Multiple-Labeled Antibodies Behave Like Single Emitters in Photoswitching Buffer.

Area of Science:

• Biophysics
• Optical Microscopy

Background:

• Antibody labeling degree (DOL) is crucial for brightness and binding in microscopy.
• The impact of DOL on blinking in direct stochastic optical reconstruction microscopy (dSTORM) is understudied.

Purpose of the Study:

• Investigate how DOL of Alexa Fluor 647 (Al647)-labeled IgG antibodies affects their spectroscopic properties and blinking behavior in dSTORM.
• Develop a model to explain and potentially improve fluorescent probes for dSTORM.

Main Methods:

• Ensemble and single-molecule spectroscopy of IgG antibodies labeled with Al647 at varying DOLs (1.1-8.3).
• Analysis of fluorescence trajectories and photon antibunching.
• dSTORM imaging to assess spatial resolution.

Main Results:

• Multiple-Al647-labeled antibodies exhibited quenching in aqueous buffer but enabled dSTORM imaging with ~20 nm resolution regardless of DOL.
• Single-molecule studies revealed complex photophysics in aqueous buffer, simplifying to single-emitter behavior in photoswitching buffer, independent of DOL.
• A model was developed to explain blinking in multiply labeled antibodies.

Conclusions:

• The DOL of Al647-labeled antibodies does not impede dSTORM resolution, with blinking behavior becoming DOL-independent in photoswitching buffer.
• The developed model aids in designing improved fluorescent probes for enhanced dSTORM performance.