Probing microscopic diffusion by 2-photon flash photolysis: measurement of isotropic and anisotropic diffusion in...

Two-photon excited flash photolysis (TPEFP) was used to photorelease caged fluorescein in test solutions and inside fiber cells of the eye lens. Accurate alignment between the focus of the IR beam and the probe beam from the confocal microscope was achieved with an accessory focussing lens and computer models of diffusion were fit to experimental data to extract apparent diffusion coefficients. Inside a fiber cell, the diffusion coefficient for fluorescein was 4 x 10(-7) cm(2)/s at 21 degrees C, a value an order of magnitude lower than observed in free solution. Fluorescence also diffused between fiber cells via gap junctions. In the periphery, diffusion between cells occurred mainly in a radial direction while deep in the lens the diffusion between cells appeared more isotropic. Diffusion between cells was slower than inside cells and corresponded to less than approximately 1% of the area between cells being available for diffusion. This value is in good agreement with that expected from measurements of gap junction structure and packing density if a 1-1.5-nm aqueous gap junction pore is nearly always open.