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Proceedings Paper

Time-resolved multicolor two-photon excitation fluorescence microscopy of cells and tissues
Author(s): Wei Zheng
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Paper Abstract

Multilabeling which maps the distribution of different targets is an indispensable technique in many biochemical and biophysical studies. Two-photon excitation fluorescence (TPEF) microscopy of endogenous fluorophores combining with conventional fluorescence labeling techniques such as genetically encoded fluorescent protein (FP) and fluorescent dyes staining could be a powerful tool for imaging living cells. However, the challenge is that the excitation and emission wavelength of these endogenous fluorophores and fluorescent 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, fluorescent proteins and fluorescent dyes were excited in their optimal wavelengths 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 wavelength domains. Cellular organelles such as nucleus, mitochondria, microtubule and endoplasmic reticulum, were clearly revealed in the TPEF images. The simultaneous imaging of multiple fluorophores of cells will greatly aid the study of sub-cellular compartments and protein localization.

Paper Details

Date Published: 20 November 2014
PDF: 7 pages
Proc. SPIE 9268, Optics in Health Care and Biomedical Optics VI, 926825 (20 November 2014); doi: 10.1117/12.2071655
Show Author Affiliations
Wei Zheng, Shenzhen Institute of Advanced Technology (China)

Published in SPIE Proceedings Vol. 9268:
Optics in Health Care and Biomedical Optics VI
Qingming Luo; Xingde Li; Ying Gu; Yuguo Tang, Editor(s)

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