Single quantum dot tracking based on perceptual Grouping using minimal paths in a spatiotemporal volume

Bonneau, Stéphane; Dahan, Maxime; Cohen, Laurent D.

doi:10.1109/tip.2005.852794

Cited by 130 publications

(92 citation statements)

References 41 publications

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“…As described previously (Bonneau et al, 2005;Bannai et al, 2006;Ehrensperger et al, 2007), tracking of single QDs, which were identified by their fluorescence intermittence, was performed with MATLAB (MathWorks) using a homemade macro that accounts for blinking in the fluorescence signal. In brief, the method consists of two main steps, applied successively to each frame of the sequence.…”

Section: Methodsmentioning

confidence: 99%

Gephyrin Oligomerization Controls GlyR Mobility and Synaptic Clustering

Calamai

Specht²,

Heller³

et al. 2009

Journal of Neuroscience

149

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High local concentrations of glycine receptors (GlyRs) at inhibitory postsynaptic sites are achieved through their binding to the scaffold protein gephyrin. The N-and C-terminal domains of gephyrin are believed to trimerize and dimerize, respectively, thus contributing to the formation of submembranous gephyrin clusters at synapses. GlyRs are associated with gephyrin also at extrasynaptic locations. We have investigated how gephyrin oligomerization influences GlyR dynamics and clustering in COS-7 cells and in cultured spinal cord neurons. To this aim, we have expressed isolated N-and C-terminal domains of gephyrin that interfere with the oligomerization of the full-length protein. We also studied the effect of an endogenous splice variant, ge(2,4,5), with a decreased propensity to trimerize. A reduction of the size and number of gephyrin-GlyR clusters was found in cells expressing the various interfering gephyrin constructs. Using fluorescence recovery after photobleaching, we studied the exchange kinetics of synaptic gephyrin clusters. Real-time singleparticle tracking was used to analyze the mobility of GlyRs. We found that all the tested constructs displayed faster rates of recovery than wild-type gephyrin and increased the mobility of extrasynaptic receptors, showing that gephyrin-gephyrin interactions modulate the lateral diffusion of GlyRs. Furthermore, we observed an inverse correlation between GlyR diffusion properties and gephyrin cluster size that depended on the number of binding sites blocked by the different constructs. Since alterations in the oligomerization properties of gephyrin are related to the dynamics of GlyRs, the gephyrin splice variant ge(2,4,5) may be implicated in the modulation of synaptic strength.

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Section: Methodsmentioning

confidence: 99%

Gephyrin Oligomerization Controls GlyR Mobility and Synaptic Clustering

Calamai

Specht²,

Heller³

et al. 2009

Journal of Neuroscience

149

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“…After detecting candidate particles and linking image feature points frame-byframe, some segmented trajectories are obtained initially. SPT trajectory data from time-lapse TIRF microscopy are combined from individual truncated tracks to create much larger trajectories using a pseudo-three-dimensional volume, and then the track in this pseudo-volume space from each moving particle is obtained using a combination of a minimal energy path approach mediated through a Fast Marching method, 50 a Dynamic Programming approach, 51 and the Linear Assignment Solution. 52 To extract a particle trajectory, our method consists of three modules.…”

Section: Pinpointing Fluorescently-labelled Moleculesmentioning

confidence: 99%

Analytical tools for single-molecule fluorescence imaging in cellulo

Leake

2014

Phys. Chem. Chem. Phys.

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Recent technological advances in cutting-edge ultrasensitive fluorescence microscopy have allowed single-molecule imaging experiments in living cells across all three domains of life to become commonplace. Single-molecule live-cell data is typically obtained in a low signal-to-noise ratio (SNR) regime sometimes only marginally in excess of 1, in which a combination of detector shot noise, suboptimal probe photophysics, native cell autofluorescence and intrinsically underlying stochasticity of molecules result in highly noisy datasets for which underlying true molecular behaviour is nontrivial to discern. The ability to elucidate real molecular phenomena is essential in relating experimental single-molecule observations to both the biological system under study as well as offering insight into the fine details of the physical and chemical environments of the living cell. To confront this problem of faithful signal extraction and analysis in a noise-dominated regime, the 'needle in a haystack' challenge, such experiments benefit enormously from a suite of objective, automated, high-throughput analysis tools that can home in on the underlying 'molecular signature' and generate meaningful statistics across a large population of individual cells and molecules. Here, I discuss the development and application of several analytical methods applied to real case studies, including objective methods of segmenting cellular images from light microscopy data, tools to robustly localize and track single fluorescently-labelled molecules, algorithms to objectively interpret molecular mobility, analysis protocols to reliably estimate molecular stoichiometry and turnover, and methods to objectively render distributions of molecular parameters.

