Alternating EPI-TIRF Imaging for Phaluorin Fluorescence

EPI or TIRF illuminations were used for our experiments. In Bafilomycin A1 experiments, EPI and TIRF illuminations were alternated in real time for whole-cell pHluorin fluorescence (pHF) signal measurements in individual cells, in basal condition and upon stimulation. By imaging with excitation light at 488 nm generated by a 488 nm laser (20 mW; Laserphysics) and by a polychromator illumination system (Visichrome), the pHF signal was recorded at 10 Hz through a 100X objective lens (α-plan FLUAR 100X, 1.45 NA, Zeiss, Germany) and filtered with Zeiss filter set 10 (Zeiss) [19] (link). For TIRF illumination, the expanded beam (488/568 nm argon/krypton multilinelaser, 20 mW) (Laserphysics, USA) was passed through an AOTF laser wavelength selector (VisiTech International) synchronized with a SNAP-HQ CCD camera (Roper Scientific, Germany) under Metafluor software (Universal Imaging, USA) control and was introduced from the high numerical aperture objective lens (α-plan FLUAR 100X, 1.45 NA, Zeiss, Germany). Light entered the coverslips and underwent total internal reflection at the glass-cell interface. In our experimental conditions, penetration depth of TIRF illumination was calculated to be about 90 nm [20] (link). Light was filtered with a beam splitter (Zeiss filter set 10). Images were acquired at 20–40 Hz. Pixel size 126 nm at binning 2.