QUANTITATIVE IMAGING OF GREEN FLUORESCENT PROTEIN IN CULTURED-CELLS -COMPARISON OF MICROSCOPIC TECHNIQUES, USE IN FUSION PROTEINS AND DETECTION LIMITS

To determine the application limits of green fluorescent protein (GFP) as a reporter gene or protein tag, we expressed GFP by itself and wit h fusion protein partners, and used three different imaging methods to identify GFP fluorescence. In conventional epifluorescence photomicro scopy, GFP expressed in cells could be distinguished as a bright green signal over a yellow-green autofluorescence background, In quantitati ve fluorescence microscopy, however, the GFP signal is contaminated by cellular autofluorescence, Improved separation of GFP signal from HeL a cell autofluorescence was achieved by the combination of confocal sc anning laser microscopy using 488-nm excitation, a rapid cut-on dichro ic mirror and a narrow-bandpass emission filter, Two-photon excitation of GFP fluorescence at the equivalent of approximate to 390 nm provid ed better absorption than did 488-nm excitation, This resulted in incr eased signal/background but also generated a different autofluorescenc e pattern and appeared to increase GFP photobleaching, Fluorescence sp ectra similar to those of GFP alone were observed when GFP was express ed as a fusion protein either with glutathione-S-transferase (GST) or with glucokinase. Furthermore, purified GST . GFP fusion protein displ ayed an extinction coefficient and quantum yield consistent with value s previously reported for GFP alone, In HeLa cells, the cytoplasmic GF P concentration must be greater than approximate to mu M to allow quan tifiable discrimination over autofluorescence, However, lower expressi on levels may be detectable if GFP is targeted to discrete subcellular compartments, such as the plasma membrane, organelles or nucleus.