Fluorescence microscopy for visualizing single-molecule protein dynamics

Background: Single-molecule fluorescence imaging (smFI) has evolved into a valuable method used in biophysical and biochemical studies as it can observe the real-time behavior of individual protein molecules, enabling understanding of their detailed dynamic features. smFI is also closely related to other state-of-the-art microscopic methods, optics, and nanomaterials in that smFI and these technologies have developed synergistically. Scope of review: This paper provides an overview of the recently developed single-molecule fluorescence microscopy methods, focusing on critical techniques employed in higher-precision measurements in vitro and fluorescent nanodiamond, an emerging promising fluorophore that will improve single-molecule fluorescence microscopy. Major conclusions: smFI will continue to improve regarding the photostability of fluorophores and will develop via combination with other techniques based on nanofabrication, single-molecule manipulation, and so on. General significance: Quantitative, high-resolution single-molecule studies will help establish an understanding of protein dynamics and complex biomolecular systems.