Abstract:

In recent years three powerful optical imaging techniques have emerged that provide nanometer-scale information about the topography of membrane surfaces, whether cellular or artificial: intermembrane fluorescence resonance energy transfer (FRET), fluorescence interference contrast microscopy (FLIC), and reflection interference contrast microscopy (RICM). In intermembrane FRET, the sharp distance dependence of resonant energy transfer between fluorophores allows topographic measurements in the Ångstrom to few-nanometer range. In FLIC and RICM, interference between light from a membrane (either from fluorescent probes, or reflected illumination) and light reflected by a planar substrate provide spatial sensitivity in the few to hundreds of nanometer range, with few-nanometer resolution. All of these techniques are fairly easy to implement. We discuss the physics and optics behind each of these tools, as well as practical concerns regarding their uses. We also provide examples of their application in imaging molecular-scale structures at intermembrane junctions.

Keywords: Membrane topography; interference contrast microscopy; FRET; FLIC; RICM

Document Type: Research article

DOI: 10.1385/CBB:41:3:391

Affiliations: 1: Department of Chemistry, University of California, 94720, Berkeley, CA, 2: Department of Chemistry, University of California, 94720, Berkeley, CA, Email: JTGroves@LBL.gov