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Single Molecule Super-resolution Microscopy How-to: improve it and use it

le 18 février 2015 à 14 h
Nicolas OLIVIER, King’s College, London, UK.

Super-resolution fluorescence imaging has emerged as an attractive approach to determine the nanoscale distribution of proteins in their cellular context. Among super-resolution methods, three-dimensional stochastic optical reconstruction microscopy (3D-STORM) offers one of the highest resolutions currently achieved, by stochastically switching fluorophores between fluorescent and dark states and precisely localizing their positions.

A fundamental limit to the localization precision in STORM is the number of photons detected from each fluorophore. Therefore, one way to improve the 3D localization precision is to increase the number of photons emitted by each dye. Here, we show that STORM-buffer optimization can provide significantly increased photon yields from the commonly used dye Alexa-647, and therefore an increased localization precision enabling us to investigate the 3D nanoscale distribution of proteins on a simple microscope. In particular, the increased brightness afforded by these buffers enabled us to reveal the ring-like organization of the centriolar protein HsSAS-6 in cells using 3D-STORM with near-isotropic resolution of ~30 nanometer. Moreover, we show that alternate STORM buffers with Vectashield can be used that simplify the protocols, increase the reproducibility and improve index-matching for 3D imaging.

Finally we will discuss the limits of the methods in terms of live imaging and data analysis, in particular how "super" the resolution of the images are.

[1] N. Olivier, D. Keller et al. PLoS One 8(7) e69004 (2013)

[2] N. Olivier et al. Biomedical Optics Express 4 (6), 885-99 (2013)

[3] D. Keller et al. The Journal of cell biology 204.5: 697-712. (2014)

[4] G. Lukinavicius et al. Nature chemistry 5.2 132-139. (2013)

[5] A. Benke, N. Olivier et al. Nano Letters 12 (5), 2619-2624 (2012)

[6] T. Pengo, et al. arXiv preprint arXiv:1501.05807 (2015).

 


Lieu(x) :         Amphi Gay Lussac, Ecole Polytechnique


Contact :       Emmanuel Beaurepaire
                      emmanuel.beaurepaire at polytechnique.edu