7, 8, 9 Nevertheless, the errors in diffusion coefficients measured by FRAP and SPT have not been quantitatively analyzed at the same platform. 8 The disagreement of the diffusion coefficients is caused by interlaboratory or intersystem variations of several factors, including the condition of sample, labeling marker, detecting platform and microenvironment. They found that D FRAP was biased to larger or smaller values compared with D SPT depending on the length scales and temporal resolution. 8 measured lateral lipid diffusion in supported lipid bilayers and giant unilamellar vesicles by SPT and FRAP on the same samples using different instruments. Saxton and Jacobson 7 reported that D FRAP was 4 − 7 times higher than the apparent D SPT for the mobile fraction of lipids and GPI (glycosylphosphatidylinositol)-linked proteins at the membrane of cells such as fibroblast and myoblast. 6ĭespite many researches, diffusion coefficients determined by FRAP ( D FRAP ) were found to be inconsistent with SPT measurement ( D SPT ). SPT also has been studied for many years and applied to various areas, such as measurement of diffusion coefficient of quantum dot-labeled protein 5 and membrane receptors conjugated with gold nanoparticles. 2, 3, 4 As opposed to FRAP, SPT traces the trajectory of each molecule conjugated with nanoparticles and reveals the structure of cell membrane as well as the dynamic behavior of the molecule. 1 and has been improved by a number of researchers. FRAP had been originally devised by Axelrod et al. Subsequently, fluorescence is recovered by the diffusion of both bleached and unbleached molecules and diffusion coefficient is determined by analysis of the fluorescence recovery curve.
#Particle tracker imagej full
In the FRAP system, a micron-sized region full of fluorophores is instantaneously bleached by a high-intensity laser irradiation. In addition, quantitative measurement of diffusion of solutes is crucial in drug delivery system and cell morphogenesis. Our i FRAP-SPT technique can be potentially used for not only cellular membrane dynamics but also for quantitative analysis of the spatiotemporal distribution of the solutes in small scale analytical devices.įluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) have been used to estimate the diffusion coefficients of macromolecules in biological media because diffusion process is a key mechanism for biomolecular transport in micro-environment such as scaffolds and membranes. Our results reveal that diffusion coefficient overestimated by FRAP is caused by inaccurate estimation of the bleaching spot size and can be corrected by simple image analysis. The combined i FRAP-SPT technique allowed us to measure the diffusion coefficient of the same fluorescent particle by utilizing both techniques in a single platform and to scrutinize inherent errors and artifacts of FRAP.
Here, we designed an image-based FRAP-SPT system and made a direct comparison between FRAP and SPT for diffusion coefficient of submicron particles with known theoretical values derived from Stokes–Einstein equation in aqueous solution. Lateral diffusion coefficients measured by FRAP and SPT techniques for the same biomolecule on cell membrane have exhibited inconsistent values across laboratories and platforms with larger diffusion coefficient determined by FRAP, but the sources of the inconsistency have not been investigated thoroughly.
Fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) techniques determine the diffusion coefficient from average diffusive motion of high-concentration molecules and from trajectories of low-concentration single molecules, respectively.