title: High-content 3D multicolor super-resolution localization microscopy.
abstract: Super-resolution (SR) methodologies permit the visualization of cellular structures at near-molecular scale (1-30 nm), enabling novel mechanistic analysis of key events in cell biology not resolvable by conventional fluorescence imaging (∼300-nm resolution). When this level of detail is combined with computing power and fast and reliable analysis software, high-content screenings using SR becomes a practical option to address multiple biological questions. The importance of combining these powerful analytical techniques cannot be ignored, as they can address phenotypic changes on the molecular scale and in a statistically robust manner. In this work, we suggest an easy-to-implement protocol that can be applied to set up a high-content 3D SR experiment with user-friendly and freely available software. The protocol can be divided into two main parts: chamber and sample preparation, where a protocol to set up a direct STORM (dSTORM) sample is presented; and a second part where a protocol for image acquisition and analysis is described. We intend to take the reader step-by-step through the experimental process highlighting possible experimental bottlenecks and possible improvements based on recent developments in the field.
authors: MRC Laboratory for Molecular Cell Biology and Department of Cell and Developmental Biology, University College London, London, UK.
authors: Pedro M
authors: PM
authors: Pereira
authors: MRC Laboratory for Molecular Cell Biology and Department of Cell and Developmental Biology, University College London, London, UK.
authors: Pedro
authors: P
authors: Almada
authors: MRC Laboratory for Molecular Cell Biology and Department of Cell and Developmental Biology, University College London, London, UK.
authors: Ricardo
authors: R
authors: Henriques
mesh_terms: D000818:Animals; D002460:Cell Line; D003116:Color; D006801:Humans; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008853:Microscopy / Q000379:methods*
keywords: 3D; High-content screening; Immunofluorescence; Localization microscopy; Multicolor; Super-resolution; dSTORM