Determination of the Mobile / Immobile Fraction
If the whole population of the investigated molecule is freely mobile the fluorescence intensity (background subtracted and corrected for loss of fluorescence due to the bleaching pulse) recovery curve should reach a plateau at 100% of the initial fluorescence of the prebleach. Binding of a fraction of the molecules of interest to slow or immobile structures (e.g. Nuclear envelope) reduces the recovered level of the fluorescence, the fractions can be calculated with the following equations.
Mobile fraction Fm = (IE - I0) / (II - I0)
With: IE: Endvalue of the recovered fluorescence intensity
An additional method to measure the mobile and immobile fractions exemplified for the nucleus is described by Houtsmuller 2001. A spot in the compartment of interest is bleached over an extended period of time with relatively low laser intensity. During this extended bleaching time a large percentage of mobile molecules passes through the bleaching spot and partially will be bleached. Subsequently, the mobile molecules are allowed to completely redistribute through the nucleus (depending on their diffusion coefficient). The ratio of fluorescence intensity of confocal images before and after this procedure is then plotted against distance to the laser spot. To accurately calculate the immobile fraction from this plot one should obtain two reference curves, representing the situations in which all molecules are immobile (fixed sample) and in which all molecules are mobile (e.g. in an inducible system).
contact: Stefan Terjung last update: 02/06/04