ABSTRACT
Over the years, uorescence microscopy has become the workhorse of almost every biology laboratory around the world. Far-eld imaging with visible light provides several advantages over methods such as electron microscopy. The immense toolbox of uorescence probes, in particular the revolution that has led to the development of a large palette of uorescent proteins, has given us the ability to label almost anything inside cells with high molecular specicity and in many colors. The noninvasive quality of visible light allows us to study dynamic biological processes in real time inside living cells or even living animals. Key technological developments have extended the capabilities of uorescence microscopes and have provided us with several different imaging modalities from total internal reection uorescence (TIRF) to confocal and two-photon microscopy. These methods have overcome barriers such as reduction of background uorescence and deep tissue imaging. However, one major barrier has remained impenetrable until recently-the diffraction limit. It has been known since the time of Ernst Abbe that structures smaller than the wavelength of light become blurred when imaged with a light microscope. As a result, two objects that are closer in distance than the wavelength of light cannot be resolved as two separate objects. The resolving power of an optical microscope can be approximated by λ/(2NA) in the lateral (x-y) and (2λn)/NA2 in the axial (z) direction, where NA is the numerical aperture of the microscope objective, λ is the wavelength of light, and n is the refractive index of the medium. For visible light and high-NA objectives, the resolution of conventional optical microscopes is limited to ~200 and ~500 nm in the lateral and axial directions, respectively. This limitation is highly problematic in biology because many structures of interest are below the diffraction limit (e.g., protein complexes, DNA, cytoskeletal laments, and viruses), and these structures are densely packed inside the crowded environment of the cell.
