Robust single-particle tracking in live-cell time-lapse sequences

Jaqaman, Khuloud; Loerke, Dinah; Mettlen, Marcel; Kuwata, Hirotaka; Grinstein, Sergio; Schmid, Sandra L.; Danuser, Gaudenz

doi:10.1038/nmeth.1237

Cited by 1,797 publications

(2,042 citation statements)

References 36 publications

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“…This was similar to previous estimates in BSC1 cells, wherein productive CCSs were defined as spot-like CCSs having lifetimes anywhere from tens to hundreds of seconds (average 87 s) [3,27,28]. Because the size and lifetimes of scission-detected CCSs were so variable ( Figure 1H and 1I), in our subsequent investigation of late events in CME we made measurements over a time window of 680 s, centred on scission.…”

Section: Author Summarysupporting

confidence: 81%

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A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis

Perrais²,

2011

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Dual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein-tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated structure (CCS), but the precision of spot-like CCS disappearance as a marker for canonical CME remained unknown. Here we have used an imaging assay based on total internal reflection fluorescence microscopy to detect scission events with a resolution of ,2 s. We found that scission events engulfed comparable amounts of transferrin receptor cargo at CCSs of different sizes and CCS did not always disappear following scission. We measured the recruitment dynamics of 34 types of endocytic protein to scission events: Abp1, ACK1, amphiphysin1, APPL1, Arp3, BIN1, CALM, CIP4, clathrin light chain (Clc), cofilin, coronin1B, cortactin, dynamin1/ 2, endophilin2, Eps15, Eps8, epsin2, FBP17, FCHo1/2, GAK, Hip1R, lifeAct, mu2 subunit of the AP2 complex, myosin1E, myosin6, NECAP, N-WASP, OCRL1, Rab5, SNX9, synaptojanin2b1, and syndapin2. For each protein we aligned ,1,000 recruitment profiles to their respective scission events and constructed characteristic ''recruitment signatures'' that were grouped, as for yeast, to reveal the modular organization of mammalian CME. A detailed analysis revealed the unanticipated recruitment dynamics of SNX9, FBP17, and CIP4 and showed that the same set of proteins was recruited, in the same order, to scission events at CCSs of different sizes and lifetimes. Collectively these data reveal the fine-grained temporal structure of CME and suggest a simplified canonical model of mammalian CME in which the same core mechanism of CME, involving actin, operates at CCSs of diverse sizes and lifetimes.

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Section: Author Summarysupporting

confidence: 81%

“…Therefore, scission events defined a class of larger, longer-lived CCSs. The shorter-lived scission-undetected CCSs most likely correspond to the ''abortive'' CCSs described previously [3,27,28], although some of these structures may have represented endosomal clathrin.…”

Section: Author Summarymentioning

confidence: 58%

A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis

Perrais²,

2011

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“…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. Considering the particle intensity models from fluorescence microscopy, first a set of filters are applied to the image data to suppress noise and enhance the contrast of moving particles in each image frame.…”

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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“…Recent advancements in tracking algorithms designed for high particle densities might prove to be beneficial in this respect. 31,32 Theoretically there is no lower limit to the particle concentration as the size is calculated for individual particles. However, it is clear that there should be a sufficient number of particles in the system to obtain good statistics within a reasonable time period.…”

mentioning

confidence: 99%

Sizing Nanomatter in Biological Fluids by Fluorescence Single Particle Tracking

Braeckmans¹,

Buyens²,

Bouquet³

et al. 2010

Nano Lett.

142

104

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Accurate sizing of nanoparticles in biological media is important for drug delivery and biomedical imaging applications since size directly influences the nanoparticle processing and nanotoxicity in vivo. Using fluorescence single particle tracking we have succeeded for the first time in following the aggregation of drug delivery nanoparticles in real time in undiluted whole blood. We demonstrate that, by using a suitable surface functionalization, nanoparticle aggregation in the blood circulation is prevented to a large extent.

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Robust single-particle tracking in live-cell time-lapse sequences

Cited by 1,797 publications

References 36 publications

A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis

A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis

Analytical tools for single-molecule fluorescence imaging in cellulo

Sizing Nanomatter in Biological Fluids by Fluorescence Single Particle Tracking

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