Single-Molecule Imaging Measurements of Protein-Protein Interactions in Living Cells

Even though we have several techniques, represented by the electron microscopy, to obtain images of single molecules, in this chapter, we use ‘single-molecule imaging’ (SMI) for a lim‐ ited means—that is, imaging of fluorescently labeled biological molecules at work for ana‐ lyzing their behaviors. To observe biological molecules at work, imaging in aqueous conditions is essential. Therefore, optical microscopy is the main technology in SMI. Fluores‐ cence labeling is good to use for imaging in optical microscopy, because it allows high con‐ trast and selective imaging of molecules that we are interested in. SMI provides information of dynamics and kinetics of molecular reactions. In 1995, two groups firstly and independ‐ ently realized SMI of biological molecules in aqueous conditions [1,2]. In the early days, SMI was used mainly for the in vitro studies of protein motors [1,2] and metabolic enzymes [3]. Detection of enzymatic reaction (reaction kinetics) [1,3] and detection of protein dynamics (lateral and rotational movements) [2] have been the two main usages of SMI since the first development of this technology. After that, the application of SMI has been extended, and in 2000, SMI became to be used in living cells [4,5].