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“…When combined with ultrasensitive optical techniques, QDs allow tracking of individual biomolecules in live cells with high signal-to-noise ratio and over unprecedented durations (25)(26)(27). We examined retrograde transport of NGF-containing vesicles by using QD-NGF to mark these organelles.…”

Section: Qd-ngf Is Fully Biologically Activementioning

confidence: 99%

One at a time, live tracking of NGF axonal transport using quantum dots

Cui¹,

Chen

et al. 2007

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

340

336

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Retrograde axonal transport of nerve growth factor (NGF) signals is critical for the survival, differentiation, and maintenance of peripheral sympathetic and sensory neurons and basal forebrain cholinergic neurons. However, the mechanisms by which the NGF signal is propagated from the axon terminal to the cell body are yet to be fully elucidated. To gain insight into the mechanisms, we used quantum dot-labeled NGF (QD-NGF) to track the movement of NGF in real time in compartmentalized culture of rat dorsal root ganglion (DRG) neurons. Our studies showed that active transport of NGF within the axons was characterized by rapid, unidirectional movements interrupted by frequent pauses. Almost all movements were retrograde, but short-distance anterograde movements were occasionally observed. Surprisingly, quantitative analysis at the single molecule level demonstrated that the majority of NGFcontaining endosomes contained only a single NGF dimer. Electron microscopic analysis of axonal vesicles carrying QD-NGF confirmed this finding. The majority of QD-NGF was found to localize in vesicles 50 -150 nm in diameter with a single lumen and no visible intralumenal membranous components. Our findings point to the possibility that a single NGF dimer is sufficient to sustain signaling during retrograde axonal transport to the cell body.live imaging ͉ nerve growth factor ͉ single molecule imaging ͉ NGF signaling ͉ retrograde transport N erve growth factor (NGF) is produced and released by target tissues to activate specific receptors at the axon terminals of innervating neurons. In order for NGF to regulate gene expression and the survival of target neurons, a signal must be moved a considerable distance, in some cases Ͼ1,000-fold the diameter of the neuron cell body. The elucidation of the mechanism(s) used to transmit NGF signal from the terminals of axons to cell bodies of neurons is yet to be fully defined. In that retrograde NGF signaling is critical for the survival and maintenance of neurons of both the peripheral and central nervous systems, and the underlying mechanisms are likely to be shared by related neurotrophic factors, the issue remains one of the most significant and intriguing questions in neurobiology (1-16).In the past, radio-labeled NGF ( 125 I-NGF) has been used to study the binding, internalization, and axonal transport of NGF. These studies facilitated the measurement of transport rate and provided insights into the endocytic pathways used for NGF transport (8,(16)(17)(18)(19). Fluorescent labels such as rhodamine (20), Texas red (21), and Cy3 (22) were also used to track NGF movement in neurons. However, because these previous methods have limited spatial and temporal resolution, they provide only a coarse look at what is expected to be a very dynamic process. Moreover, few studies have examined NGF-containing endosomes during axonal transit. Some have suggested that NGF is transported principally within early endosomes (4, 5, 7-14) whereas others suggest that NGF is transported in organelles with com...

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Single quantum dot tracking based on perceptual Grouping using minimal paths in a spatiotemporal volume

Cited by 130 publications

References 41 publications

Gephyrin Oligomerization Controls GlyR Mobility and Synaptic Clustering

Gephyrin Oligomerization Controls GlyR Mobility and Synaptic Clustering

Analytical tools for single-molecule fluorescence imaging in cellulo

One at a time, live tracking of NGF axonal transport using quantum dots

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