This method achieves simultaneous and spatially colocalized excitation of three fluorophores with distinct spectra, doing so via two-photon microscopy using a femtosecond laser and an optical parametric oscillator and by temporally overlapping the beams. Imaging of 'Brainbow'-labeled mouse and chicken nervous tissue and of developing fly embryos is shown.
- Pierre Mahou
- Maxwell Zimmerley
- Emmanuel Beaurepaire