Axiovert 200m

Following plating on gelatin-coated Lab-Tek I chambers, cells were maintained in phenol red-deprived DMEM-F12 (supplemented with low serum growth supplement and 10% FBS) for epifluorescence video microscopy experiments. For bright field video microscopy, cells were observed over a 30 min period using a Zeiss Axiovert 200M equipped with a Coolsnap camera (Roper Scientific, Trenton, NJ, USA). Cells were maintained at 37°C in 5% CO₂ atmosphere using a temperature-controlled chamber (PECON, Erbach, Germany). Data were acquired using MetaMorph software (Roper Scientific). Timespan between image capture was 4 s. Confocal microscopy images of fixed cells were collected with a Zeiss (Oberkochen, Germany) LSM710 Meta confocal microscope using either a ×63 Plan Apochromat objective (oil immersion, 1.40 NA, DIC) at a 132 nm.pixel^-1 resolution, leading to a slight XY oversampling. Optical slices were spaced by 1 μm so as to limit Z oversampling. Fluorescence filters were chosen and tested for each combination of plasmid construction and fluorescent dye so as to abolish spectral overlap. Zen software program was used to acquire images.

For US28-pHluorin TIRF imaging, HeLa cells on poly-l-lysine-coated round 18 mm coverslips in a 12 wells plate were transfected with 500 ng HA-US28-pHluorin plasmid using Lipofectamine 2000 (Invitrogen). 24 hours after transfection, coverslips were placed in an imaging chamber, perfused with Tyrode’s solution and imaged on a microscope (Zeiss, Axiovert 200M) equipped with an EMCCD camera (Cascade, Roper Scientific). For TIRF imaging, a laser beam from an air-cooled argon ion laser was coupled into a 100 × 1.45 N.A. TIRF objective via a TIRF condenser (TILL Photonics). Images were acquired at 2 Hz with MetaMorph 6.2 software (Universal Imaging). Imaging experiments were performed at room temperature (21°C–24°C).

The transmittance spectra of Ag/Au alloyed nanoislands were measured with an inverted microscope (Carl Zeiss, Axiovert 200M) equipped with a commercial spectrometer (Princeton Instruments, MicroSpec 2300i) and a charge-coupled device camera (Princeton Instruments, PIXIS: 400BR). The transmittance spectra were collected with 50x objective lens.