Semiconductor quantum dots are quickly becoming a critical diagnostic tool for discerning cellular function at the molecular level. Their high brightness, long-lasting, sizetunable, and narrow luminescence set them apart from conventional fluorescence dyes. Quantum dots are being developed for a variety of biologically oriented applications, including fluorescent assays for drug discovery, disease detection, single protein tracking, and intracellular reporting. This review introduces the science behind quantum dots and describes how they are made biologically compatible. Several applications are also included, illustrating strategies toward target specificity, and are followed by a discussion on the limitations of quantum dot approaches. The article is concluded with a look at the future direction of quantum dots.

The presynaptic, cocaine- and amphetamine-sensitive dopamine (DA) transporter (DAT, SLC6A3) controls the intensity and duration of synaptic dopamine signals by rapid clearance of DA back into presynaptic nerve terminals. Abnormalities in DAT-mediated DA clearance have been linked to a variety of neuropsychiatric disorders, including addiction, autism, and attention deficit/hyperactivity disorder (ADHD). Membrane trafficking of DAT appears to be an important, albeit incompletely understood, post-translational regulatory mechanism; its dysregulation has been recently proposed as a potential risk determinant of these disorders. In this study, we demonstrate a link between an ADHD-associated DAT mutation (Arg615Cys, R615C) and variation on DAT transporter cell surface dynamics, a combination only previously studied with ensemble biochemical and optical approaches that featured limited spatiotemporal resolution. Here, we utilize high-affinity, DAT-specific antagonist-conjugated quantum dot (QD) probes to establish the dynamic mobility of wild-type and mutant DATs at the plasma membrane of living cells. Single DAT-QD complex trajectory analysis revealed that the DAT 615C variant exhibited increased membrane mobility relative to DAT 615R, with diffusion rates comparable to those observed after lipid raft disruption. This phenomenon was accompanied by a loss of transporter mobilization triggered by amphetamine, a common component of ADHD medications. Together, our data provides the first dynamic imaging of single DAT proteins, providing new insights into the relationship between surface dynamics and trafficking of both wild-type and disease-associated transporters. Our approach should be generalizable to future studies that explore the possibilities of perturbed surface DAT dynamics that may arise as a consequence of genetic alterations, regulatory changes, and drug use that contribute to the etiology or treatment of neuropsychiatric disorders.

1À3 Impaired DA signaling has been linked to a number of neurodegenerative and psychiatric disorders such as attention-deficit hyperactivity disorder (ADHD), bipolar disorder, major depression, Tourette's syndrome, Parkinson's disease, and schizophrenia. 4À8 The synaptic DA concentration influences postsynaptic DA signal transduction capacity and is modulated by the activity of a presynaptic D 2 DA receptor, that modulates DA release, and the DA transporter (DAT), 1,9 that clears DA to achieve DA inactivation and recycling.10 DAT (SLC6A3) is a member of a family of Na þ -coupled solute transporters whose substrates include neurotransmitters, nutrients, osmolytes, and amino acids. Several reports have demonstrated that experimental DAT deficiency results in pronounced changes in dopaminergic tone and locomotor hyperactivity.10À12 In addition, DAT is the primary target for widely used psychostimulants, such as amphetamine and cocaine that acutely elevate synaptic DA concentrations. Cocaine is a competitive DAT inhibitor and attenuates DA clearance by occupying the DA binding site on DAT, whereas amphetamine promotes DAT-mediated DA efflux that also results in the increased DA synaptic concentration. 13 DAT activity has also been demonstrated to be a subject of acute, dynamic regulation by several post-translational mechanisms, such as constitutive endocytosis, protein-kinase-C (PKC)-dependent internalization, proteinÀprotein interactions, and substrate-induced changes in surface expression level. 13,14 The spatial organization and temporal control of these mechanisms remain largely unknown and, when disrupted, may influence risk for disorders linked to compromised DA signaling.The investigation of DAT regulation has thus far trailed similar efforts directed at membrane receptors and channels due a number of important challenges. First, the lack of an efficient antibody against an extracellular epitope does not allow direct localization and visualization of DAT molecules in living cells without prior chemical processing (fixation and permeabilization). 15,16 Second, the use of popular fusion tags, such as green fluorescent protein (GFP) and hemagglutinin (HA), requires genetic perturbation of DAT and thus does not allow direct visualization of endogenous DAT. Third, traditional autoradiographic, biochemical, and optical techniques to monitor DAT expression, function, and cellular distribution suffer from suboptimal ABSTRACT: The presynaptic dopamine (DA) transporter is responsible for DA inactivation following release and is a major target for the psychostimulants cocaine and amphetamine. Dysfunction and/or polymorphisms in human DAT (SLC6A3) have been associated with schizophrenia, bipolar disorder, Parkinson's disease, and attention-deficit hyperactivity disorder (ADHD). Despite the clinical importance of DAT, many uncertainties remain regarding the transporter's regulation, in part due to the poor spatiotemporal resolution of conventional methodologies and the relative lack of efficient DAT-specific fluorescent...

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The use of nanometer-sized semiconductor crystals, known as quantum dots, allows us to directly observe individual biomolecular transactions through a fluorescence microscope. Here, we review the evolution of single quantum dot tracking over the past two decades, highlight key biophysical discoveries facilitated by quantum dots, briefly discuss biochemical and optical implementation strategies for a single quantum dot tracking experiment, and report recent accomplishments of our group at the interface of molecular neuroscience and nanoscience.