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Time-Resolved Long-Lived Luminescence Imaging Method Employing Luminescent Lanthanide Probes with a New Microscopy System
journal contribution
posted on 2007-11-07, 00:00 authored by Kenjiro Hanaoka, Kazuya Kikuchi, Shigeru Kobayashi, Tetsuo NaganoSuperior 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.