But there was scope for further improvement. In particular, tracking protein movement in real time was difficult if the tagged protein became evenly distributed in the cell under steady-state conditions. Writing in Science (297, 1873–1877; 2002), George H. Patterson and Jennifer Lippincott-Schwartz now describe a possible solution to this problem. They have produced a GFP variant that shows greatly increased fluorescence if it is activated by light; and it functions in physiological conditions. Using this photoactivatable GFP, tagged proteins in a small area of the cell can be selectively marked by light activation, so that their movement through the cell can be followed against a dark background by fluorescence microscopy.
Normal GFP contains a mixed population of neutral and anionic chromophores — molecules that absorb light then re-emit it. These are respectively associated with a major light-absorption peak at a wavelength of 397 nm and a minor peak at 475 nm. Intense illumination at 400 nm shifts the population to the anionic form, thereby increasing the absorbance of the minor peak. Patterson and Lippincott-Schwartz set out to develop a GFP variant that had a negligible 475-nm peak. They reasoned that illumination at 400 nm would then produce a much greater proportional increase in 475-nm absorbance compared with the normal protein, and therefore increase optical contrast.
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