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Time-Resolved Long-Lived Luminescence Imaging Method Employing Luminescent Lanthanide Probes with a New Microscopy System

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Contribution from the Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Department of Materials and Life Sciences, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita City, Osaka 565-0871, and Products Development Department 2, Micro-Imaging Systems Division, OLYMPUS CORPORATION, 2951 Ishikawa-cho, Hachioji-shi, Tokyo 192-8507, Japan
Cite this: J. Am. Chem. Soc. 2007, 129, 44, 13502–13509
Publication Date (Web):October 10, 2007
Copyright © 2007 American Chemical Society

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    Abstract Image

    Superior fluorescence imaging methods are needed for detailed studies on biological phenomena, and one approach that permits precise analyses is time-resolved fluorescence measurement, which offers a high signal-to-noise ratio. Herein, we describe a new fluorescence imaging system to visualize biomolecules within living biological samples by means of time-resolved, long-lived luminescence microscopy (TRLLM). In TRLLM, short-lived background fluorescence and scattered light are gated out, allowing the long-lived luminescence to be selectively imaged. Usual time-resolved fluorescence microscopy provides fluorescence images with nanosecond resolution and has been used to image interactions between proteins, protein phosphorylation, the local pH, the refractive index, ion or oxygen concentrations, etc. Luminescent lanthanide complexes (especially europium and terbium trivalent ions (Eu3+ and Tb3+)), in contrast, have long luminescence lifetimes on the order of milliseconds. We have designed and synthesized new luminescent Eu3+ complexes for TRLLM and also developed a new TRLLM system using a conventional fluorescence microscope with an image intensifier unit for gated signal acquisition and a xenon flash lamp as the excitation source. When the newly developed luminescent Eu3+ complexes were applied to living cells, clear fluorescence images were acquired with the TRLLM system, and short-lived fluorescence was completely excluded. By using Eu3+ and Tb3+ luminescent complexes in combination, time-resolved dual-color imaging was also possible. Furthermore, we monitored changes of intracellular ionic zinc (Zn2+) concentration by using a Zn2+-selective luminescent Eu3+ chemosensor, [Eu-7]. This new imaging technique should facilitate investigations of biological functions with fluorescence microscopy, complementing other fluorescence imaging methodologies.

     The University of Tokyo.

     Osaka University.




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    Detailed descriptions of synthetic procedures for luminescent Eu3+ complexes and the luminescent Tb3+ complex, photograph of solutions of luminescent Eu3+ complexes, photobleaching profiles of solutions of Eu3+ complexes and fluorescein, comparative singlet oxygen generation plots of Rose Bengal, fluorescein, and Eu3+ complexes, and time-resolved long-lived luminescence images of intracellular Zn2+ in HeLa cells which were stained with rhodamine 6G. This material is available free of charge via the Internet at

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