Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope

Abstract

Fluorescence photobleaching methods have been widely used to study diffusion processes in the plasma membrane of single living cells and other membrane systems. Here we describe the application of a new photobleaching technique, scanning microphotolysis. Employing a recently developed extension module to a commercial confocal microscope, an intensive laser beam was switched on and off during scanning according to a user definable image mask. Thereby the location, geometry, and number of photolysed spots could be chosen arbitrarily, their size ranging from tens of micrometers down to the diffraction limit. Therewith we bleached circular areas on the surface of single living 3T3 cells labeled with the fluorescent lipid analog NBD-HPC. Subsequently, the fluorescence recovery process was observed using the attenuated laser beam for excitation. This yielded image stacks representing snapshots of the spatial distribution of fluorescent molecules. From these we computed the radial distribution functions of the photobleached dye molecules. The variance of these distributions is linearly related to the diffusion constant, time, and the mobile fraction of the diffusing species. Furthermore, we compared directly the theoretically expected and measured distribution functions, and could thus determine the diffusion coefficient from each single image. The results of these two new evaluation methods (D = 0.3 +/- 0.1 micron 2/s) agreed well with the outcome of conventional fluorescence recovery measurements. We show that by scanning microphotolysis information on dynamical processes such as diffusion of lipids or proteins can be acquired at the superior spatial resolution of a confocal laser scanning microscope.

The analysis of intermolecular interactions in concentrated hyaluronan solutions suggest no evidence for chain–chain association

“…At low counterion concentrations ( 10 mM), sample fluorescence was reduced, consistent with previous observations [21]. For all samples, analysis of the recovery series showed that the expected linear dependence of the second moment of the radial distribution of bleached fluorophore on time and recovery kinetics were consistent with first-order behaviour of a single component [12].…”

“…The confocal-FRAP instrumentation and variance recovery analysis, for determining self and tracer lateral-diffusion coefficients, was as previously described [8,12,13]. Lateral selfand tracer-diffusion coefficients were calculated from the timedependence of plots of the second moment of the radially averaged distribution of bleached fluorophores.…”

“…This provides measurements of lateral self-and tracer-diffusion coefficients and is a powerful method for determining concentrated solution properties in the absence of flow and shear forces and with no concentration gradients [12,13]. In previous work using confocal-FRAP analysis it was shown that HA solution properties at high concentrations could be predicted from the behaviour in dilute solution [8].…”

Diffusion of α-Chymotrypsin in Solution-Crowded Media. A Fluorescence Recovery after Photobleaching Study

“…The intersection between the slope of the linear decreasing (anomalous diffusion regime) and the line corresponding to the final and constant value of the diffusion coefficient (normal diffusion in a dense medium) yields the crossover time between these two behaviours 56 . Although a confocal laser scanning microscope was used, the uniform circular disc profile approximation 38,[43][44]48 , described first in 1983 by Soumpasis 47 , is a suitable model to study our confocal FRAP curves. In fact, according to Pucadyil and…”

“…An important number of manuscripts explain how, from Axelrod et al equation 42 (Gaussian profile approximation) for stationary lasers, it is possible to study the diffusion of tracer particles (e.g. proteins) in 2D or 3D media when a confocal FRAP is employed [38][39][40][41][43][44][45][46]48 . But, normally, in 2D media confocal FRAP experimental curves can be examined using Soumpasis equation 47 (uniform circular disc profile approximation) for nonscanning microscope 48 .…”