(601ae) Fluorescent Dendrimer Nanoconjugates As Advanced Probes for Biological Imaging

Recent advances in optical microscopy have enabled tremendous improvements in the spatial and temporal resolution of fluorescence-based imaging techniques. However, there is a strong need for the development of advanced fluorescent probes to study biological events in living cells, which will enable a molecular-scale understanding of biological processes. To this end, we are developing a new class of fluorescent probes based on dye-conjugated dendrimers called fluorescent dendrimer nanoconjugates (FDNs). We utilize molecular-scale dendritic scaffolds as fluorescent probes, thereby enabling conjugation of multiple fluorescent dyes and chemical linkers to the scaffold periphery. In particular, we use polyamidoamine (PAMAM) dendrimers as molecular scaffolds for organic dyes and “helper” molecules, and we characterize these probes using single molecule fluorescence imaging and total internal reflection fluorescence microscopy (TIRF-M). We observe that single FDNs are much brighter compared to a single fluorophore, which allows for high-resolution localization and detection.

In addition, we link photoprotective molecules such as triplet state quenchers (TSQs) directly onto FDNs, thereby generating extremely photostable fluorescent probes. A major problem in fluorescence imaging involves the fast and irreversible photobleaching of dyes, which limits the timescale of experiments. Moreover, single molecule experiments are limited by dye “blinking” and transient dark periods, which confounds single molecule tracking and Forster resonance energy transfer experiments. Traditionally, improvements in photostability have been achieved by adding photostabilizing compounds such as reducing/oxidizing agents (beta-mercaptoethanol, trolox, ascorbic acid, methyl viologen) directly to the imaging solution. However, in our work, we link multiple TSQs (trolox) directly onto FDNs as a way to avoid addition of harsh chemicals into biological imaging buffers and to ensure a local high concentration of the photoprotective group near the dye molecules. In this way, we observe major improvements in photostability and decreased blinking events that surpasses the enhancements possible with trolox added to solution. We test these probes in two biological experiments, cell immunofluorescence and single DNA molecule labeling. In future work, we are developing a new class of sequence-defined multi-dye probes with precise control over the number and distribution of dyes and “helper” molecules, which will increase the uniformity and performance of these probes. In addition, these sequence defined probes will allow a fundamental understanding of how Cy5 photophysics, such as self-quenching and blinking, change when dye number is increased from 1 to 2 or above. Overall, we anticipate that these new fluorescent probes will enable new vistas and mechanistic understanding in molecular and cellular biology.