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Fig. 4
Multicolor two-photon efficient imaging of multiple endogenous fluorophores during early stages of zebrafish embryo development (a) Energy diagrams of two-photon excited fluorescence (2PEF) for blue and red fluorophores, two-color two-photon excited fluorescence (2c-2PEF) for green fluorophores, second harmonic generation (SHG), sum frequency generation (SFG) and four wave mixing (FWM). (b) Bandpass filters are chosen to select the emission of blue, green and red fluorophores rejecting coherent signals such as SHG, SFG and FWM. (c) Images of the blue, green, red fluorescence channel and SHG of the same zebrafish embryo at three different time points of development. Images of merged channels represent a shift in the spectroscopic characteristics of the yolk. (d) Multicolor two-photon and SHG images of the zebrafish embryo at 4 cell stage. Time per pixel, 40 µs. This experiment has been repeated in three independent samples.