Figure 1.

Evanescent field effects on FRET calibrations and TIRF-FRET. (a) Representative raw and pseudocolored images of cYFP-coated beads imaged under epifluorescence or TIRF (514-nm laser line). Characteristic TIRF depth is indicated below each image. (b) Schematic depicting determination of z-distance using beads. A fluorophore-coated bead with radius, R, rests on a glass coverslip. Each pixel of the TIRF bead footprint is at a specific radial distance, d, corresponding to fluorescence from a z-distance, z₁, from the coverslip. (c) Average characteristic TIRF depths were calculated from beads imaged with the 442-nm line (purple markers) using five incident angles, and plotted against the incident TIRF angle, which was measured using a prism. (Solid curves) Theoretical relationships between D₄₄₂ and angle of incidence (n₁ = 1.515, n₂ = 1.37 or 1.38). Beads were imaged in a solution where n ∼ 1.372. (d) Similar to panel c, except that beads were imaged using the 514-nm laser line; theoretical relationships were determined for a 514-nm laser. (Markers) Mean ± SE. For panels c and d, n = number of beads. (e–j) Effects of three TIRF illumination conditions on FRET constants and FRET efficiency. (Black) D₄₄₂, D₅₁₄ ∼ 270 nm. (Purple) D₄₄₂ ∼ 280 nm, D₅₁₄ ∼ 140 nm. (Green) D₄₄₂ ∼ 160 nm, D₅₁₄ ∼ 270 nm. Plots were normalized to the value at z = 0. For panels e–g, α, γ, and ξ versus z-distance, respectively. The value β does not vary with z-distance (data not shown). (h–j) EA, ED, and Ratio versus z-distance, respectively. Error-free measurements would be evident as horizontal lines with EA, ED = 0.4, and Ratio = 1. (Markers) Mean ± SE (n = purple, green, black): α (7,7,12); β (6,8,6); and ξ, γ, EA, ED, Ratio (7,4,10).