Three-Dimensional Fluorescence Recovery after Photobleaching with the Confocal Scanning Laser Microscope

Abstract

Confocal scanning laser microscopes (CSLMs) are equipped with the feature to photobleach user-defined regions. This makes them a handy tool to perform fluorescence recovery after photobleaching (FRAP) measurements. To allow quantification of such FRAP experiments, a three-dimensional model has been developed that describes the fluorescence recovery process for a disk-shaped geometry that is photobleached by the scanning beam of a CSLM. First the general mathematical basis is outlined describing the bleaching process for an arbitrary geometry bleached by a scanning laser beam. Next, these general expressions are applied to the bleaching by a CSLM of a disk-shaped geometry and an analytical solution is derived that describes three-dimensional fluorescence recovery in the bleached area as observed by the CSLM. The FRAP model is validated through both the Stokes-Einstein relation and the comparison of the measured diffusion coefficients with their theoretical estimates. Finally, the FRAP model is used to characterize the transport of FITC-dextrans through bulk three-dimensional biological materials: vitreous body isolated from bovine eyes, and lung sputum expectorated by cystic fibrosis patients. The decrease in the diffusion coefficient relative to its value in solution was dependent on the size of the FITC-dextrans in vitreous, whereas it was size-independent in cystic fibrosis sputum.

Ultrasound and microbubble mediated drug delivery: Acoustic pressure as determinant for uptake via membrane pores or endocytosis

“…Dextrans of two molecular weights were tested, i.e. 4 kDa and 2 MDa FITC-dextran with an estimated radius of 1.23 nm and 32.78nm [22], respectively. The Opticell TM was flipped to allow the microbubbles to rise against the cell monolayer and submerged in the water tank.…”

A spectrophotometer-based diffusivity assay reveals that diffusion hindrance of small molecules in extracellular matrix gels used in 3D cultures is dominated by viscous effects

“…The latter was assessed using an empirical relation [21] that elicited 1.21 nm for Dex4, 4.12 nm for Dex40 and 5.55 nm for Dex70.…”

pH-dependent Intraluminal Organization of Mucin Granules in Live Human Mucous/Goblet Cells

“…35. As applied to the granule, the model assumed that recovery of the fluorescence within the bleached spot was caused by radial (rather than axial) diffusion of SHGFP-MUC5AC/CK, which is a reasonable assumption (46,47) considering that (a) FRAP analysis was carried out with a 1.4 numerical aperture objective, and hence, a substantial region in the axial direction was also bleached during the photobleaching pulse; and (b) SHGFP-MUC5AC/CK had a low intragranular mobility (see "Results"). Only granules with diameters of 1.1 m or larger in which the fluorescence was homogeneously distributed were chosen for D eff analysis.…”