Fluorescence Microscopy with Nanometer Resolution

Throughout the twentieth century, it was widely accepted that a light microscope relying on propagating light waves and conventional optical lenses could not discern details that were much finer than about half the wavelength of light, or 200−400nm, due to diffraction. However, in the 1990s, the potential for overcoming the diffraction barrier was realized, and microscopy concepts were defined that now resolve fluorescent features down to molecular dimensions. This chapter discusses the simple yet powerful principles that make it possible to neutralize the resolution-limiting role of diffraction in far-field fluorescence nanoscopy methods such as STED, RESOLFT, PALM/"​"​STORM, or PAINT. In a nutshell, feature molecules residing closer than the diffraction barrier are transferred to different (quantum) states, usually a bright fluorescent state and a dark state, so that they become discernible for a brief period of detection. With nanoscopy, the interior of transparent samples, such as living cells and tissues, can be imaged at the nanoscale. A fresh look at the foundations shows that an in-depth description of the basic principles spawns powerful new concepts. Although they differ in some aspects, these concepts harness a local intensity minimum (of a doughnut-shaped or a standing wave pattern) for determining the coordinate of the fluorophore(s) to be registered. Most strikingly, by using an intensity minimum of the excitation light to establish the fluorophore position, MINFLUX nanoscopy has obtained the ultimate (super)resolution: the size of a molecule (≈1nm)