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Optimized sample preparation for single-molecule localization-based superresolution microscopy in yeast.

Charlotte Kaplan1, Helge Ewers2

  • 1Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.

Nature Protocols
|June 13, 2015
PubMed
Summary
This summary is machine-generated.

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This study presents an optimized protocol for superresolution microscopy in yeast, improving structural preservation using nanobodies and enzymatic labeling for high-resolution imaging of cellular structures.

Area of Science:

  • Cell Biology
  • Microscopy
  • Biochemistry

Background:

  • Single-molecule localization microscopy (SMLM) offers nanoscale resolution but faces challenges with structural preservation in yeast.
  • Current yeast labeling protocols hinder SMLM due to issues with maintaining cellular integrity.

Purpose of the Study:

  • To develop an optimized sample preparation protocol for high-resolution SMLM in Saccharomyces cerevisiae.
  • To enhance structural preservation during superresolution imaging of yeast cells.

Main Methods:

  • Utilized nanobodies as small binders for efficient dye delivery.
  • Employed an enzymatic labeling strategy to target proteins with organic dyes.
  • Optimized sample preparation to avoid yeast cell wall degradation.

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Main Results:

  • Achieved high-resolution SMLM with improved structural preservation in yeast.
  • Nanobodies facilitated dye penetration without compromising cell structure.
  • Reduced localization error due to the proximity of dyes to target proteins.

Conclusions:

  • The developed protocol enables advanced superresolution imaging in yeast.
  • This method offers a faster (2-4 hours) and more effective approach for yeast cell preparation.
  • Requires expertise in yeast molecular biology, immunolabeling, and access to SMLM equipment.