Variable-angle total internal reflection fluorescence microscopy of intact cells of Arabidopsis thaliana

Here, we present theoretical and experimental evidence supporting the use of variable-angle TIRFM in observations of intact plant cells. We show that when total internal reflection occurs at the cell wall/cytosol interface with an appropriate angle of incidence, an evanescent wave field of constant depth is produced inside the cytosol. Results of experimental TIRFM observations of the dynamic behaviors of phototropin 1 (a membrane receptor protein) and clathrin light chain (a vesicle coat protein) support our theoretical analysis.These findings demonstrate that variable-angle TIRFM is appropriate for quantitative live imaging of cells in intact tissues of Arabidopsis thaliana.Total internal reflection fluorescence microscopy (TIRFM), also known as evanescent wave microscopy (EWM), is a powerful tool for observing the distribution and movement of fluorescently labeled molecules in an aqueous environment. This technique can be used when the molecules of interest are very close to the boundary between the aqueous environment and another medium with a higher refractive index (n). It is based on the physical phenomenon of total internal reflection (TIR), which occurs when a ray of light strikes a boundary between two materials with different n values and the incident angle (θi) is greater than the critical angle of incidence (θc) [1]. Under these conditions, all of the light is reflected back into the medium with the higher n value (Formula 1) [1]. When TIR occurs, a nearfield light wave forms at the boundary; this "evanescent wave" (EW) can penetrate the surface of the medium to a depth (d) approximately equal to 1/3 of the wavelength of the incident light (Formula 2) [1].Formula for calculating critical angle of incidence:Formula for calculating the depth of an EW field:The TIRFM can provide a few hundred nanometers of excitation volume near the plasma membrane; this feature is particularly useful because the excitation volume is the site of many important events assoc