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Proceedings Paper

Two-photon microscopy of living cells by simultaneously exciting multiple endogenous fluorophores and fluorescent proteins
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Paper Abstract

Endogenous fluorophores, such as reduced nicotinamide adenine dinucleotide (NADH), keratin, and tryptophan, have been used as contrast agents for imaging metabolism and morphology of living cells and tissues. Multilabeling which maps the distribution of different targets is an indispensable technique in many biomedical and biochemical studies. Therefore, two-photon excitation fluorescence (TPEF) microscopy of endogenous fluorophores combining with in vivo fluorescence labeling techniques such as genetically encoded fluorescent protein could be a powerful tool for imaging living cells and tissues. However, the challenge is that the excitation and emission wavelengths of these endogenous fluorophores and fluorescence labels are very different. A multi-color ultrafast source is required for the excitation of multiple fluorescence molecules. In this study, we developed a two-photon imaging system with excitations from the pump femtosecond laser and the selected Supercontinuum generated from a photonic crystal fiber (PCF). Multiple endogenous fluorophores and fluorescent proteins such as NADH, tryptophan, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) were excited in their optimal wavelengths alternately or simultaneously. A time- and spectral-resolved detection system was used to record the TPEF signals. This detection technique separated the TPEF signals from multiple sources in time and spectral domains. Cellular organelles such as nucleus, mitochondria, microtubule and Endoplasmic Reticulum (ER), were clearly revealed in the TPEF images.

Paper Details

Date Published: 24 February 2010
PDF: 6 pages
Proc. SPIE 7568, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VIII, 756806 (24 February 2010); doi: 10.1117/12.841428
Show Author Affiliations
Wei Zheng, Hong Kong Univ. of Science and Technology (Hong Kong, China)
Dong Li, Hong Kong Univ. of Science and Technology (Hong Kong, China)
Jianan Y. Qu, Hong Kong Univ. of Science and Technology (Hong Kong, China)


Published in SPIE Proceedings Vol. 7568:
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VIII
Daniel L. Farkas; Dan V. Nicolau; Robert C. Leif, Editor(s)

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