Confocal, Three-Dimensional Tracking of Individual Quantum Dots in High-Background Environments

Wells, Nathan P.; Lessard, Guillaume A.; Werner, James H.

doi:10.1021/ac8021899

Cited by 66 publications

(146 citation statements)

References 35 publications

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“…On the other hand, as for single-particle tracking in general (19,(31)(32)(33)(34), too low a brightness of the single molecules, which in our case may result from too low an excitation power, also leads to an overestimation of the values of α because short traps can no longer be discerned from free diffusion. Consequently, we have chosen a laser power of 20 μW to elicit strong fluorescence response and limit bleaching.…”

Section: Resultsmentioning

confidence: 80%

“…S1). The use of several distinct detection pinholes to obtain localizations was suggested (29,30) and also demonstrated (31,32) with individual quantum dot probes in conjunction with fast translational feedback motion of the sample. Other sophisticated apparatus can track particles "on-the-fly" in 3D by scanning one or several laser spots in elliptical or circular orbits using feedback loops to keep the particle at the center of the observation field (33,34).…”

Section: Resultsmentioning

confidence: 99%

“…Since the detection PSF (A PSF ) can hardly be reduced anymore, future improvements of the spatio-temporal resolution will mainly rely on higher detection rates Q. This can be achieved by brighter fluorophores (currently, Atto647N with Q ≤ 1 MHz), as implemented in numerous single-particle tracking experiments by applying large signal markers (19,(31)(32)(33)(34), and/or an increased collection efficiency afforded by one or two opposing objectives of high NA, such as provided by a 4Pi microscope (38). Increasing Q by increasing the excitation power may be challenged by bleaching.…”

Section: Discussion Conclusion and Outlookmentioning

confidence: 99%

“…Arrays of more than three detectors may be used, covering a larger observation area, and axial (z) sensitivity could be achieved by a tetrahedral arrangement (30)(31)(32) or similar. Employing photoswitchable probe molecules within this method to control the density of fluorescing entities could enable the study of dynamics of individual molecules at comparatively high concentrations (29,(39)(40)(41).…”

Section: Discussion Conclusion and Outlookmentioning

confidence: 99%

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Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids

Sahl

Leutenegger

Hilbert

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

175

218

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We describe an optical method capable of tracking a single fluorescent molecule with a flexible choice of high spatial accuracy (∼10-20 nm standard deviation or ∼20-40 nm full-width-at-halfmaximum) and temporal resolution (<1 ms). The fluorescence signal during individual passages of fluorescent molecules through a spot of excitation light allows the sequential localization and thus spatio-temporal tracking of the molecule if its fluorescence is collected on at least three separate point detectors arranged in close proximity. We show two-dimensional trajectories of individual, small organic dye labeled lipids diffusing in the plasma membrane of living cells and directly observe transient events of trapping on <20 nm spatial scales. The trapping is cholesterolassisted and much more pronounced for a sphingo-than for a phosphoglycero-lipid, with average trapping times of ∼15 ms and <4 ms, respectively. The results support previous STED nanoscopy measurements and suggest that, at least for nontreated cells, the transient interaction of a single lipid is confined to macromolecular dimensions. Our experimental approach demonstrates that fast molecular movements can be tracked with minimal invasion, which can reveal new important details of cellular nano-organization. M any open questions in biology can be tackled only if the dynamics of individual molecules can be observed noninvasively in vivo and at the appropriate spatial and temporal scale (1-5). Over the years, specific labeling of the cell's constituent parts with fluorescent markers has enabled deeper understanding in many areas of cell biology and allowed, for example, the spatiotemporal tracking of single particles (6, 7). However, to reach the desired spatial and temporal accuracy, single-particle tracking often applies bright but large and clumsy signal markers, which potentially influence the system under study. One notable example is the dynamics of proteins and lipids in cellular membranes and their organization into nanodomains, so-called "lipid rafts" (8-12). Since their proposal, the question of the existence and functional role of such cholesterol-mediated lipid assemblies has caused much controversy (13-16). Owing to the lack of suitable noninvasive techniques to detect these nanodomains in living cells, their spatial extent has been estimated at somewhere in the range of 5-200 nm (17), that is between molecular dimensions and the resolution limit of conventional fluorescence microscopy. Camera-based tracking of single lipids could provide more detailed insight (18)(19)(20). However, so far it either lacked temporal resolution or it made use of rather large gold beads as lipid labels, implying that the measurements may not give comprehensive answers to the open questions. Recently, stimulated-emission-depletion (STED) nanoscopy (21, 22), delivering subdiffraction resolution in live cells, provided direct evidence that certain lipids are transiently trapped in cholesterol-assisted molecular complexes (23, 24). In those experiments, which are evaluate...

