Confocal Fluctuation Spectroscopy and Imaging

Földes-Papp, Zeno; Liao, Shih-Chu Jeff; You, Tiefeng; Terpetschnig, Ewald; Barbieri, Beniamino

doi:10.2174/138920110792246618

Cited by 5 publications

(3 citation statements)

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“…Measurements of single-molecule fluctuations have a long history as an essential tool for studying diffusive and kinetic properties in confocal microscopy and spectroscopy [1]. For example, in dilute solutions fluctuations in the fluorescence intensity are caused by fluctuations in the local concentration of fluorescent molecules.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence Molecule Counting for Single-Molecule Studies in Crowded Environment of Living Cells without and with Broken Ergodicity

Földes-Papp¹,

Baumann

2011

CPB

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We present a new approach to distinguish between non-ergodic and ergodic behavior. Performing ensemble averaging in a subpopulation of individual molecules leads to a mean value that can be similar to the mean value obtained in an ergodic system. The averaging is carried out by minimizing the variation between the sum of the temporal averaged mean square deviation of the simulated data with respect to the logarithmic scaling behavior of the subpopulation. For this reason, we first introduce a kind of Continuous Time Random Walks (CTRW), which we call Limited Continuous Time Random Walks (LCTRW) on fractal support. The random waiting time distributions are sampled at points which fulfill the condition N < 1, where N is the Poisson probability of finding a single molecule in the femtoliter-sized observation volume ΔV at the single-molecule level. Given a subpopulation of different single molecules of the same kind, the ratio T/ Tm between the measurement time T and the meaningful time Tm, which is the time for observing just one and the same single molecule, is the experimentally accessible quantity that allows to compare different molecule numbers in the subpopulation. In addition, the mean square displacement traveled by the molecule during the time t is determined by an upper limit of the geometric dimension of the living cell or its nucleus.

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Section: Introductionmentioning

confidence: 99%

Fluorescence Molecule Counting for Single-Molecule Studies in Crowded Environment of Living Cells without and with Broken Ergodicity

Földes-Papp¹,

Baumann

2011

CPB

Self Cite

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“…The platforms are combined with analytical methods such as fluorescence resonance energy transfer (FRET), fluorescence auto-or dual color cross-correlation spectroscopy (FCS), fluorescence polarizing anisotropy, fluorescence quenching and fluorescence lifetime measurements. The concept of fluctuations due to the same molecule was theoretically and experimentally developed in recent years [55,57,8,9,18,35,36,27,12,13,62,63,1,2]. Under conditions in which only one molecule is present in the observation volume being sampled, we have shown that it is possible to measure correlations due to the re-entry of the same molecule in the volume of illumination.…”

Section: Resultsmentioning

confidence: 96%

Anomalous Subdiffusive Measurements by Fluorescence Correlations Spectroscopy and Simulations of Translational Diffusive Behavior in Live Cells

2014

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Using a combination of optical experiments and computer simulations, we found that live cells act as traps to produce anomalous subdiffusive translational motion of molecules exemplified by glucocorticoid receptor α. Glucocorticoid receptor α was expressed as a fusion protein with the green fluorescent protein in living mammalian U2OS cells. The measurements were carried out by fluorescence correlation spectroscopy. The GFP-tagged glucocorticoid receptor α was either a homodimer or a monomer in the nucleoplasm depending on the presence or absence of the stimulus dexamethasone. Our simulations showed that the experimentally measured rates of translational diffusion could be attributed to spatial and temporal traps. Thus, traps acted by spatial and temporal heterogeneity and randomness, respectively. As we prove here for the first time, that anomalous translational diffusion caused by spatial randomness was different from anomalous translational diffusion caused by heterogeneous temporal randomness. For this purpose, we explored two classes of transformations in the time domain for which the assumption that they are stable and have probability density functions was satisfied. The first one was the Inverse Gamma distribution and the second class was a stable Levy distribution. The spatial exponent α that was extracted from time series expressed scale invariance in the space domain. The time exponent γ measured limited time scaling of the embedding complex dynamics. Hence, both exponents must not be mixed up by a single exponent. Fluorescence correlation spectroscopy is the method of choice for measuring the exponents of anomalous subdiffusive translational motion of molecules in live cells.

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“…ZEISS confocal laser-scanning microscope LSM510 META [3,26] was used for the scanning measurements. The microscope came with four confocal channels for reflected light, plus one for transmitted light.…”

Section: Confocal Laser Scanning Microscopymentioning

confidence: 99%

Visualization of subdiffusive sites in a live single cell

Földes-Papp

Baumann

2021

J Biol Methods

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We measured anomalous diffusion in human prostate cancer cells which were transfected with the Alexa633 fluorescent RNA probe and co-transfected with enhanced green fluorescent protein-labeled argonaute2 protein by laser scanning microscopy. The image analysis arose from diffusion based on a “two-level system”. A trap was an interaction site where the diffusive motion was slowed down. Anomalous subdiffusive spreading occurred at cellular traps. The cellular traps were not immobile. We showed how the novel analysis method of imaging data resulted in new information about the number of traps in the crowded and heterogeneous environment of a single human prostate cancer cell. The imaging data were consistent with and explained by our modern ideas of anomalous diffusion of mixed origins in live cells. Our original research presented in this study is significant as we obtained a complex diffusion mechanism in live single cells.

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Confocal Fluctuation Spectroscopy and Imaging

Cited by 5 publications

References 0 publications

Fluorescence Molecule Counting for Single-Molecule Studies in Crowded Environment of Living Cells without and with Broken Ergodicity

Fluorescence Molecule Counting for Single-Molecule Studies in Crowded Environment of Living Cells without and with Broken Ergodicity

Anomalous Subdiffusive Measurements by Fluorescence Correlations Spectroscopy and Simulations of Translational Diffusive Behavior in Live Cells

Visualization of subdiffusive sites in a live single cell

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