Characterization of Photophysical Properties of the Photoactivatable Fluorescent Proteins for Superresolution Microscopy

Photoactivatable fluorescent proteins (PA-FPs) are widely used in photoactivated localization microscopy (PALM), and information about their blinking properties is important for precise assembling of PALM images and for quantitative molecular counting with PALM. As the photophysical properties of fluorescent proteins depend sensitively on the local physiochemical environment, we developed a series of procedures to characterize the properties of the PA-FPs in fixed cells. As an example, we characterized the properties of Dendra2 in E. coli, and found that they are appreciably different from those in vitro. This highlights the importance of charactering photophysical properties of PA-FPs under the same experimental conditions as subsequent PALM imaging or molecular counting experiments. Other PA-FPs could be characterized using this strategy.