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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.
Insights
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.
Abstract:
In single-molecule localization microscopy (SMLM), immunofluorescence (IF) staining affects the quality of the reconstructed superresolution images. However, optimizing IF staining remains challenging because IF staining is a one-step, irreversible process. Sample labeling through reversible binding presents an alternative strategy, but such techniques require significant technological advancements to enhance the dissociation of labels without sacrificing their binding specificity. In this article, we introduce time-lapse imaging of single-antibody labeling. Our versatile technique utilizes commercially available dye-conjugated antibodies. The method controls the antibody concentrations to capture single-antibody labeling of subcellular targets, thereby achieving SMLM through the labeling process. We further demonstrate dual-color single-antibody labeling to enhance the sample labeling density. The new approach allows the evaluation of antibody binding at the single-antibody level and within the cellular environment. This comprehensive guide offers step-by-step instructions for time-lapse imaging of single-antibody labeling experiments and enables the application of the single-antibody labeling technique to a wide range of targets. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for single-antibody labeling Basic Protocol 2: Data acquisition for single-molecule localization microscopy Alternate Protocol: Dual-color single-antibody labeling using OptoSplit II equation Basic Protocol 3: Image analysis.
