Recent developments in GSDIM microscopy

Marcus Dyba; Giulio A. Simonutti; Jonas Fölling

doi:10.1117/12.912748

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13 February 2012 Recent developments in GSDIM microscopy

Marcus Dyba, Giulio A. Simonutti, Jonas Fölling

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Proceedings Volume 8228, Single Molecule Spectroscopy and Superresolution Imaging V; 82280R (2012) https://doi.org/10.1117/12.912748
Event: SPIE BiOS, 2012, San Francisco, California, United States

Abstract

In the presented study we characterized the suitability of 15 conventional fluorescence dyes for GSDIM microscopy. For all dyes involved in the screening labeled secondary antibodies for immunohistochemistry are commercially available. The dye performance was tested after staining to fixed mammalian cells. Chemical environments were chosen to be compatible with the applicative and spectroscopic demands. Investigated watery environments are suitable for TIRF based applications. To the best of our knowledge, we present for the first time systematic screening for configurations of dyes embedded in solid polymer. The polymer mounting matches well to the refractive index of oil immersion optics. This is crucial for applications at high penetration depth into the sample and suitable for long-term sample storage. We rated the final super-resolution image quality additional to quantitative characterization of important spectroscopic parameters. Therefore, this dye screening is optimized for various biological imaging applications. Control of the single molecule blinking rate by 405nm light exposure is quantified, as well. It is shown that this important effect is applicable to numerous fluorescent dyes. Thus, the controlled application of low intensities of 405nm light allows to maximize recording speed. As this option is already included in commercial GSDIM microscopes the results of our study allow optimized super-resolution imaging down to ~20nm with multiple dyes and multi-color staining.

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Marcus Dyba, Giulio A. Simonutti, and Jonas Fölling "Recent developments in GSDIM microscopy", Proc. SPIE 8228, Single Molecule Spectroscopy and Superresolution Imaging V, 82280R (13 February 2012); https://doi.org/10.1117/12.912748

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