In vitro and In vivo Applications of Stimulated Emission Depletion and Fluorescence Lifetime Imaging Microscopy with Fluorescent Nanodiamonds as Biomarkers

• Main Authors: Yung Kuo, 郭雍
• Other Authors: Huan-Cheng Chang
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/76163453371765934213

碩士 === 國立臺灣大學 === 化學研究所 === 100 === Far-field fluorescence microscopy has become a powerful tool for imaging biological systems because of its high sensitivity, high signal-to-noise ratio, high specificity and non-invasiveness. The spatial resolution, however, is limited by light diffraction to ~200 nm, which is larger than most sub-cellular organelles. Therefore, developing sub-diffraction microscopy is an important task to image delicate biological structures. Among many superresolution imaging techniques, stimulated emission depletion (STED) microscopy is purely lens-based and has the shortest image acquisition time. In this thesis, a home-built STED microscope has been constructed and applied to image 35-nm albumin-coated fluorescent nanodiamonds (FNDs) taken up by HeLa cells through endocytosis or electroporation. A resolution of ~40 nm has been achieved for a single FND in cells. In bioimaging, cell and tissue autofluorescence is another big obstacle which decreases the signal-to-noise ratio and restricts in vivo applications of fluorescence microscopy. FNDs with nitrogen-vacancy (N-V) centers as fluorophores have longer fluorescence lifetimes than autofluorescence. Using fluorescence lifetime imaging microscopy (FLIM) and a model organism, Caenorhabditis elegans (C. elegans), we studied the fate of carboxylated and albumin-coated FNDs microinjected into the worm and performed real-time tracking of single FNDs in vivo over an extended period of time.