Imaging at the single molecule level has been pivotal for advancing our knowledge of dopamine transporter (DAT) function and regulation by visualization of transporter membrane dynamics at the cell surface. These studies are limited to heterologous expression systems and cultured neurons, leaving native 3D neuronal architecture an unexplored arena. In the interest of transitioning to a more physiologically relevant environment, we report progress toward development of a compact antagonist-conjugated quantum dot (QD) probe tailored for single DAT imaging in acute mouse brain slices. Despite our current QD probes offering nanomolar affinity to DAT in cell cultures, the probe framework includes commercial streptavidin-coated QDs that are limited to large particle sizes and display a broad distribution of blinking dynamics. Our compact probes are made with blinking-suppressed CdSe/Cds QDs customized to (i) reach maximum penetration depths and (ii) yield higher photon count per particle 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 single molecule imaging of dopamine transporters in acute brain slices

256th National Meeting (2018)

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