Straightforward FRAP for quantitative diffusion measurements with a laser scanning microscope

Abstract

Confocal or multi-photon laser scanning microscopes are convenient tools to perform FRAP diffusion measurements. Despite its popularity, accurate FRAP remains often challenging since current methods are either limited to relatively large bleach regions or can be complicated for non-specialists. In order to bring reliable quantitative FRAP measurements to the broad community of laser scanning microscopy users, here we have revised FRAP theory and present a new pixelbased FRAP method relying on the photobleaching of rectangular regions of any size and aspect ratio. The method allows for fast and straightforward quantitative diffusion measurements due to a closed-form expression for the recovery process utilising all available spatial and temporal data. After a detailed validation, its versatility is demonstrated by diffusion studies in heterogeneous biopolymer mixtures. 2010 Optical Society of America

Effect of pre-crystallization process and solid particle addition on microstructure in chocolate model systems

“…The size of the ROI was set to a 10 × 10 μm square, according to the rectangular FRAP model developed by Deschout et al (2010). This relatively small ROI allowed measurement of the local diffusion rate of BODIPY solely in the fat crystal structure, so the ROI was always placed on the fat crystal networks visible in the frames.…”

“…Calculations of the local diffusion rate of BODIPY, expressed in μm 2 /s, in the cocoa butter matrix were made according to the methods of rectangular FRAP developed by Deschout et al (2010) using 50 post-bleached images. To extract as much information as possible from the recovery, every third post-bleached image was used for samples with a markedly slow recovery, whereas the first 50 were used for samples with a fast recovery.…”

“…A recent development in FRAP methodology is the pixel-based rectangle FRAP (rFRAP) method, which measures the change in intensity in each pixel of the bleached area instead of averaging it over the entire surface. This approach makes the rFRAP method a sensitive technique with extremely high precision (Deschout et al, 2010). The rFRAP technique permits further investigation of the structure-process relationship in chocolate, since the structure density, and its ability to retard or hinder fat migration, obtained through optimized precrystallization can be quantified by local migration measurements using FRAP.…”

Signal Integration by Lipid-Mediated Spatial Cross Talk between Ras Nanoclusters

“…The TopFluor-PS (TF-PS) labeling protocol and fluorescence recovery after photobleaching (FRAP) protocol followed those in previous studies (26)(27)(28), except that cells were allowed to equilibrate for 1 h after being pulse-labeled. Wild-type BHK cells or BHK-Cav-1KD cells were seeded on fibronectin-coated glass-bottom dishes overnight.…”

FRAP in Pharmaceutical Research: Practical Guidelines and Applications in Drug Delivery

“…It was only in the 1990s, with enough computer memory and processing power becoming available in standard computers, that FRAP models were developed that started to exploit more of the available spatial information (20,21). This development is ongoing and allows to extract increasingly more information with better accuracy from the observed fluorescence recovery (22)(23)(24).…”