathway in shikimate biosynthesis, they were able to select for bacterial dehydratases with activities over a 50,000-fold range.

Kleeb, A.C. et al. Proc. Natl. Acad. Sci. USA 104, 13907–13912 (2007).

Imaging and visualization

A molecular thermometer based on EGFP

Fluorescent proteins such as enhanced green fluorescent protein (EGFP) fluctuate between fluorescent and non-fluorescent states, a process known as blinking. Wong et al. observed that fluorescence blinking is strongly correlated to temperature and, as such, demonstrate that EGFP can be used as a molecular thermometer.

Wong, F.H.C. et al. J. Am. Chem. Soc. 129, 10302–10303 (2007).