Single molecules of two different mRNAs imaged in cells. Credit: A. Raj

In their quest to understand the function of biological systems some researchers have determined that classical ensemble measurements, by relying on averages, are missing spatial and temporal heterogeneity that could be crucial for understanding biological function. Now a new quest is on: to rectify this situation with experiments on single cells and single molecules.

Most single-molecule experiments, however, have been limited to in vitro experiments in which the object under study is removed from the cellular environment. This is often a necessity, but scientists are increasingly conducting single-molecule experiments in whole cells.

One process being investigated using these methods is transcriptional activity. Over the past few years researchers developed several different methods capable of detecting single mRNA molecules in cells. These methods rely on a variety of different fluorescent probing techniques that push the limits of sensitivity, and they are beginning to produce valuable biological results that could not be obtained using classical ensemble measurements.

It turns out that transcription does not produce a steady stream of mRNA molecules as might be expected based on results from ensemble experiments. In fact, mRNA molecules are transcribed during large, random bursts of gene activity. Several groups discovered this in bacteria and yeast using GFP-based methods and Arjun Raj, Sanjay Tyagi and colleagues used highly optimized fluorescence in situ hybridization to extend and expand these results to mammalian cells (PLoS Biol. 4, e309; 2006). In 2007, Sunney Xie's laboratory extended single-molecule studies of gene expression in living cells to the measurement of transcription-factor binding to DNA (Science 316, 1191–1194; 2007).

The difficulties in conducting single-molecule experiments in cells require the development of new methods though. The new super-resolution fluorescence microscopy techniques are certainly a step forward, but more developments are needed. We believe however, that the ability of single-molecule measurements to shine a light into the dark areas of biology that are invisible to traditional methods makes it a certainty that these methods will be developed in the future.