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 Cae- norhabditis elegans C. elegans. Moreover, we show that the THG signal is obtained in all the embryo development stages, showing dif- ferent 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 ef- ficiency 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 un- stained C. elegans in vivo samples. © 2010 Society of Photo-Optical Instrumen-