Intensity-Based Estimation of Monomeric Brightness for Fluorescent Proteins

Fluorescence fluctuation spectroscopy (FFS) techniques rely on determination of monomeric molecular brightness, i.e., the fluorescence intensity of a single, non-aggregated fluorophore, as a critical reference for estimating protein oligomer size. By comparing measured molecular brightness of fluorescently labeled proteins of interest to this monomeric brightness benchmark, FFS enables inference of oligomerization states. However, widely used fluorescent proteins often exhibit self-association, compromising monomeric brightness calibration and introducing errors in brightness-derived oligomer-size estimates. This study compares two strategies for determining monomeric brightness: the conventional fluctuation-based method and a more recently proposed average-intensity-based alternative. The comparison uses two model fluorophores, a fluorescent protein (mCitrine) and the small-molecule dye Janelia Fluor 525 (JF₅₂₅) conjugated to HaloTag. Our results show strong agreement between intensity- and fluctuation-derived brightness values only in the minimally aggregating JF₅₂₅-HaloTag benchmark. In contrast, in the mCitrine samples, where aggregation is more prevalent, only the intensity-based method maintains a consistent estimate across sample preparation, while the fluctuation-based method overestimates brightness when aggregation effects become pronounced. This robustness makes the intensity-based approach a valuable cross-check for monomeric brightness calibration. Our results support a combined strategy, using both methods to improve the reliability of monomeric brightness calibration and protein oligomerization analysis in FFS.