Now you see it, now you don't: A biochemist's primer for advances in high-resolution microscopy: Comment on “Biomolecular dynamics and binding studies in the living cell” by Stephan Diekmann and Christian Hoischen

Abstract

For many scientists, the first glimpse of the inner workings of the world around us comes from the use of a simple light microscope. Indeed, ground glass was used to make lenses for magnifying glasses as early as the time of the Romans (Seneca, Pliny). In the mid-15th century, the Janssens put together a series of lenses in a tube, discovering the ability of compound microscopes to magnify objects greatly. This advance allowed Hooke and Leeuewenhoek to conclude that life was composed of cells, and resulted in Schleiden, Schwann, Virchows’ observations that cells regenerated new cells. Warp speed to 400 years later, with a hop in between for Coon’s tagging of antibodies, and for the development of GFP-tags by Shimomura, Chalfie, Tsien, and the humble light microscope has transformed our understanding of the natural and physical world. In recent years, revolutionary changes to microscopes and the application of powerful computational algorithms to analyze data have yielded information far surpassing the physical limits set by the diffraction of light. In this comprehensive review, Diekmann and Hoeschien discuss the application of new technologies such as FRET, FRAP, FCS, STORM/PALM, FLIM-FRET, now commonly used to illuminate the inner mysteries of the sanctum sanctorum of the cell – the nucleus. They demonstrate that with careful and rigorous measurements, an almost quantitative knowledge of the nucleus is well within our grasp. For the biochemist, geneticist, or molecular biologist, confounded by the bewildering array of acronyms denoting different techniques in light microscopy, this review is both timely, and a time saver. It describes in great detail how each advance named above can be applied to the quantitative and qualitative measurement of protein–protein interactions, positional information, and dynamics of multi-protein complexes in living cells, down to 10 nm resolution. These technological advances, as Diekmann and Hoeschien explain, are powerful in their ability to gather every pixel of information available, and when carefully analyzed, able to yield tremendous insights on the behavior of proteins in a biologically meaningful setting. The review is particularly useful in systematically and objectively comparing the benefits and the limitations of each approach, as well as highlighting the potential artifacts associated with the use of fluorophores or covalently-attached