In vivo harmonic generation microscopy:Long term observations of vertebrate embryos development
Date Issued
2007
Date
2007
Author(s)
Hsieh, Cho-Shuen
DOI
en-US
Abstract
In this thesis, I discuss the role optical harmonic generation microscopy plays in morphant technology and embryogenesis of embryology. A previous optical harmonic generation microscopy system is built based on a modified optical scanning microscope and a Cr:forsterite laser. Second harmonic generation (SHG) and third harmonic generation (THG) have been used as imaging modalities. I present the in vivo long-term observation of morphant type zebrafish using HGM from the very beginning of embryogenesis, thus identify that the gene not only functions after nerve fiber formation in the nervous system, but up to the gastrula period of the embryo. With the continuous observation of the same morphant type embryo, optical harmonic generation microscopy is demonstrated as an excellent least invasive imaging tool for revealing gene expression in embryos. Combined with the near-infrared femtosecond source and a high numerical aperture (NA) objective, optical harmonic generation microscopy can offer >1-mm penetration depth and sub-micrometer spatial resolution in live biological samples with much-reduced photodamage and phototoxicity, and therefore it can reveal many organs and organelles in various vertebrate embryos and construct the whole 3D structure mouse embryo. Obviously HGM can be used to select mouse-even human- embryos as well. Combining SHG and THG imaging modalities, optical harmonic generation microscopy can provide biological critical information without invasive treatment such as dye staining or fluorescence protein labeling and contribute a better spatial resolution in embryology studies.
Previously, our lab had successfully demonstrated the nerve fiber development through harmonic generation microscopy, but we cannot identify any gene function. With the aid of morphant technique, specific genes blocked by morpholinos have become a powerful tool in developmental biology studies. Based on a high-order harmonic generation microscope, images with high 3-dimensional (3D) resolution can be obtained due to its nonlinear nature. We have used higher-harmonic generation microscopy based on the femtosceond Cr:forsterite laser with the aid of zARNT2A and HIF 1
Subjects
倍頻顯微術
胚胎
基因阻斷
harmonics generation microscopy
embryo
morphant
Type
thesis
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