Prospects & Overviews Super-resolution imaging for cell biologists Concepts, applications, current challenges and developments Eugenio F. Fornasiero 1)2) and Felipe Opazo 1)2) The recent 2014 Nobel Prize in chemistry honored an era of discoveries and technical advancements in the field of super-resolution microscopy. However, the applications of diffraction-unlimited imaging in biology have a long road ahead and persistently engage scientists with new challenges. Some of the bottlenecks that restrain the dissemination of super-resolution techniques are tangible, and include the limited performance of affinity probes and the yet not capillary diffusion of imaging setups. Likewise, super-resolution microscopy has introduced new para- digms in the design of projects that require imaging with nanometer-resolution and in the interpretation of biological images. Besides structural or morphological characteri- zation, super-resolution imaging is quickly expanding towards interaction mapping, multiple target detection and live imaging. Here we review the recent progress of biologists employing super-resolution imaging, some pitfalls, implications and new trends, with the purpose of animating the field and spurring future developments. Keywords: .affinity-probes; cell imaging; PALM; SIM; STED; STORM; sub-diffraction imaging Introduction: Sub-diffraction imaging and its application to cell biology From the dawn of microscopy, celebrated pioneers such as Robert Hook and Antoni van Leeuwenhoek grasped the impact that imaging technologies could exert on the study of life. In modern times microscopy techniques are broadly used. Imaging in cell biology serves at least two main purposes: first, it illustrates concisely what many words would describe less precisely, and second, it constitutes a primary source of data that can be analyzed and interpreted [1]. Although imaging in biology is largely employed for descriptive purposes, in recent years we have witnessed a shift towards a more quantitative use of imaging data, substantially changing the nature of microscopy (see [2] and Box 1). A second prominent advancement is the development of techniques that provide sub-diffraction resolution and a substantially higher level of detail (for an historical perspective concerning the develop- ment of super-resolution techniques refer to [3]). The maximum resolution of lens-based microscopy is constrained by the diffraction limit of light. The principle, proposed by Ernst Abbe, states that the minimum distance between two objects in the same plane at which they can be discriminated as separate elements depends on the wave- length of light and the numerical aperture of the lens used [4]. In practical terms the best lateral resolution attainable using visible light is 200 nm. Despite the apparent barrier imposed DOI 10.1002/bies.201400170 1) STED Microscopy Group, European Neuroscience Institute, Go ¨ ttingen, Germany 2) Department of Neuro- and Sensory-physiology, University of Go ¨ ttingen, Go ¨ ttingen, Germany *Corresponding authors: Eugenio F. Fornasiero E-mail: efornas@gwdg.de Felipe Opazo E-mail: fopazo@gwdg.de Abbreviations: CLEM, correlative light electron microscopy; FRET, Fo ¨ rster resonance energy transfer; PAINT, point accumulation for imaging in nanoscale topography; PALM, photoactivation localization microscopy; PSF, point spread function; RESOLFT, Reversible saturated optical fluorescent transitions; SIM, struc- tured illumination microscopy; SPDM, spectral precision distance microsco- py; STED, stimulated emission depletion microscopy; STORM, stochastic optical reconstruction microscopy; TIRF, total internal reflection microscopy. www.bioessays-journal.com 1 Bioessays 37: 0000–0000, ß 2015 WILEY Periodicals, Inc. Methods, Models & Techniques