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Section: Resultsmentioning

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Section: Discussion Conclusion and Outlookmentioning

confidence: 99%

Section: Discussion Conclusion and Outlookmentioning

confidence: 99%

See 2 more Smart Citations

Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids

Sahl

Leutenegger

Hilbert

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

175

218

View full text Add to dashboard Cite

show abstract

“…Whereas SPT has made important discoveries that change our view of plasma membrane organization (17,19) and molecular motor dynamics (20), the use of SPT in monitoring ''intracellular'' processes is rather limited because of the lack of three-dimensional (3D) tracking capacity that can follow a single particle inside a live cell for a long period of time. In the past decade, new SPT techniques have been developed to visualize molecular motion in the 3D space (termed 3D-SPT), including multiple imaging planes (21,22), orbital tracking (23)(24)(25), point spread function engineering (26,27), and confocal tracking (28,29). Although allowing for direct observation of transport processes from membrane to cytoplasm, current 3D-SPT methods often suffer from shallow imaging depth (because of the use of one-photon excitation) and limited z-tracking range (e.g., astigmatismbased, nonfeedback tracking systems (27)), which prevent these methods from tracking single molecules inside multicellular models such as spheroids (see our review in (8)).…”

Section: Introductionmentioning

confidence: 99%

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking

Liu

Perillo

Liu

et al. 2016

Biophysical Journal

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Whereas important discoveries made by single-particle tracking have changed our view of the plasma membrane organization and motor protein dynamics in the past three decades, experimental studies of intracellular processes using singleparticle tracking are rather scarce because of the lack of three-dimensional (3D) tracking capacity. In this study we use a newly developed 3D single-particle tracking method termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) to investigate epidermal growth factor receptor (EGFR) trafficking dynamics in live cells at 16/43 nm (xy/z) spatial resolution, with track duration ranging from 2 to 10 min and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, we developed a trajectory analysis algorithm, which reaches 81% segment classification accuracy in control experiments (termed simulated movement experiments). When analyzing 95 EGF-stimulated EGFR trajectories acquired in live skin cancer cells, we find that these trajectories can be separated into three groups-immobilization (24.2%), membrane diffusion only (51.6%), and transport from membrane to cytoplasm (24.2%). When EGFRs are membrane-bound, they show an interchange of Brownian diffusion and confined diffusion. When EGFRs are internalized, transitions from confined diffusion to directed diffusion and from directed diffusion back to confined diffusion are clearly seen. This observation agrees well with the model of clathrin-mediated endocytosis.

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3‐Dimensional Tracking of Non‐blinking ‘Giant’ Quantum Dots in Live Cells

Keller

Ghosh

DeVore

et al. 2014

Adv Funct Materials

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While semiconductor quantum dots (QDs) have been used successfully in numerous single particle tracking (SPT) studies due to their high photoluminescence efficiency, photostability, and broad palette of emission colors, conventional QDs exhibit fluorescence intermittency or ‘blinking,’ which causes ambiguity in particle trajectory analysis and limits tracking duration. Here, non-blinking ‘giant’ quantum dots (gQDs) are exploited to study IgE-FcεRI receptor dynamics in live cells using a confocal-based 3D SPT microscope. There is a 7-fold increase in the probability of observing IgE-FcεRI for longer than 1 min using the gQDs compared to commercially available QDs. A time-gated photon-pair correlation analysis is implemented to verify that selected SPT trajectories are definitively from individual gQDs and not aggregates. The increase in tracking duration for the gQDs allows the observation of multiple changes in diffusion rates of individual IgE-FcεRI receptors occurring on long (>1 min) time scales, which are quantified using a time-dependent diffusion coefficient and hidden Markov modeling. Non-blinking gQDs should become an important tool in future live cell 2D and 3D SPT studies, especially in cases where changes in cellular dynamics are occurring on the time scale of several minutes.

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Confocal, Three-Dimensional Tracking of Individual Quantum Dots in High-Background Environments

Cited by 66 publications

References 35 publications

Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids

Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking

3‐Dimensional Tracking of Non‐blinking ‘Giant’ Quantum Dots in Live Cells

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