Protocol

Live Cell Imaging

Volume 591 of the series Methods in Molecular Biology pp 185-199

Date:

Sample Preparation for STED Microscopy

  • Christian A. WurmAffiliated withMitochondrial Structure and Dynamics / Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry
  • , Daniel NeumannAffiliated withMitochondrial Structure and Dynamics / Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry
  • , Roman SchmidtAffiliated withDepartment of NanoBiphotonics, Max Planck Institute for Biophysical Chemistry
  • , Alexander EgnerAffiliated withDepartment of NanoBiphotonics, Max Planck Institute for Biophysical Chemistry
  • , Stefan JakobsAffiliated withMitochondrial Structure and Dynamics / Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry

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Abstract

Since the discovery of the diffraction barrier in the late nineteenth century, it has been commonly accepted that with far-field optical microscopy it is not possible to resolve structural details considerably finer than half the wavelength of light. The emergence of STED microscopy showed that, at least for fluorescence imaging, these limits can be overcome. Since STED microscopy is a far-field technique, in principle, the same sample preparation as for conventional confocal microscopy may be utilized. The increased resolution, however, requires additional precautions to ensure the structural preservation of the specimen. We present robust protocols to generate test samples for STED microscopy. These protocols for bead samples and immunolabeled mammalian cells may be used as starting points to adapt existing labeling strategies for the requirements of sub-diffraction resolution microscopy.

Key words

Fluorescence microscopy Stimulated emission depletion microscopy Nanoscopy Superresolution Immunofluorescence Sample preparation