α plan fluar 100x

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.

TIRF illumination (TIRFi) was used for our experiments. The expanded beam of a 488/568 nm argon/krypton multiline laser (20 milliwatts, Laserphysics, Germany) passed through an AOTF laser wavelength selector (VisiTech International, UK) synchronized with a SNAP-HQ CCD camera (Roper Scientific, Germany) under Metafluor software (Universal Imaging, USA) control and was introduced to the coverslip from the high numerical aperture objective lens (Zeiss α-plan FLUAR 100X). Light entered the coverslip and underwent total internal reflection at the glass-cell interface. In our experimental conditions, penetration depth of TIRFi was calculated to be about 90 nm [17 (link), 25 (link)]. In single-wavelength TIRFi experiments (488 nm) the laser beam was filtered via the Zeiss filter set 10 and images were acquired at 20–40 Hz (Zeiss, Switzerland). In dual-wavelength TIRF illumination (488/568 nm), laser beams were combined by a dichroic mirror from the Zeiss filter 24 at 20–40 Hz. The pixel size was 126 nm (at binning 2).