Variable-angle TIRF microscopy

Cells were transfected and plated on 35-mm glass-bottom dishes (MatTek) coated with 0.1 mg/ml poly-dl-lysine (Sigma-Aldrich). After 72–96 h, cells were imaged on a Nikon N-STORM Imaging System with an Apo TIRF 100×/NA 1.49 objective lens. EGFP fluorescence was excited with a 488-nm diode laser. The laser angle was set using a motorized TIRF illuminator programmed through Elements imaging software. Images were captured sequentially with 100-ms exposures using an iXon DU897 EM CCD camera (Andor, Concord, MA). We calculated the TIRF field penetration depth using the equation d = λ/[4π(n₁² sin²Θ – n₂²)^1/2], where sin Θ = 2rm/200,000n₁, r is the distance of the laser illumination position from the center (in micrometers), n₁ and n₂ are the refractive indices of the glass and medium, respectively, and m is the magnification of the objective; TIRF angle positions (r) were found relative to the critical angle. The position illuminator angle used in live cells was calculated at a 1/e value of 90, 130, and >130 nm. These penetration depths were confirmed using fluorescent beads (0.05, 0.1, 0.2, and 0.4-μm diameter) absorbed to glass (MatTek) and imaged in PSS-Na plus 15% BSA. Bead images were obtained with the laser illuminator at 90, 130, >130, or vertical positions. Total fluorescence (vertical position) was compared with bead fluorescence at each position. Total fluorescence of 0.05- and 0.1-μm beads was evident at ≥90 nm, whereas total fluorescence of the 0.2-μm bead was evident at ≥130 nm.