Genetic labeling techniques allow for noninvasive lineage tracing of cells in vivo.Two-photon inducible activators provide spatial resolution for superficial cells,but labeling cells located deep within tissues is pre...Genetic labeling techniques allow for noninvasive lineage tracing of cells in vivo.Two-photon inducible activators provide spatial resolution for superficial cells,but labeling cells located deep within tissues is precluded by scattering of the far-red illumination required for two-photon photolysis.Three-photon illumination has been shown to overcome the limitations of two-photon microscopy for in vivo imaging of deep structures,but whether it can be used for photoactivation remains to be tested.Here we show,both theoretically and experimentally,that three-photon illumination overcomes scattering problems by combining longer wavelength excitation with high uncaging three-photon cross-section molecules.We prospectively labeled heart muscle cells in zebrafish embryos and found permanent labeling in their progeny in adult animals with negligible tissue damage.This technique allows for a noninvasive genetic manipulation in vivo with spatial,temporal and cell-type specificity,and may have wide applicability in experimental biology.展开更多
基金supported by a pre-doctoral fellowship from MINECO and the I3 program,respectivelysupport was provided by grants from MINECO(SAF2012-33526,SAF2015-69706-R and BFU2012-38146)+3 种基金ISCIII/FEDER(Red de Terapia Celular—TerCel RD12/0019/0019)AGAUR(2014-SGR-1460)FundacióLa Maratóde TV3(201534-30)ERC(Grant Agreement 242993).
文摘Genetic labeling techniques allow for noninvasive lineage tracing of cells in vivo.Two-photon inducible activators provide spatial resolution for superficial cells,but labeling cells located deep within tissues is precluded by scattering of the far-red illumination required for two-photon photolysis.Three-photon illumination has been shown to overcome the limitations of two-photon microscopy for in vivo imaging of deep structures,but whether it can be used for photoactivation remains to be tested.Here we show,both theoretically and experimentally,that three-photon illumination overcomes scattering problems by combining longer wavelength excitation with high uncaging three-photon cross-section molecules.We prospectively labeled heart muscle cells in zebrafish embryos and found permanent labeling in their progeny in adult animals with negligible tissue damage.This technique allows for a noninvasive genetic manipulation in vivo with spatial,temporal and cell-type specificity,and may have wide applicability in experimental biology.
