Abstract
In these last two decades , fluorescent proteins (FPs) have become highly valued imaging tools for cell biology, owing to their compatibility with living samples, their low levels of invasiveness and the possibility to specifically fuse them to a variety of proteins of interest. Remarkably, the recent development of phototransformable fluorescent proteins (PTFPs) has made it possible to conceive optical imaging experiments that were unimaginable only a few years ago. For example, it is nowadays possible to monitor intra- or intercellular trafficking, to optically individualize single cells in tissues or to observe single molecules in live cells. The tagging specificity brought by these genetically encoded highlighters leads to constant progress in the engineering of increasingly powerful, versatile and non-cytotoxic FPs. This review is focused on the recent developments of PTFPs and highlights their contribution to studies within cells, tissues and even living organisms. The aspects of single-molecule localization microscopy, intracellular tracking of photoactivated molecules, applications of PTFPs in biotechnology/optobiology and complementarities between PTFPs and other microscopy techniques are particularly discussed.
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Notes
To attain the highest possible resolution, a correct sampling is required. Sampling is defined by the Nyquist–Shannon theorem (Shannon 1949), which for PALM imaging in two dimensions corresponds to the observation of molecules spatially separated by intervals finer than the observed feature. For a resolution d, the intermolecular interval d/2 within a spot of diameter D defines the minimal molecular density \(\rho = \left[ {\pi \times \left( \frac{D}{2} \right)^{2} } \right]/\left( \frac{d}{2} \right)^{2} = \pi \times \left( \frac{D}{d} \right)^{2}\), D and d being expressed in nanometers. Below the value ρ, the feature is said under-sampled and above, it is said over-sampled. Reversely, the maximal attainable Nyquist resolution d of a correctly sampled feature is given by \(d = D \times \sqrt {\frac{\pi }{\rho }}\). In PALM conditions, this translates to the sampling of molecular densities ranging from 3 × 102 to 3 × 104 molecules/µm2 in order to reach Nyquist resolutions comprised between 100 and 10 nm.
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Acknowledgments
VA is grateful to Dominique Bourgeois for stimulating and fruitful discussions and to Jacques-Philippe Colletier for critical reading of the manuscript. Institutional Grants from the CNRS, CEA and UJF and financial support by the ANR (ANR-2011-BSV5-012-01 NOBLEACH) are acknowledged.
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Adam, V. Phototransformable fluorescent proteins: which one for which application?. Histochem Cell Biol 142, 19–41 (2014). https://doi.org/10.1007/s00418-014-1190-5
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DOI: https://doi.org/10.1007/s00418-014-1190-5