Imaging at the single molecule level has played a pivotal role in advancing our knowledge of dopamine transporter (DAT) function and regulation. Typically, these studies are limited to heterologous expression systems and cultured neurons, leaving native 3D neuronal architecture an unexplored arena. In the interest of transitioning from cell culture to a more physiologically relevant environment, we report progress toward development of a compact antagonist-conjugated quantum dot (QD) probe tailored for single protein imaging in acute brain slices. Despite our current QD probes offering nanomolar affinity to DAT in cell cultures, the probe framework includes commercial streptavidin-coated QDs at the expense of large particle sizes and broad distribution of blinking dynamics. Our next-generation probes are made with blinking-suppressed CdSe/CdS QDs customized to (i) reach maximum penetration depths, (ii) yield higher photon count per particle in oxygenated artificial cerebrospinal fluid such that we have obtained signal-to-noise ratios suitable for high-speed single particle tracking experiments in live, intact brain tissue. Once our imaging platform is established, endogenous DAT diffusion could then be linked to animal model pathology providing a more nuanced understanding of behavioral disorders.

Generation of a compact quantum dot conjugate for imaging dopamine transporter membrane dynamics in acute brain slices

2019 Fall National Meeting

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