Js. Marchant et al., Multiphoton-evoked color change of DsRed as an optical highlighter for cellular and subcellular labeling, NAT BIOTECH, 19(7), 2001, pp. 645-649
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.