New tools for real-time study of embryonic development.

摘要

Embryonic development represents one of the most complex and dynamic cellular processes in biology, and plays vital roles in understanding of functions of embryonic stem cells (ESCs) and design of ESC-based therapy. Conventional assays and fluorescence-based imaging methods have been widely used for the study of embryonic development. These conventional methods cannot effectively provide spatial and temporal resolutions with sufficient sensitivity and selectivity that are required to depict embryonic development in vivo in real-time at single-cell and single-molecule resolutions. In this dissertation, we have developed a wide range of innovative tools for real-time study of embryonic development. These new tools include biocompatible and photostable plasmonic gold (Au) and silver (Ag) nanoparticle (NP) imaging probes, dark-field optical microscopy and spectroscopy (DFOMS), and ultrashort electric pulses. We have designed and synthesized a mini-library of Au and Ag NPs with different sizes and chemical properties. We have used developing zebrafish embryos as in vivo model organisms to study embryonic development and as in vivo assays to study size- and chemical-dependent nanotoxicity. We found that these multicolored imaging probes can passively diffuse into embryos and enter into embryos non-invasively. These NPs exhibit superior photostability and enable us to study embryonic environments for a desired period of time. They can be illuminated under a standard microscope halogen lamp and characterized simultaneously using DFOMS equipped with a multi-spectral imaging system to achieve real-time multiplexing imaging. Our studies show that Au NPs are much more biocompatible than Ag NPs, while Ag NPs are much more sensitive and colorful than Au NPs. Notably, we can make Ag NPs nearly as biocompatible as Au NPs by functionalizing their surfaces with biocompatible peptides. Furthermore, Ag NPs can incite stage-specific embryonic phenotypes, and enable us to generate distinctive mutants for further identification of biomarkers for better understanding of embryonic development and for potential diagnosis of birth defects. We have developed new methods to effectively culture and sustain ESCs of zebrafish, mouse and human, laying down the foundation for real-time study of differentiation of ESCs both in vitro and in vivo for a wide variety of biomedical applications.