Multiple-labeled antibodies behave like single emitters in photoswitching buffer

The degree of labeling (DOL) of antibodies has so far been optimized for high brightness and specific and efficient binding. The influence of the DOL on the blinking performance of antibodies used in direct stochastic optical reconstruction microscopy (dSTORM) has so far attained limited attention. Here, we investigated the spectroscopic characteristics of IgG antibodies labeled at DOLs of 1.1- 8.3 with Alexa Fluor 647 (Al647) at the ensemble and single-molecule level. Multiple-Al647-labeled antibodies showed weak and strong quenching interactions in aqueous buffer but could all be used for dSTORM imaging with spatial resolutions of [~] 20 nm independent of the DOL. Photon antibunching experiments in aqueous buffer demonstrate that the emission of multiple-Al647-labeled antibodies switches from classical to non-classical light in photoswitching buffer. We developed a model that explains the observed blinking of multiple-labeled antibodies and can be used advantageously to develop improved fluorescent probes for dSTORM experiments.