BPS2026 - Comparing the photophysical properties of mStayGold, eGFP, and Alexa Fluor 488 labeled SNAP-tag2

The ability to visualize biological molecules at a single-molecule level is often facilitated by fluorescent proteins or dyes. Dyes are often brighter, more photostable, and more versatile than fluorescent proteins. More advanced fluorescent proteins are being engineered to catch up with fluorescent dyes, such as mStayGold. Rigorous comparison of fluorophores is often useful for finding the optimal tools for specific experiments. In this study, mStayGold and Alexa Fluor 488 labeled SNAP-tag2 are compared with eGFP, a standard fluorescent protein. SNAP-tag2 is a self-labeling protein (SLP) that covalently binds to an organic dye, in this case Alexa Fluor 488. These fluorophores are studied with the purpose of comparing their photophysical properties: brightness, durability to photobleaching, and presence of temporary transitions to dark states known as photoblinking. The fluorophores all share similar emission and excitation wavelengths, allowing for easier comparison. The techniques used to study their photophysical properties include single molecule TIRF fluorescence microscopy-which provides information on the average time until photobleaching occurs, photoblinking, and signal-to-noise ratio for immobilized molecules-and confocal fluorescence correlation spectroscopy (FCS), which measures the count rate and brightness of the fluorophore for diffusing molecules. Current results show mStayGold to have improved brightness, photodurability, and signal-to-noise ratio over the other proteins tested. Alexa Fluor 488 and eGFP show similar results in terms of signal-to-noise ratio and lifetimes of the fluorophore, with a lower propensity to photoblink than mStayGold.