Noninvasive harmonics optical microscopy for long-term observation of embryonic nervous system development in vivo.
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.
Chen
Szu-Yu
SY
Department of Electrical Engineering and Graduate Institute of Electro-Optical Engineering, National Taiwan University, Taipei 10617, Taiwan.
Hsieh
Cho-Shuen
CS
Chu
Shi-Wei
SW
Lin
Cheng-Yung
CY
Ko
Ching-Yi
CY
Chen
Yi-Chung
YC
Tsai
Huai-Jen
HJ
Hu
Chin-Hwa
CH
Sun
Chi-Kuang
CK
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*