Using FRAP to Determine the Diffusivity of
Molecules in the Eye
Chris Breward, Oxford University
The lens is a transparent component of the eye which focuses light rays entering the eye. It is sandwiched between two aqueous components and relies on diffusion of nutrients and waste products for survival. Diffusion in the lens is also important for lens development and growth, drug delivery, ocular inflammation and for cataract formation and treatment.
Fluorescence Recovery After Photo-bleaching (FRAP), a technique used to quantify diffusion of fluorescently labelled tracer molecules through a matrix, has been used to quantify the diffusivity of various molecules through the lens capsule. In these experiments, the lens is placed in a chamber containing a bath of tissue media containing the fluorescent molecules, and is left there for sufficient time to ensure that the molecules have diffused into the lens and that the system is in equilibrium. Some of the molecules are free to diffuse around and others become bound to the matrix scaffold. A confocal microscope is focused in the matrix at a depth of 5 microns and primary and secondary regions of interest (ROI), both with 5 micron radius, are defined within the field of view. Under the excitation of a laser, the tracer molecules emit a signal which is detected and turned into a fluorescence intensity value. Baseline intensity is measured for several frames in both ROIs and then the laser is briefly focused at a higher power on the primary ROI, which irreversibly bleaches the tracer molecules in this region. The secondary ROI is not bleached and is used to normalize the data in the primary ROI. Recovery of the fluorescence intensity in the primary ROI represents the passive exchange of bleached and unbleached tracer molecules and is assumed to correlate with planar diffusion.
The data are fitted using a piece of software that uses a single exponential and a double exponential. The exponentials are used to back out the “half recovery time” for the fluorescence in the bleached ROI, which is used to calculate the diffusion coefficient for the mobile fraction of the tracer molecule in the matrix.
Experimental concerns include whether neglecting diffusion in the third dimension is justified, whether neglecting the aurora at the edge of the ROI alters the results, and how the truncation of the time series affects the value of the fitted parameters.
The aim of the week will be to more accurately quantify the experimental results by: