Photoswitchable Dendrimer Nanoconjugates as Fluorescent Probes for Super-Resolution Microscopy

Abstract

We demonstrate photoswitchable dendrimer nanoconjugates as a new class of imaging probes for general fluorescence and super-resolution microscopy. Fluorescence-based imaging techniques currently rely on a limited set of fluorescent probes, including fluorescent proteins, organic dyes and quantum dots. Recent advances in super-resolution imaging techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) have broken the diffraction barrier for optical imaging, thereby enabling observation of biological processes at nanometer-scale resolution. However, the performance of these super-resolution imaging techniques is intimately related to the photophysical properties of the “standard set” of current fluorescent probes, including photoswitchable fluorescent proteins and organic dyes. Overall, there is a strong need for development of new bright and photoswitchable fluorescent probes to enable biological imaging with enhanced spatiotemporal resolution.In this work, we developed photoswitchable dendrimer nanoconjugates (PDNs) as a new class of probes for fluorescence microscopy, which enables high-resolution fluorescence imaging. Polyamidoamine (PAMAM) dendrimers were engineered to contain multiple cyanine dyes, and the photophysical properties were characterized by single molecule fluorescence microscopy using total internal reflection microscopy (TIRF-M). We observe that collective interactions in the multichromophoric nanoprobe system result in extended photobleaching lifetimes, enhanced brightness and improved localization precision compared to single organic dyes. In this way, we engineered PDNs as enhanced fluorescent probes for super-resolution imaging. PDNs are bright, photostable and nanometer-sized macromolecular probes that can be synthesized to contain a variety of surface chemical functionalities for biological labeling and imaging. Based on these advantages, PDN probes hold strong promise to find pervasive applications as a new class of probes for fluorescence microscopy.