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Direct stochastic optical reconstruction microscopy with standard fluorescent probes

Abstract

Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of 20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.

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Figure 1: The dSTORM concept.
Figure 2: Reversible photoswitching of Alexa Fluor and ATTO dyes in the presence of thiols.
Figure 3: dSTORM acquisition procedure exemplified for Alexa Fluor 647–labeled β-tubulin in a COS-7 cell.
Figure 4: Simulated data demonstrating the influence of the ratio of photoswitching rates, r, on different complexities.
Figure 5: Scheme of experimental setup for single-molecule based localization microscopy with standard synthetic fluorophores.
Figure 6: dSTORM images of fixed COS-7 and living HeLa cells.

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Acknowledgements

We gratefully acknowledge support from G. Wiebusch, B. Vogel, U. Endesfelder, T. Holm and S. Proppert. We thank V.W. Cornish and R. Wombacher for providing the TMP substrates and the H2B-eDHFR plasmid. This work was supported by the Biophotonics and the Systems Biology Initiative (FORSYS) of the German Ministry of Research and Education (BMBF).

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S.v.d.L., A.L., T.K., M. Heidbreder, S.W., M. Heilemann and M.S. conceived and designed the experiments; S.v.d.L., A.L., T.K., M. Heidbreder and S.W. performed the experiments; M. Heilemann and M.S. supervised the project; and M. Heilemann, S.v.d.L. and M.S. wrote the paper.

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Correspondence to Markus Sauer.

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van de Linde, S., Löschberger, A., Klein, T. et al. Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc 6, 991–1009 (2011). https://doi.org/10.1038/nprot.2011.336

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