Imaging embryonic development with ultrashort pulse microscopy
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We report the application of ultrashort pulse microscopy (UPM) for integrated imaging of embryonic development at the tissue, cell, and molecular length scales. The UPM is a multimodal imaging platform that utilizes the broad-power spectrum and high-peak power of 10-fs pulses to render two-photon excited signals and the short coherence gate of such pulses to render optical coherence signals. We show that ultrashort pulses efficiently excite cellular autofluorescence in developing zebrafish embryos such that tissues are readily visualized and individual cells can be monitored, providing a potential method for label-free cell tracking. We also show the ability of ultrashort pulses, without tuning, to excite a broad spectrum of fluorescent protein variants for tracking genetically labeled cell lineages in live embryos, with no apparent damage to the embryos. Molecular information at the mRNA transcript level can also be obtained from embryos that have been stained to reveal the localization of the expression of a gene using NBT/BCIP, which we show can be detected with three-dimensional resolution using a combination of twophoton and optical coherence signals. From this demonstration, we conclude that UPM is an efficient and a powerful tool for elucidating the dynamic multiparameter and multiscale mechanisms of embryonic development. The Authors.