Single Molecule Fluorescence Spectroscopy at Ambient Temperature

Perillo

et al. 2016

Biophysical Journal

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.

Section: Introductionmentioning

confidence: 99%

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

Perillo

et al. 2016

Biophysical Journal

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

“…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)(2)(3)(4)(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).…”

mentioning

confidence: 99%

Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids

Sahl

Leutenegger

Hilbert

et al. 2010

175

218

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...

“…3 The population mean and CV were determined from replicate simulations of data sets containing 10 4 measurements for each value of CV exp . The number of measurements used to determine pop and CV pop , 10 4 , was based on the number required to obtain a 1% CV for Poisson-limited measurements (8) and the typical size of flow cytometry data sets used below. The N min values defined above correspond to a range of CV pop values from 2% to 20%.…”

Section: Resultsmentioning

confidence: 99%

“…The Gaussian cumulative distribution function (CDF), shown in equation 2 (35,36), derived from Gaussian statistics and the error function, 4 can be used, without binning of the data, to obtain estimates of pop and pop from unnormalized data sets. *The minimum number of single-molecule measurements required to obtain an estimate of the population mean was determined through Monte Carlo simulations.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Statistics of single‐molecule measurements: Applications in flow‐cytometry sizing of DNA fragments

Ferris¹,

Habbersett²,

Wolinsky³

et al. 2004

Cytometry Pt A

Self Cite

Background:The measurement of physical properties from single molecules has been demonstrated. However, the majority of single-molecule studies report values based on relatively large data sets (e.g., N Ͼ 50). While there are studies that report physical quantities based on small sample sets, there has not been a detailed statistical analysis relating sample size to the reliability of derived parameters. Methods: Monte Carlo simulations and multinomial analysis, dependent on quantifiable experimental parameters, were used to determine the minimum number of singlemolecule measurements required to produce an accurate estimate of a population mean. Simulation results were applied to the fluorescence-based sizing of DNA fragments by ultrasensitive flow cytometry (FCM). Results: Our simulations show, for an analytical technique with a 10% CV, that the average of as few as five