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Single-Molecule Localization Microscopy Using Time-Lapse Imaging of Single-Antibody Labeling
Thilini Perera1, Hirushi Gunasekara1, Ying S Hu1
1Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, Illinois.
Current Protocols
|October 13, 2023
Summary
This study introduces a new method for single-molecule localization microscopy (SMLM) using time-lapse imaging of single-antibody labeling. This technique improves superresolution image quality by optimizing the antibody labeling process for subcellular targets.
Area of Science:
- Biotechnology
- Microscopy
- Cell Biology
Background:
- Immunofluorescence (IF) staining impacts superresolution image quality in single-molecule localization microscopy (SMLM).
- Optimizing IF staining is difficult due to its irreversible nature.
- Reversible binding methods require advanced technology for label dissociation without compromising specificity.
Purpose of the Study:
- To develop a novel technique for SMLM utilizing time-lapse imaging of single-antibody labeling.
- To enable precise control over antibody binding for improved SMLM.
- To provide a comprehensive guide for applying this technique to various targets.
Main Methods:
- Utilized commercially available dye-conjugated antibodies.
- Controlled antibody concentrations to achieve single-antibody labeling of subcellular targets.
- Implemented time-lapse imaging and dual-color labeling strategies.
Main Results:
- Achieved SMLM through optimized single-antibody labeling.
- Demonstrated dual-color single-antibody labeling to increase sample labeling density.
- Enabled evaluation of antibody binding at the single-antibody level within cells.
Conclusions:
- The developed technique offers a versatile approach to SMLM by optimizing the labeling process.
- This method allows for detailed analysis of antibody binding dynamics.
- Provides a practical guide for researchers to apply single-antibody labeling in SMLM experiments.
