Figure 2

Optical analysis of light paths in VAEM and TIRFM observations in plant cells. (A) When the aqueous film between the cover glass and the cell wall is thick (> 100 nm) and θ _i is greater than 61.0° (θ _c ), all light energy is reflected at the g/am interface. The EW field forms in the aqueous medium but cannot penetrate into the cytosol. (B) When θ _i is less than 61.0° (θ _c ), a VAE field forms, illuminating the cytosol with variable depth. Angles of refraction for all optical boundaries with θ _i = 60.0° are shown. (C) When the aqueous film is sufficiently thin (< 100 nm), and θ _i is smaller than θ _c for g/cw (θ _c2 ) but greater than θ _c for g/c (θ _c1 ) [(i.e., θ _c1 <θ _i < θ _c2 )], an EW field of constant depth is created inside the cytosol at the cw/c interface. (D) When θ _i > θ _c2 , the light is completely reflected at the cell wall interface, and the EW field is not deep enough to penetrate into the cytosol. (E) Optical parameters of plant-cell peripheral boundaries. In the situation shown in (A), the EW field is 105 nm deep and cannot penetrate the cell wall. In the situation shown in (C) and with θ _i = 67.0°, the EW field penetrates 159 nm into the cytosol. CW: cell wall; EW: evanescent wave; PM: plasma membrane; VAE: variable-angle epifluorescence.