| Method |
To quantify the diffusion constant of small molecules in the plant cell wall, fluorescence from carboxyfluorescein (CF) in the intact roots of Arabidopsis thaliana was recorded. Roots were immersed in a solution of the fluorescent dye and viewed through a confocal fluorescence microscope. These roots are sufficiently transparent that much of the apoplast can be imaged. The diffusion coefficient, D(cw), of CF in the cell wall was probed using two protocols: fluorescence recovery after photobleaching and fluorescence loss following perfusion with dye-free solution. Diffusion coefficients were obtained from the kinetics of the fluorescent transients and modelling apoplast geometry. To determine the aqueous diffusion coefficient of CF,
a small volume of the aqueous CF solution was placed
between two coverslips, forming a thin film between
20 µm and 100 µm thick. Under the confocal microscope,
a circular spot with a radius of ~200 µm and an optical
thickness greater than the fluid thickness (to avoid
measurement artefacts due to diffusion in the z-direction)
was bleached. Then the half-time for fluorescence recovery,
t1/2, was determined as the time needed for the intensity
at the centre of the disc to reach 50% of the asymptotic
recovery value. The exact solution to the diffusion equation,
assuming an initial bleached disc of radius R, gives
D=R^2/[4ln(2)t1/2]=(0.36R^2)/t1/2 (Crank, 1985). The aqueous
diffusion coefficient for CF determined in this way
was D=(4.87±0.22)×10^-10 m^2 s^-1 (mean±SE, n=4). |