Super-resolution microscopy is the term commonly given to fluorescence microscopy techniques with resolutions that are not limited by the diffraction of light. Since their conception a little over a decade ago, these techniques have quickly become the method of choice for many biologists studying structures and processes of single cells at the nanoscale. In this review, we present the three main approaches used to tackle the diffraction barrier of ∼200 nm: stimulated-emission depletion (STED) microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM). We first present a theoretical overview of the techniques and underlying physics, followed by a practical guide to all of the facets involved in designing a super-resolution experiment, including an approachable explanation of the photochemistry involved, labeling methods available, and sample preparation procedures. Finally, we highlight some of the most exciting recent applications of and developments in these techniques, and discuss the outlook for this field.Keywords Super-resolution microscopy . Photophysics and photochemistry of fluorophores . Live cell imaging . Quantitative cell biology Spatial and temporal scales in the life sciences and microscopyThe timescales and spatial scales of the processes and molecules associated with life span extremely broad ranges, covering many orders of magnitude (Fig. 1). For instance, intracellular regulation (e.g., conformational changes or biochemical reactions within molecules) takes place at submillisecond timescales, nanosized molecules such as ATP (which serves the energy demands of cells) diffuse in milliseconds through cell volumes ranging from several micrometers up to millimeters, while (clustered) membrane receptors move at speeds that are about a magnitude slower. Large multicomponent machineries realize and control complex multilayered cellular functions that occur in seconds to hours. The ribosome, a large macromolecule which consists of two functional subunits of several dozen proteins on nucleic acid chain scaffolds, takes a matter of seconds to synthesize new peptide chains comprising hundreds of amino acids, which then quickly fold up into functional proteins. On the other hand, the replication of a full genome requires at least about 40 min for the 4.6 million nucleic acid base pairs of the bacterium Escherichia coli, and the cellular division cycle ranges from tens of minutes for E. coli to several hours for mammalian cells.Observing and understanding all of these components of life requires us to be, at best, passive witnesses of undisturbed processes, but also to demand hard observational data that can allow us to quantitatively measure and trace all of the players involved-ranging from small molecules up to the interactions of whole cells in cellular communities-with the highest specificity and precision.To achieve this, instrumentation is needed that permits a wide three-dimensional view but also allows details to be

Photoconversion of fluorescent proteins by blue and complementary near-infrared light, termed primed conversion (PC), is a mechanism recently discovered for Dendra2. We demonstrate that controlling the conformation of arginine at residue 66 by threonine at residue 69 of fluorescent proteins from Anthozoan families (Dendra2, mMaple, Eos, mKikGR, pcDronpa protein families) represents a general route to facilitate PC. Mutations of alanine 159 or serine 173, which are known to influence chromophore flexibility and allow for reversible photoswitching, prevent PC. In addition, we report enhanced photoconversion for pcDronpa variants with asparagine 116. We demonstrate live-cell single-molecule imaging with reduced phototoxicity using PC and record trajectories of RNA polymerase in Escherichia coli cells.

Super-resolution fluorescence microscopy plays a major role in revealing the organization and dynamics of living cells. Nevertheless, single-molecule localization microscopy imaging of multiple targets is still limited by the availability of suitable fluorophore combinations. Here, we introduce a novel imaging strategy which combines primed photoconversion (PC) and UV-photoactivation for imaging different molecular species tagged by suitable fluorescent protein combinations. In this approach, the fluorescent proteins can be specifically photoactivated/-converted by different light wavelengths using PC and UV-activation modes but emit fluorescence in the same spectral emission channel. We demonstrate that this aberration-free, live-cell compatible imaging method can be applied to various targets in bacteria, yeast and mammalian cells and can be advantageously combined with correlative imaging schemes.