The cloning and expression of GFP in heterologous systems introduced a fantastic tool for studying specific gene expression and protein localization inside living cells. However, one aspect of GFP that has not been exploited to its full potential is its use as a quantitative imaging tool. To determine its quantitative usefulness, we have addressed five points that are important in GFP imaging: detectable signal over background, photostability, pH stability of the molecule, temperature dependence of chromophore formation, and estimation and normalization of GFP levels.

To determine the quantitative limits of GFP in cells, several GFP versions (wtGFP, αGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F)) were compared by imaging of GFP expressing cells or by spectroscopic measurements of purified proteins. When imaged, the GFP signals are contaminated by the naturally occurring background autofluorescence, but improved detection can be achieved for each green GFP by combination of confocal microscopy using 488 nm excitation, a rapid cut-on dichroic mirror, and a narrow bandpass emission filter (Figure l).