Live-Cell Super-Resolution with dSTORM: A detailed microscopic characterization of cellular structures is important to understand cellular function. Conventional microscopy in some cases is limited by the achievable spatial resolution of about 200 nm in the imaging plane, which is not sufficient to reveal details at the near-molecular level. This is important if the organization of proteins in small organelles, clusters or machineries are studied. Here, advanced microscopic techniques that are capable to bypass the resolution limit can offer additional and valuable information and pave the way to a new level of understanding. Methods that employ stochastic activation and read out of photoactivatable or photoswitchable fluorophores are in particular interesting because they can achieve near-molecular resolution, can be operated with moderate irradiation intensities and even applied inside live cells.

Super-Resolution Microscopy of ­Cellular Structures

The limit in light microscopy is to about 200 nm, such that objects which are closer cannot be discerned. In the past years, a variety of super-resolution fluorescence imaging techniques have been developed which can bypass the resolution limit in microscopy [1]. These technologies have made the important step from pure demonstration towards real-life applications, with a particular focus on cellular structures. Among the different super-resolution methods available today, so-called stochastic methods benefit from a simple experimental realization and provide near-molecular spatial resolution. The key of these techniques are photoactivatable or photoswitchable fluorophores: a controllable number of only a very few fluorophores is activated stochastically e.g. by light, the fluorescence signal is read-out with sensitive detectors (such as single-photon sensitive EMCCD cameras), and the position of individual fluorophores is determined to a precision of a few nanometers. This procedure is repeated until a sufficient number of single-molecule coordinates were collected, from which a "pointillistic" image is finally reconstructed (fig. 1).

Direct Stochastic Optical Recon­struction Microscopy (dSTORM)

A very simple approach for stochastic super-resolution imaging is direct stochastic optical reconstruction microscopy [2, 3]. dSTORM operates a large variety of unmodified and widely used organic fluorophores as photoswitches, on the basis of light-induced photophysical transitions and redox-chemical processes that are inherent to these probes.

The main advantage of dSTORM is that it can easily be combined with well-established labeling techniques such as immunofluorescence to visualize cellular structures (fig. 2). Additionally, small and specific markers for cellular structures can be used, such as the fluorophore-labeled heptapeptide phalloidin to label actin structures. Organic fluorophores are brighter than fluorescent proteins, which leads to a better spatial resolution reaching 20 nm in the imaging plane. Additionally, organic fluorophores can be cycled many times between a fluorescent and a non-fluorescent state and imaged repeatedly without irreversible photobleaching. This fact is in particular helpful for studies of dynamic processes, which in contrast to static samples require more than one read out of a fluorophore to follow. Likewise, the photoswitching kinetics of organic fluorophores can readily be controlled, which enables fast imaging [4] that is essential for the observation of faster dynamics. For example, a spatial resolution of 30 nm together with a temporal resolution of 1 Hz has been used to study actin filament dynamics on a myosin surface [5].

dSTORM in Live Cells

The challenge for all super-resolution technologies is their application in living cells under moderate excitation conditions and reasonable acquisition times. Here, stochastic methods have the advantage of applying comparably low irradiation intensities on the one hand, and providing a spatial resolution of about 20 nm on the other hand. A first demonstration of live cell imaging used photoactivatable fluorescent proteins which were genetically attached to a protein of interest [6]. However, fluorescent proteins are less bright than organic fluorophores, and in most cases can only be read out once and with relatively low imaging speed. These parameters limit both, the achievable resolution (which is directly related to the number of photons emitted by a single fluorophore) as well as the temporal resolution and thus their suitability to observe dynamic processes. At this point, organic fluorophores represent a promising alternative for stochastic imaging inside living cells [3, 7], making use of their inherent photophysical and photochemical properties in reducing experimental conditions as they are found with e.g. the tripeptide glutathione in the cytosol and nucleus in all kinds of cells (fig. 3).