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 interest as an expression tracer and fusion partner for multicolor imaging. We report that three-photon excitation (lambda <760 nm) rapidly changes the fluorescence of DsRed from red to green when viewed subsequently by conventional (one-photon) epifluorescence. Mechanistically, three-photon excitation (lambda <760 nm) selectively bleaches the mature, red-emitting form of DsRed, thereby enhancing emission from the immature green form through reduction 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 viability. This technique allows individual cells, organelles, and fusion proteins to be optically marked and has potential utility for studying cell lineage, organelle dynamics, and protein trafficking, as well as for selective retrieval of cells from a population. We describe optimal parameters to induce the color change of DsRed, and demonstrate applications that show the potential of this optical highlighter.
Marchant
J S
JS
Laboratory of Cellular and Molecular Neurobiology, University of California, Irvine, CA 92697-4550, USA.
Stutzmann
G E
GE
Leissring
M A
MA
LaFerla
F M
FM
Parker
I
I
D016475:3T3 Cells; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006801:Humans; D008164:Luminescent Proteins / Q000494:pharmacology*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D017785:Photons; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C413663:fluorescent protein 583