Dual color detection of cyan and yellow derivatives of green fluorescent protein using conventional fluorescence microscopy and 35-mm photography

The GFP has been used to monitor protein localization in living cells. The early development of the reporter improvements have been made by mutating the sequence of the original gene to obtain brighter fluorescence and improved expression. Brighter versions of the green fluorescent protein have been widely used in many experimental applications. The chapter explores the spectral characteristics and fluorescence intensities of enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) that are well suited for dual-color labeling experiments using standard epifluorescence microscopy and image documentation with a 35mm camera. The two fluorescent proteins fulfills two requirements for documentation on film: they are distinguishable in color and are strong enough in fluorescence for the relatively long exposure times needed for conventional photography. Although EBFP and EGFP are well distinguishable in color, ECFP proved to be superior to EBFP with respect to fluorescence intensity and photo-bleaching properties. The use of EYFP does not result in any disadvantage in comparison with EGFP because its fluorescence intensity and photo-bleaching characteristics are similar.