(273f) Characterization of New Classes of Bright and Photostable Fluorescent Nanoconjugates 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 have developed a new class of fluorescent probes based on dye-conjugated dendrimers called fluorescent dendrimer nanoconjugates (FDNs). In particular, we use polyamidoamine (PAMAM) dendrimers as molecular scaffolds for the attachment of multiple organic dyes and “helper” molecules. We then characterize these probes using single molecule fluorescence imaging and total internal reflection fluorescence microscopy (TIRF-M). We observe that FDNs are much brighter compared to single fluorophores, which allows for high-resolution localization and detection. In addition, we link photoprotectant molecules such as triplet state quenchers (TSQs) directly onto FDNs, thereby generating “self-healing” probes which have enhanced photostability when compared with native fluorescent dyes. In this work, we characterized two separate synthetic strategies for creating self-healing dye conjugates, by calculating important photophysical parameters such as total accumulated photons and stability of single molecule intensity over time. In the first generation of probes, we attached the fluorescent dye Cy5 and photoprotective Trolox molecules to the PAMAM dendrimer, which allowed us to vary the Trolox:dye ratio. This led to an increased stabilization with the higher synthesized Trolox:dye ratios. However, while this strategy allows for control over the Trolox:dye ratio, it does not allow for precise control over the distance between the molecules which has been shown to be an important parameter. Thus, in a second generation of self-healing FDNs, we first covalently attached the Cy5 and Trolox into one dye conjugate, which we then labeled onto the dendrimer in varying amounts. This keeps the Trolox:dye ratio at 1:1, and it also ensures constant close contact between the two species and results in large increases in brightness, total photon output and stabilization of fluorescence. We tested these probes in vitro under a variety of different solution conditions to gain insights into the mechanisms of stabilization, along with testing in a cell immunofluorescence experiment as a proof-of-principle demonstration of biological utility. Overall, we anticipate that these new fluorescent probes will enable new insight and mechanistic understanding in molecular and cellular biology.