Single-molecule microscopy of the green fluorescent protein using simultaneous two-color excitation.

In vivo microscopy of the Green Fluorescent Protein (GFP), the most important label in cell biology, with single-molecule sensitivity is hampered by an insufficient signal-to-noise ratio. A significant improvement is obtained with a novel two-color excitation technique. The picture clearly shows the increased brightness of GFP in in vitro single-molecule assays and in live-cell microscopy under two-color illumination (upper cell) as compared to normal illumination (lower cell).