FLIM was done as described previously (McCullock et al., 2020 (link)). Cells were transfected in either 35 or 60 mm culture dishes and imaged using a water immersion 25× objective (XL Plan N, 1.05 NA) mounted on an Olympus IX61WI upright microscope. A Mai Tai Ti:Sapphire multi-photon laser (Spectra Physics) was used for excitation with an 860 nm wavelength, a repetition rate of 80 MHz and pulse width of approximately 100 fs. Donor emission was filtered by a 480–20 filter and measured by a H72422P Hamamatsu hybrid avalanche photodiode. Time-correlated single photon counting was done using a Becker and Hickl card with a resolution of 25 ps. Using VistaVision software (ISS), donor fluorescence from the plasma membrane from individual cells was binned and fit with a single exponential function, consistent with the lifetime of CFP variant mTurquoise2 (Goedhart et al., 2012 (link)).
For confocal imaging, cells were transfected with Lck-CFP or CFP-tagged cASIC1 in 35 mm dishes. After 2 days, cells were stained with 2 mL of 7.5 µM FM1–43 (Invitrogen) immediately prior to imaging with an Olympus FV1000MP microscope using a 60× water immersion objective (U Plan SApo, 1.20 NA). CFP and FM1–43 were simultaneously excited with a 440 nm laser and emissions between 465 and 495 nm collected as CFP and 575 and 675 nm collected as FM1–43.
For confocal imaging, cells were transfected with Lck-CFP or CFP-tagged cASIC1 in 35 mm dishes. After 2 days, cells were stained with 2 mL of 7.5 µM FM1–43 (Invitrogen) immediately prior to imaging with an Olympus FV1000MP microscope using a 60× water immersion objective (U Plan SApo, 1.20 NA). CFP and FM1–43 were simultaneously excited with a 440 nm laser and emissions between 465 and 495 nm collected as CFP and 575 and 675 nm collected as FM1–43.