Em ccd digital camera

Imaging experiments were performed using a Zeiss Axiovert 200M inverted epifluorescence microscope equipped with an EM-CCD digital camera (Hamamatsu) and a MS200 XY Piezo Z stage (Applied Scientific Instruments). The light source was an X-Cite 120 metal-halide lamp (EXFO) and the fluorescence images were obtained using an oil-immersion objective at 63× magnification. The fluorescence filters sets used are defined as blue: excitation G 365 nm, beamsplitter FT 395 nm, emission BP 445/50 nm; red: BP 550/25 nm, beamsplitter FT 570 nm, emission BP 605/70 nm. The microscope was operated using Volocity software (Perkin-Elmer).
The exposure time for acquisition of fluorescence images were kept constant for each series of images at each channel. To measure analyte-induced fluorescence changes, the cells were treated with dye-free DMEM containing 20 μM ZnCl₂ and 50 μM pyrithione for 10 min. To reverse the effect of zinc, the cells were exposed to dye-free DMEM containing 50 μM TPEN for 15 min. All these experiments were carried out on the stage of the microscope. Quantification of fluorescence intensity was performed using ImageJ (version 1.45, NIH). The whole cell was selected as the region of interest. The integrated fluorescence from the background region was subtracted from the cell body region.