Dye-Conjugated Dendrimers as Bright and Photostable Fluorescent Labels for Single Molecule Microscopy

Abstract

In this work, we report a new class of bright and photostable fluorescent labels for single molecule fluorescence microscopy based on chemically functionalized dendrimers. We synthesized polymeric dendrimers containing multiple fluorophores and versatile functional groups, thereby enabling facile fluorescent labeling of biomolecules (proteins, DNA, RNA) via specific chemical conjugation. We characterized the photophysical properties of single dye-conjugated dendrimer molecules immobilized to chemically modified surfaces using total internal reflection fluorescence microscopy (TIRF-M). Fluorescence photobleaching lifetimes of dye-conjugated dendrimers were observed to far exceed the lifetimes of single conventional organic dye molecules, both in the presence or absence of oxygen scavengers. In addition, single dendrimer molecules are significantly brighter than single conventional organic dye molecules. Furthermore, we determined the precision of centroid position determination for single molecule localization by fitting the fluorescence emission from single dendrimers to a 2-dimensional Gaussian function. Dye-conjugated dendrimers exhibit several advantages as polymeric fluorophores for labeling biological molecules. First, single dendrimer molecules exhibit a relatively small size which is comparable to common GFP-family fluorescent proteins and smaller than inorganic quantum dots. Second, fluorescently-labeled dendrimers are synthesized to contain multiple fluorophores, thereby overcoming blinking in single dyes and short fluorescence “on” times before photobleaching, which are common limitations of single organic dyes for applications in single molecule microscopy. Third, a wide variety of commonly used fluorophores can be conjugated to polymeric dendrimers, thereby increasing their utility for labeling. Overall, we anticipate that dye-conjugated dendritic polymers will be a versatile tool for single molecule microscopy applications, including fine-scale characterization of protein structural dynamics and super-resolution microscopy.