Techniques for Single-Molecule mRNA Imaging in Living Cells

Typical measurement of macromolecules in a biological sample typically averages the result over all the cells or molecules within the sample, and while these types of measurements provide very useful information, they completely miss heterogeneity among the components within the sample that could be a very important aspect of the sample's function. These techniques are also limited in their ability to examine intracellular spatial orientation of molecular activity, which is often a critical component to the regulation of biological processes, particularly in cells with unique spatial relationships, such as neurons. This makes a strong case for single-cell and single-molecule analysis that allows similar novel insight into complex molecular machinery that would not be possible when pooling heterogeneous molecular states. mRNA has proven to be quite tractable to molecular analysis in single cells. Almost two decades of single-molecule studies of mRNA processing both in situ and in live cells have been facilitated by microscopy of mRNA. This has been made possible by multiplexing fluorophores in situ hybridization probes or fluorescent RNA-tag-binding protein probes. The purpose of this chapter is to describe the approaches that have made single-molecule mRNA imaging accessible, as well as to give an overview of the state of the art for techniques that are available to track mRNA in real time in living cells, highlighting the application to neuroscience.