Quantitative Analysis of Single Quantum Dot Trajectories

Kovtun, Oleg; Thal, Lucas B.; Josephs, Travis; Rosenthal, Sandra J.

doi:10.1007/978-1-0716-0463-2_6

Cited by 4 publications

(4 citation statements)

References 36 publications

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“…For objective comparison, several popular open-access algorithms were employed to determine the center position of individual Qdots with sub-pixel accuracy (spatial detection stage), connect obtained Qdot coordinates into continuous trajectory segments (temporal detection stage), and estimate the diffusion coefficient for individual trajectories [ 44 , 45 ]. Qdot-D2L trajectories were reconstructed via (1) Crocker–Weeks algorithm that is based on work by Crocker and Grier [ 46 ], (2) u-track suite developed by Jaqaman et al [ 47 ] (only trajectories of blinking Qdots with a minimum duration of 50 frames were retained for diffusion analysis), and (3) ImageJ TrackMate plug-in developed by Tinevez et al [ 48 ].…”

Section: Methodsmentioning

confidence: 99%

Membrane Nanoscopic Organization of D2L Dopamine Receptor Probed by Quantum Dot Tracking

et al. 2021

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The role of lateral mobility and nanodomain organization of G protein-coupled receptors in modulating subcellular signaling has been under increasing scrutiny. Investigation of D2 dopamine receptor diffusion dynamics is of particular interest, as these receptors have been linked to altered neurotransmission in affective disorders and represent the primary target for commonly prescribed antipsychotics. Here, we applied our single quantum dot tracking approach to decipher intrinsic diffusion patterns of the wild-type long isoform of the D2 dopamine receptor and its genetic variants previously identified in several cohorts of schizophrenia patients. We identified a subtle decrease in the diffusion rate of the Val96Ala mutant that parallels its previously reported reduced affinity for potent neuroleptics clozapine and chlorpromazine. Slower Val96Ala variant diffusion was not accompanied by a change in receptor-receptor transient interactions as defined by the diffraction-limited quantum dot colocalization events. In addition, we implemented a Voronoї tessellation-based algorithm to compare nanoclustering of the D2 dopamine receptor to the dominant anionic phospholipid phosphatidylinositol 4,5-bisphosphate in the plasma membrane of live cells.

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Section: Methodsmentioning

confidence: 99%

Membrane Nanoscopic Organization of D2L Dopamine Receptor Probed by Quantum Dot Tracking

et al. 2021

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“…Image analysis and trajectory construction were performed using ImageJ TrackMate plugin ( Jaqaman et al, 2008 ; Tinevez et al, 2017 ). Intermittency of QD fluorescence was used to verify that single fluorophores were analyzed, and extracted trajectories were at least 50 frames in length to increase the robustness of statistical analysis ( Chang and Rosenthal, 2011 , 2013 ; Kovtun et al, 2020 ). Trajectories were considered continuous if a blinking QD was rediscovered within a 1 μm distance during the 10-frame time window.…”

Section: Methodsmentioning

confidence: 99%

A Novel Biotinylated Homotryptamine Derivative for Quantum Dot Imaging of Serotonin Transporter in Live Cells

Tomlinson

Kovtun

Torres

et al. 2021

Front. Cell. Neurosci.

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The serotonin transporter (SERT) is the primary target for selective serotonin reuptake inhibitor (SSRI) antidepressants that are thought to exert their therapeutic effects by increasing the synaptic concentration of serotonin. Consequently, probes that can be utilized to study cellular trafficking of SERT are valuable research tools. We have developed a novel ligand (IDT785) that is composed of a SERT antagonist (a tetrahydro pyridyl indole derivative) conjugated to a biotinylated poly ethylene glycol (PEG) via a phenethyl linker. This compound was determined to be biologically active and inhibited SERT-mediated reuptake of IDT307 with the half-maximal inhibitory concentration of 7.2 ± 0.3 μM. We demonstrated that IDT785 enabled quantum dot (QD) labeling of membrane SERT in transfected HEK-293 cultures that could be blocked using the high affinity serotonin reuptake inhibitor paroxetine. Molecular docking studies suggested that IDT785 might be binding to the extracellular vestibule binding site rather than the orthosteric substrate binding site, which could be attributable to the hydrophilicity of the PEG chain and the increased loss of degrees of freedom that would be required to penetrate into the orthosteric binding site. Using IDT785, we were able to study the membrane localization and membrane dynamics of YFP-SERT heterologously expressed in HEK-293 cells and demonstrated that SERT expression was enriched in the membrane edge and in thin cellular protrusions.

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“…Neuronal protein-specific affinity handles can be conjugated to biocompatible QDs of different emission wavelengths, allowing for multiplexed labeling. Since QDs exhibit narrow Gaussian emission spectra, 25 multiplexed imaging can be more easily implemented than organic dyes or fluorescent proteins 77 to yield high resolution spatiotemporal data 78 for several principal proteins during the entire signaling event, as depicted in Figure 6 . The “ideal” QD possesses the ability to reach sterically hindered spaces while maintaining high PL intensity; however, atomic structure photophysical correlation is needed to develop the “ideal” QD.…”

Section: Prospects For the Futurementioning

confidence: 99%

Quantum Dot Fluorescent Imaging: Using Atomic Structure Correlation Studies to Improve Photophysical Properties

Torres,

Thal,

McBride

et al. 2024

J. Phys. Chem. C

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Efforts to study intricate, higher-order cellular functions have called for fluorescence imaging under physiologically relevant conditions such as tissue systems in simulated native buffers. This endeavor has presented novel challenges for fluorescent probes initially designed for use in simple buffers and monolayer cell culture. Among current fluorescent probes, semiconductor nanocrystals, or quantum dots (QDs), offer superior photophysical properties that are the products of their nanoscale architectures and chemical formulations. While their high brightness and photostability are ideal for these biological environments, even state of the art QDs can struggle under certain physiological conditions. A recent method correlating electron microscopy ultrastructure with single-QD fluorescence has begun to highlight subtle structural defects in QDs once believed to have no significant impact on photoluminescence (PL). Specific defects, such as exposed core facets, have been shown to quench QD PL in physiologically accurate conditions. For QD-based imaging in complex cellular systems to be fully realized, mechanistic insight and structural optimization of size and PL should be established. Insight from single QD resolution atomic structure and photophysical correlative studies provides a direct course to synthetically tune QDs to match these challenging environments.

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Quantitative Analysis of Single Quantum Dot Trajectories

Cited by 4 publications

References 36 publications

Membrane Nanoscopic Organization of D2L Dopamine Receptor Probed by Quantum Dot Tracking

Membrane Nanoscopic Organization of D2L Dopamine Receptor Probed by Quantum Dot Tracking

A Novel Biotinylated Homotryptamine Derivative for Quantum Dot Imaging of Serotonin Transporter in Live Cells

Quantum Dot Fluorescent Imaging: Using Atomic Structure Correlation Studies to Improve Photophysical Properties

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