Quantitative Fluorescence Microscopy Analysis

Observations were made using E-600 epifluorescence microscope (Nikon) equipped with phase-contrast optics, U2 filter (UVB light; λ = 340–380 nm) for DAPI and Hoechst 33342, B2 filter (blue light; λ = 465–496 nm) for Alexa Fluor^® 488, or G2 filter (green light; λ = 540/25 nm) for Alexa Fluor® 555 and PI-stained cell nuclei. All images were recorded at exactly the same time of integration using a DS-Fi1 CCD camera (Nikon). Quantitative analyses and nuclear DNA fluorescence measurements were made after converting color images into greyscale and expressed in arbitrary units as mean pixel value (pv) spanning the range from 0 (dark) to 255 (white) according to described methods (Żabka et al. 2010 (link), 2012 (link)). The obtained data were expressed as the mean values ± standard deviation of the mean (SD). Student's t tests for paired data were used to compare individual variables.