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Semiconductor quantum dots (QDs) have demonstrated utility in long-term single particle tracking of membrane proteins in live cells in culture. To extend the superior optical properties of QDs to more physiologically relevant cell platforms, such as acute brain slices, we examine the photophysics of compact ligand-conjugated CdSe/CdS QDs using both ensemble and single particle analysis in brain tissue media. We find that symmetric core passivation is critical for both photostability in oxygenated media and for prolonged single particle imaging in brain slices. We then demonstrate the utility of these QDs by imaging single dopamine transporters in acute brain slices, achieving 20 nm localization precision at 10 Hz frame rates. These findings detail design requirements needed for new QD probes in complex living environments, and open the door to physiologically relevant studies that capture the utility of QD probes in acute brain slices.
The presynaptic dopamine transporter mediates rapid reuptake of synaptic dopamine. Although cell surface DAT trafficking recently emerged as an important component of DAT regulation, it has not been systematically investigated. Here, we apply our single quantum dot (Qdot) tracking approach to monitor DAT plasma membrane dynamics in several heterologous expression cell hosts with nanometer localization accuracy. We demonstrate that Qdot-tagged DAT proteins exhibited highly heterogeneous membrane diffusivity dependent on the local membrane topography. We also show that Qdot-tagged DATs were localized away from the flat membrane regions and were dynamically retained in the membrane protrusions and cell edges for the duration of imaging. Single quantum dot tracking of wildtype DAT and its conformation-defective coding variants (R60A and W63A) revealed a significantly accelerated rate of dysfunctional DAT membrane diffusion. We believe our results warrant an in-depth investigation as to whether compromised membrane dynamics is a common feature of brain disorder-derived DAT mutants.
We have synthesized 3 analogs of the dopamine D2 receptor (D2 DR) antagonist spiperone that can be conjugated to streptavidin-coated quantum dots via a pegylated biotin derivative. Using fluorescent imaging we demonstrate that substitution on the spiro position is tolerated, whilst the length and rigidity of a spacer arm attached to spiperone is important in controlling specific labeling as well as minimizing nonspecific labeling to cells and the surface of cell culture dishes. The ligand with the most rigid linker IDT772 (4) had the best binding profile and had high specific binding to D2 DR expressing HEK-293T cells with low nonspecific binding to plates and HEK-293T cells that lacked the D2 DR.
Copyright © 2019. Published by Elsevier Ltd.
Quantum dots are nanometer-sized semiconductors that have size-tunable, narrow emission bands, high quantum yields, and are resistant to photobleaching. Ligand-conjugated quantum dots enable the real time visualization of membrane proteins and have revealed that membrane diffusion dynamics are intrinsic to protein regulation, are susceptible to the level of membrane cholesterol, and are altered in genetic variants linked to disease, suggesting a mise en place approach to neuropsychopharmacology.
Single particle tracking (SPT) experiments have provided the scientific community with invaluable single-molecule information about the dynamic regulation of individual receptors, transporters, kinases, lipids, and molecular motors. SPT is an alternative to ensemble averaging approaches, where heterogeneous modes of motion might be lost. Quantum dots (QDs) are excellent probes for SPT experiments due to their photostability, high brightness, and size-dependent, narrow emission spectra. In a typical QD-based SPT experiment, QDs are bound to the target of interest and imaged for seconds to minutes via fluorescence video microscopy. Single QD spots in individual frames are then linked to form trajectories that are analyzed to determine their mean square displacement, diffusion coefficient, confinement index, and instantaneous velocity. This chapter describes a generalizable protocol for the single particle tracking of membrane neurotransmitter transporters on cell membranes with either unmodified extracellular antibody probes and secondary antibody-conjugated quantum dots or biotinylated extracellular antibody probes and streptavidin-conjugated quantum dots in primary neuronal cultures. The neuronal cell culture, the biotinylation protocol and the quantum dot labeling procedures, as well as basic data analysis are discussed.
We present an approach toward dynamic nanoimaging: live fluorescence of cells encapsulated in a bionanoreactor is complemented with in situ scanning electron microscopy (SEM) on an integrated microscope. This allows us to take SEM snapshots on-demand, that is, at a specific location in time, at a desired region of interest, guided by the dynamic fluorescence imaging. We show that this approach enables direct visualization, with EM resolution, of the distribution of bioconjugated quantum dots on cellular extensions during uptake and internalization.
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.
Biomolecule detection using quantum dots (Qdots), nanometer-sized semiconductor crystals, effectively addresses the limitations associated with conventional optical and biochemical techniques, as Qdots offer several key advantages over traditional fluorophores. In this minireview, we discuss the role of Qdots as a central nanoscaffold for the polyvalent assembly of multifunctional biomolecular probes and describe recent advances in Qdot-based biorecognition. Specifically, we focus on Qdot applications in target-based, drug screening assays and real-time active biosensing of cellular processes.
Real-time microscopic visualization of single molecules in living cells provides a molecular perspective of cellular dynamics, which is difficult to be observed by conventional ensemble techniques. Among various classes of fluorescent tags used in single-molecule tracking, quantum dots are particularly useful due to their unique photophysical properties. This chapter provides an overview of single quantum dot tracking for protein dynamic studies. First, we review the fundamental diffraction limit of conventional optical systems and recent developments in single-molecule detection beyond the diffraction barrier. Second, we describe methods to prepare water-soluble quantum dots for biological labeling and single-molecule microscopy experimental design. Third, we provide detailed methods to perform quantum dot-based single-molecule microscopy. This technical section covers three protocols including (1) imaging system calibration using spin-coated single quantum dots, (2) single quantum dot labeling in living cells, and (3) tracking algorithms for single-molecule analysis.
Over the last decade, research has identified a class of bone marrow-derived circulating stem cells, termed endothelial progenitor cells (EPCs), that are capable of homing to vascular lesions in the eye and contributing to pathological ocular neovascularization (NV). In preclinical and biological studies, EPCs are -frequently identified and tracked using a intracellularly loaded fluorescent tracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbo cyanine perchlorate-labeled acetylated LDL (DiI-acLDL). However, this method is limited by photobleaching and insufficient quantum efficiency for long-term imaging applications. We have developed a method for conjugation of high quantum efficiency, photostable, and multispectral quantum dot nanocrystals (QD) to acLDL for long-term tracking of EPCs with improved signal-to-noise ratios. Specifically, we conjugated QD to acLDL (QD-acLDL) and used this conjugated fluorophore to label a specific CD34(+) subpopulation of EPCs isolated from rat bone marrow. We then utilized this method to track CD34(+) EPCs in a rat model of laser-induced choroidal neovascularization (LCNV) to evaluate its potential for tracking EPCs in ocular angiogenesis, a critical pathologic feature of several blinding conditions.