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We report here the use of a simple washing approach to reduce the ionic strength of the solution, which increased the thickness of the electric double layer on the surface of silver (Ag) nanoparticles and thereby enhanced their surface zeta-potential. This approach allowed us to prepare optically uniform (75-99%) and purified Ag nanoparticles (11.3 +/- 2.3 nm) that are stable (nonaggregation) in solution for months, permitting them to become robust and widely used single nanoprobes for in vivo optical imaging. These Ag nanoparticles show remarkable photostability and serve as single nanoparticle photonic probes for continuous imaging nanoenvironments of segmentation-stage zebrafish embryos for hours. Unlike other particle tracking experiments, we utilized size-dependent localized surface plasmon resonance spectra (LSPRS) (colors) of single Ag nanoparticles to determine given colored (sized) nanoparticles in situ and used the monodisperse color (size) of nanoparticles to simultaneously measure viscosities and flow patterns of multiple proximal nanoenvironments in segmentation-stage zebrafish embryos in real time. We found new interesting counterclockwise flow patterns with rates ranging from 0.06 to 1.8 microm/s and stunningly high viscosity gradients spanning two orders of magnitude in chorion space of the embryos, with the highest viscosity observed around the center of chorion space and the lower viscosity at the interfacial areas near the surface of both chorion layers and inner mass of the embryos. This study demonstrates the possibility of using individual monodisperse nanophotonics to probe the roles of embryonic fluid dynamics in embryonic development.

Design of Stable and Uniform Single Nanoparticle Photonics for In Vivo Dynamics Imaging of Nanoenvironments of Zebrafish Embryonic Fluids