Third-harmonic generation for the study of Caenorhabditis elegans embryogenesis

Live microscopy techniques (i.e., differential interference contrast, confocal microscopy, etc.) have enabled the understanding of the mechanisms involved in cells and tissue formation. In long-term studies, special care must be taken in order to avoid sample damage, restricting the applicability of the different microscopy techniques. We demonstrate the potential of using third-harmonic generation (THG) microscopy for morphogenesis/embryogenesis studies in living Caenorhabditis elegans (C. elegans). Moreover, we show that the THG signal is obtained in all the embryo development stages, showing different tissue/structure information. For this research, we employ a 1550-nm femtosecond fiber laser and demonstrate that the expected water absorption at this wavelength does not severely compromise sample viability. Additionally, this has the important advantage that the THG signal is emitted at visible wavelengths (516 nm). Therefore, standard collection optics and detectors operating near maximum efficiency enable an optimal signal reconstruction. All this, to the best of our knowledge, demonstrates for the first time the noninvasiveness and strong potential of this particular wavelength to be used for high-resolution four-dimensional imaging of embryogenesis using unstained C. elegans in vivo samples.