Multiphoton-evoked color change of DsRed as an optical highlighter for cellular and subcellular labeling

DsRed, a recently cloned red fluorescent protein, has attracted great inter est as an expression tracer and fusion partner for multicolor imaging. We r eport that three-photon excitation (lambda <760 nm) rapidly changes the flu orescence of DsRed from red to green when viewed subsequently by convention al (one-photon) epifluorescence. Mechanistically, three-photon excitation ( <lambda> <760 nm) selectively bleaches the mature, red-emitting form of DsR ed, thereby enhancing emission from the immature green form through reducti on of fluorescence resonance energy transfer (FRET). The "greening" effect occurs in live mammalian cells at the cellular and subcellular levels, and the resultant color change persists for >30 h without affecting cell viabil ity. This technique allows individual cells, organelles, and fusion protein s to be optically marked and has potential utility for studying cell lineag e, organelle dynamics, and protein trafficking, as well as for selective re trieval of cells from a population. We describe optimal parameters to induc e the color change of DsRed, and demonstrate applications that show the pot ential of this optical highlighter.