Tcs sp5 inverted laser scanning confocal microscope

The cells were examined with Leica TCS SP5 inverted laser scanning confocal microscope (Germany) equipped with solid-state lasers for excitation (405, 488 and 543 nm). QD fluorescence was excited at 405 or 488 nm and registered in the 640–670 nm channel; Cy-3 fluorescence was excited at 543 nm and registered in the 560–620 nm range. Alexa 488 and Alexa 568 were excited at 488 nm and 543 nm and registered in the 500–550 and 580–660 nm ranges, respectively. GFP was excited at 488 nm and registered in the 500–550 nm range. Hoechst fluorescence was excited at 405 nm and registered in the 430–480 nm range. Specimens were observed with a × 40 oil immersion objective, followed by a 4 digital zoom magnification with an image size of 1024 × 1024 pixels. Images were taken in one or two spectral channels by sequential scanning mode, where only one laser was active at a time, to avoid spectral overlap. To optimize the signal to noise ratio, the final image was an average of three consequent runs. Z-series optical sections were taken at 0.5-μm intervals from the bottom to the top (14–16 sections). Images were acquired for at least 5 fields of view selected randomly per coverslip. Data were collected by Leica software as raw *.lif files and transferred as a series of tiff files for further analysis.