Self-diffusion of rodlike and spherical particles in a matrix of charged colloidal spheres: A comparison between fluorescence recovery after photobleaching and fluorescence correlation spectroscopy

2004 ◽  
Vol 121 (14) ◽  
pp. 7022-7029 ◽  
Author(s):  
C. Lellig ◽  
J. Wagner ◽  
R. Hempelmann ◽  
S. Keller ◽  
D. Lumma ◽  
...  
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Fluorescence Recovery After Photobleaching ◽  
Correlation Spectroscopy ◽  
Spherical Particles ◽  
Fluorescence Recovery ◽  
Fluorescence Correlation ◽  
Self Diffusion ◽  
Colloidal Spheres

scholarly journals EGF Receptor Stalls upon Activation as Evidenced by Complementary Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching Measurements

2019 ◽  
Vol 20 (13) ◽  
pp. 3370 ◽  
Author(s):  
György Vámosi ◽  
Elza Friedländer-Brock ◽  
Shehu M. Ibrahim ◽  
Roland Brock ◽  
János Szöllősi ◽  
...  
Keyword(s):  
Time Scales ◽  
Fluorescence Correlation Spectroscopy ◽  
Time Course ◽  
Fluorescence Recovery After Photobleaching ◽  
Slow Component ◽  
Chinese Hamster ◽  
Correlation Spectroscopy ◽  
Fluorescence Recovery ◽  
Brownian Diffusion ◽  
Fluorescence Correlation

To elucidate the molecular details of the activation-associated clustering of epidermal growth factor receptors (EGFRs), the time course of the mobility and aggregation states of eGFP tagged EGFR in the membranes of Chinese hamster ovary (CHO) cells was assessed by in situ mobility assays. Fluorescence correlation spectroscopy (FCS) was used to probe molecular movements of small ensembles of molecules over short distances and time scales, and to report on the state of aggregation. The diffusion of larger ensembles of molecules over longer distances (and time scales) was investigated by fluorescence recovery after photobleaching (FRAP). Autocorrelation functions could be best fitted by a two-component diffusion model corrected for triplet formation and blinking. The slow, 100–1000 ms component was attributed to membrane localized receptors moving with free Brownian diffusion, whereas the fast, ms component was assigned to cytosolic receptors or their fragments. Upon stimulation with 50 nM EGF, a significant decrease from 0.11 to 0.07 μm2/s in the diffusion coefficient of membrane-localized receptors was observed, followed by recovery to the original value in ~20 min. In contrast, the apparent brightness of diffusing species remained the same. Stripe FRAP experiments yielded a decrease in long-range molecular mobility directly after stimulation, evidenced by an increase in the recovery time of the slow component from 13 to 21.9 s. Our observations are best explained by the transient attachment of ligand-bound EGFRs to immobile or slowly moving structures such as the cytoskeleton or large, previously photobleached receptor aggregates.

Monte Carlo simulation of fluorescence correlation spectroscopy data

2011 ◽  
Vol 76 (3) ◽  
pp. 207-222 ◽  
Author(s):  
Peter Košovan ◽  
Filip Uhlík ◽  
Jitka Kuldová ◽  
Miroslav Štěpánek ◽  
Zuzana Limpouchová ◽  
...  
Keyword(s):  
Monte Carlo Simulation ◽  
Monte Carlo ◽  
Diffusion Coefficients ◽  
Fluorescence Correlation Spectroscopy ◽  
Rotational Diffusion ◽  
Correlation Spectroscopy ◽  
Analytical Models ◽  
Spherical Particles ◽  
Fluorescence Signal ◽  
Fluorescence Correlation

We employed the Monte Carlo simulation methodology to emulate the diffusion of fluorescently labeled particles and understand the source of differences between values of diffusion coefficients (and consequently hydrodynamic radii) of fluorescently labeled nanoparticles measured by fluorescence correlation spectroscopy (FCS) and dynamic light scattering (DLS). We used the simulation program developed in our laboratory and studied the diffusion of spherical particles of different sizes, which are labeled on their surface. In this study, we focused on two complicating effects: (i) multiple labeling and (ii) rotational diffusion which affect the fluorescence signal from large particles and hinder the analysis of autocorrelation functions according to simple analytical models. We have shown that the fluorescence fluctuations can be well fitted using the analytical model for small point-like particles, but the obtained parameters deviate in some cases significantly from the real ones. It means that the current data treatment yields apparent values of diffusion coefficients and other parameters only and the interpretation of experimental results for systems of particles with sizes comparable to the size of the active illuminated volume requires great care and precaution.

scholarly journals Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages

2011 ◽  
Vol 22 (18) ◽  
pp. 3498-3507 ◽  
Author(s):  
Urszula Golebiewska ◽  
Jason G. Kay ◽  
Thomas Masters ◽  
Sergio Grinstein ◽  
Wonpil Im ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Correlation Spectroscopy ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Surface Concentration ◽  
Correlation Spectroscopy ◽  
Similar Data ◽  
Fluorescence Correlation ◽  
The Many ◽  
Green Fluorescent

To account for the many functions of phosphatidylinositol 4,5-bisphosphate (PIP2), several investigators have proposed that there are separate pools of PIP2 in the plasma membrane. Recent experiments show the surface concentration of PIP2 is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP2 are also concentrated in these regions. However, how is the PIP2 produced by these kinases prevented from diffusing rapidly away? First, proteins could act as “fences” around the perimeter of these regions. Second, some factor could markedly decrease the diffusion coefficient, D, of PIP2 within these regions. We used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to investigate these two possibilities in the forming phagosomes of macrophages injected with fluorescent PIP2. FCS measurements show that PIP2 diffuses rapidly (D ∼ 1 μm2/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP2 does not recover (>100 s) after photobleaching the entire forming phagosome but recovers rapidly (∼10 s) in a comparable area of membrane outside the cup. These results (and similar data for a plasma membrane–anchored green fluorescent protein) support the hypothesis that a fence impedes the diffusion of PIP2 into and out of forming phagosomes.

Fluorescence correlation spectroscopy applied to rotational diffusion of macromolecules

1976 ◽  
Vol 9 (1) ◽  
pp. 69-81 ◽  
Author(s):  
Måns Ehrenberg ◽  
Rudolf Rigler
Keyword(s):  
Rotational Motion ◽  
Fluorescence Correlation Spectroscopy ◽  
Rotational Diffusion ◽  
Quantitative Relationship ◽  
Correlation Spectroscopy ◽  
Spherical Particles ◽  
Fluorescence Correlation ◽  
Fluorescence Light

A quantitative relationship between polarization properties of fluorescence light and molecular rotational diffusion was first derived by Perrin (1926). His results, which concerned spherical particles, have later been refined to the more complex rotational motion of asymmetric bodies (Memming, 1961; Chuang & Eisenthal, 1972; Ehrenberg & Rigler, 1972; Belford, Belford & Weber, 1972).

Self-diffusion of a highly concentrated monoclonal antibody by fluorescence correlation spectroscopy: insight into protein–protein interactions and self-association

Soft Matter ◽  
2019 ◽  
Vol 15 (33) ◽  
pp. 6660-6676 ◽  
Author(s):  
Jessica J. Hung ◽  
Wade F. Zeno ◽  
Amjad A. Chowdhury ◽  
Barton J. Dear ◽  
Kishan Ramachandran ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Protein Interactions ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Protein Protein Interactions ◽  
Fluorescence Correlation ◽  
Self Diffusion ◽  
Self Association ◽  
Insight Into

Measurement and interpretation of self-diffusion of a highly concentrated mAb with different formulations in context of viscosity and protein self-interactions.

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