Probing the Plasma Membrane Organization in Living Cells by Spot Variation Fluorescence Correlation Spectroscopy

Total internal reflection fluorescence microscopy for determination of size of individual immobilized vesicles: Theory and experiment

“…14, 16, and 45, and can also be characterized with a decaying excitation intensity, although extending up to $500 À 600 nm. 46,47 This suggests that the range of accuracy reported (up to 500 nm using confocal microscopy) is within the boundary for discrepancy we observed between size estimations using (3) and (20). Although strongly surface-confined illumination used (e.g., in studies requiring elimination of bulk fluorescence and improved signal to noise ratio) is more critical, it remains preferable to employ physically and mathematically correct representations when experimental data are being analyzed, which is particularly true in cases when the incorrect Eq.…”

Mechanisms Underlying Anomalous Diffusion in the Plasma Membrane

“…STED is a superresolution technique where the probe volume is confined to a region not constrained by the diffraction limit and thus, it permits diffusion measurements at length scales relevant to lipid rafts with high temporal resolution (Kastrup, Blom, Eggeling, & Hell, 2005). Billaudeau et al (2013) review the use of spot variation FCS for the investigation of the nanoscale organization of the plasma membrane. These experiments reveal molecular details showing that constrained diffusion is often caused by cholesterol-assisted molecular complexes.…”

Imaging approaches for analysis of cholesterol distribution and dynamics in the plasma membrane

“…In this intricate way, one can determine whether the diffusion of a particular membrane probe becomes anomalous for the smallest spatial and shortest temporal scales [167,168]. In practice, one performs spot variation FCS by determining the mean diffusion transit time of a fluorophore or cholesterol analogue inserted into the PM of living cells through the laser as function of the width of the laser spot [169]. This can be achieved in several ways, but only by using a STED-microscope, one has directly access to spatial processes below the diffraction limit [170].…”