Functionalization and bioconjugation of nanoruby for long-term, ultrasensitive imaging of mu-opioid receptors

Sensitive and long-term fluorescence imaging of G-protein-coupled receptors enables exploration of molecular level details of these therapeutically relevant proteins, including their expression, localization, signaling, and intracellular trafficking. In this context, labeling these receptors with bright and photostable fluorescent probes is necessary to overcome current imaging problems such as optical background and photobleaching. Here, we describe the procedures to functionalize nanoruby (and other similar nanoparticles) with NeutrAvidin (a streptavidin analog) and to apply this bioconjugate for ultrasensitive, long-term imaging of μ-opioid receptors heterologously expressed in AtT-20 cells. The receptor targeting is mediated via a biotinylated primary antibody, rendering this methodology extendable to other G-protein-coupled or, more generally, cell-surface receptors. Nanoruby-based time-gated imaging enables indefinitely long visualization of single particles even in high-autofluorescence media, such as serum, by completely suppressing autofluorescence and any laser backscatter.