Passivation of Silica Substrates for TIRF Microscopy

Silica microscope slides used for TIRF experiments were prepared as described before^{57 (link)}. Briefly, fused silica slides (Plano) were cleaned in peroxomosulfuric acid (70% (v/v) sulfuric acid; Fisher Scientific, 10% (v/v) hydrogen peroxide; Sigma-Aldrich) for 30 min and washed with Millipore water under sonication. Afterwards, the slides were incubated in methanol for 20 min and sonicated for 5 min. For silane passivation, the slides were incubated in a freshly prepared N-[3-(Trimethoxysilyl)-propyl]ethyldiamine (Sigma-Aldrich) solution (2% (v/v) in methanol with 4% (v/v) acetic acid) for 20 min, rinsed with methanol five times and an additional 20 times with Millipore water. The slides were dried for 1 h at 37 °C. For PEG passivation, 100 µL of freshly prepared passivation solution (200 mg/mL methoxy-PEG succinimidyl valerate 5000 (Laysan Bio), 5 mg/mL biotin-PEG (Laysan Bio) in 1 mM NaHCO₃) was sandwiched between a slide and a coverslip, incubated for 2 h and rinsed with Millipore water 20 times. The slides and coverslips were fully dried at 37 °C, vacuum sealed in plastic tubes and stored at −20 °C.