Proem 1024 em ccd camera

Leica DMI 6000B epifluorescence microscope, Olympus UAPO/340 40x/1.35 N.A. oil immersion objective and a ProEM 1024 EM-CCD camera (Princeton Instruments) was used to measure NAD(P)H autofluorescence. A high-pressure Xenon lamp was used for illumination with 350/20 nm excitation, 400 nm long-pass dichroic and 460/50 nm emission filters. Hardware synchronization and image acquisition was controlled through a Windows PC using custom software (Spectralyzer).

A hole of 15 mm diameter was drilled into the bottom of a 35 mm falcon tissue culture dish (353001, Becton Dickinson) and a coverslip (Ø 25 mm, Menzel-Gläser, Germany), rinsed with ethanol, was glued to the dish with silicon glue (Elastosil E43, Wacker, Germany). After coating coverslips with 20 µg ml^–1 FN (Calbiochem) overnight at 4°C, cells were plated and imaged in an inverted transmission light microscope (Zeiss Axiovert 200 M, Carl Zeiss) equipped with a climate chamber. Phase contrast images were taken with a ProEM 1024 EMCCD camera (Princeton Instruments, Acton, MA, USA) through a Zeiss Plan Neofluar 100x objective (NA 1.3, Ph3). Frames were acquired at 30 sec or 1 min intervals and converted to time lapse movies using ImageJ.