Pereira, Pedro M;
Almada, Pedro;
Henriques, Ricardo;
(2015)
High-content 3D multicolor super-resolution
localization microscopy.
In: Paluch, Ewa K, (ed.)
Biophysical Methods in Cell Biology.
(95 - 117).
Elsevier: Amsterdam, The Netherlands.
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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.
| Type: | Book chapter |
|---|---|
| Title: | High-content 3D multicolor super-resolution localization microscopy |
| Location: | United States |
| ISBN-13: | 978-0-12-801103-4 |
| DOI: | 10.1016/bs.mcb.2014.10.004 |
| Publisher version: | http://dx.doi.org/10.1016/bs.mcb.2014.10.004 |
| Language: | English |
| Additional information: | This version is the version of record. For information on re-use, please refer to the publisher’s terms and conditions. |
| Keywords: | 3D; dSTORM; High-content screening; Immunofluorescence; Localization microscopy; Multicolor; Super-resolution |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Lab for Molecular Cell Bio MRC-UCL |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1467957 |
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