Fig. 12 Left : Jablonski diagram for fluorescence following two-photon excitation to the higher S n
state. Right : Isodensity surfaces of the Kohn-Sham molecular orbitals relevant to the high-energy
two-photon excitation in the RFP chromophore (HOMO-2 is the second, in energy, occupied
molecular orbital before the HOMO, and LUMO
1 is the first unoccupied molecular orbital after
the LUMO). From Nifos` and Luo [ 113 ]
at 800 nm in red/orange FPs (Fig. 11 ). Such high-energy bands were revealed first
in DsRed [ 108 , 110 ] and were instead absent in cyan and green FPs, because of the
limited spectral window examined (750-1,000 nm). Subsequently, thanks to a
broader detection window, they were revealed in blue, green, and cyan FPs, and
reported to give rise to bright and steady fluorescence, with the same spectral
characteristics of the low-energy excitation band.
Theoretical analysis reveals that such bands arise from excitation to higher excited
states of the chromophores (Fig. 12 ). Despite having low one-photon oscillator
strength, these excitations acquire some two-photon moment mainly from the first
singlet excitation, with a near-resonance enhancement mechanism [ 112 , 113 ]. The
decomposition into molecular orbitals' excitations within a TD-DFT framework
reveals that, analogously to the one-photon case, where the dominant excitation is
LUMO for all chromophores, a common molecular orbital composition is
also found for these higher excitations (see Fig. 12 ).
Marchant et al. reported an early application of multiphoton excitation in the
high-energy bands of DsRed [ 110 ]. Femtosecond-pulsed irradiation at
readily changes the fluorescence of DsRed from red to green, presumably by
bleaching the red-emitting chromophore within the DsRed tetramer. Thanks to
the three-dimensional localization of multiphoton excitation, DsRed “greening”
could be exploited to optically highlight subcellular compartments in living cells.
More generally, the availability of additional two-photon excitation bands is quite
handy, given that laboratories are only rarely equipped with more than one two-
photon laser. Drobizhev and coworkers suggest an approach for dual color two-
photon imaging, based on the simultaneous excitation of blue and red FPs at
780 nm, corresponding to the low-energy excitation band of the former and the
high-energy excitation band of the latter [ 111 ].