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Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

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Published on: June 30, 2018

Image artifacts in single molecule localization microscopy: why optimization of sample preparation protocols matters.

Donna R Whelan¹, Toby D M Bell¹

¹School of Chemistry, Monash University, Clayton, Victoria 3800, Australia.

Scientific Reports

|January 22, 2015

Summary

Single molecule localization microscopy (SMLM) provides high-resolution imaging. This study investigates sample preparation methods to ensure reliable SMLM image quality and avoid artifacts.

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Area of Science:

Biophysics
Cell Biology
Microscopy

Background:

Single molecule localization microscopy (SMLM) achieves nanoscale resolution.
Commercial SMLM systems are available, reducing hardware/software development needs.
SMLM image reliability and preparation artifacts are under-discussed.

Purpose of the Study:

To systematically investigate sample preparation steps for SMLM.
To identify and mitigate potential artifacts in SMLM imaging.
To optimize protocols for reliable ultrastructural visualization.

Main Methods:

Systematic investigation of fixation, permeabilization, and staining parameters.
Evaluation of fixatives, concentrations, timing, and antibody concentrations.
Development of optimized protocols for microtubules, mitochondria, and actin.

Main Results:

Identified critical parameters in sample preparation affecting SMLM image quality.
Presented three optimized protocols for specific cellular targets.
Demonstrated potential artifacts arising from sample preparation steps.

Conclusions:

Sample preparation significantly impacts SMLM reliability and image quality.
Careful optimization is crucial to avoid overlooked artifacts in SMLM.
New parameters are needed to define 'good' SMLM images beyond confocal standards.