title: Noninvasive harmonics optical microscopy for long-term observation of embryonic nervous system development in vivo.
abstract: Nervous system development is a complicated dynamic process, and many mechanisms remain unknown. By utilizing endogenous second-harmonic-generation as the contrast of polarized nerve fibers and third-harmonic-generation (THG) to reveal morphological changes, we have successfully observed the vertebrate embryonic nervous development from the very beginning based on a 1230-nm light source. The dynamic development of the nerve system within a live zebrafish embryo can be recorded continuously more than 20 hr without fluorescence markers. Since the THG process is not limited by the time of gene expression and differentiation as fluorescence-based techniques are, the observable stages can be advanced to the very beginning of the development process. The complete three-dimensional brain development from a neural plate to a neural tube can be uncovered with a submicron lateral resolution. We have, for the first time, also reported the generation of SHG from myelinated nerve fibers and the outer segment of the photoreceptors with a stacked membrane structure. Our study clearly indicates the fact that higher-harmonics-based optical microscopy has the strong potential to long-term in vivo study of the nervous system, including genetic disorders of the nervous system, axon pathfinding, neural regeneration, neural repair, and neural stem cell development.
authors.affiliation: Department of Electrical Engineering and Graduate Institute of Electro-Optical Engineering, National Taiwan University, Taipei 10617, Taiwan.
authors.forename: Szu-Yu
authors.initials: SY
authors.lastname: Chen
authors.forename: Cho-Shuen
authors.initials: CS
authors.lastname: Hsieh
authors.forename: Shi-Wei
authors.initials: SW
authors.lastname: Chu
authors.forename: Cheng-Yung
authors.initials: CY
authors.lastname: Lin
authors.forename: Ching-Yi
authors.initials: CY
authors.lastname: Ko
authors.forename: Yi-Chung
authors.initials: YC
authors.lastname: Chen
authors.forename: Huai-Jen
authors.initials: HJ
authors.lastname: Tsai
authors.forename: Chin-Hwa
authors.initials: CH
authors.lastname: Hu
authors.forename: Chi-Kuang
authors.initials: CK
authors.lastname: Sun
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology* / Q000196:embryology*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000295:instrumentation* / Q000379:methods; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015027:Zebrafish / Q000033:anatomy & histology* / Q000196:embryology*