Time-resolved delayed luminescence image microscopy using an europium ion chelate complex

Abstract

Improvements and extended applications of time-resolved delayed luminescence imaging microscopy (TR-DLIM) in cell biology are described. The emission properties of europium ion complexed to a fluorescent chelating group capable of labeling proteins are exploited to provide high contrast images of biotin labeled ligands through detection of the delayed emission. The streptavidin-based macromolecular complex (SBMC) employs streptavidin cross-linked to thyroglobulin multiply labeled with the europium-fluorescent chelate. The fluorescent chelate is efficiently excited with 340-nm light, after which it sensitizes europium ion emission at 612 nm hundreds of microseconds later. The SBMC complex has a high quantum yield orders of magnitude higher than that of eosin, a commonly used delayed luminescent probe, and can be readily seen by the naked eye, even in specimens double-labeled with prompt fluorescent probes. Unlike triplet-state phosphorescent probes, sensitized europium ion emission is insensitive to photobleaching and quenching by molecular oxygen; these properties have been exploited to obtain delayed luminescence images of living cells in aerated medium thus complementing imaging studies using prompt fluorescent probes. Since TR-DLIM has the unique property of rejecting enormous signals that originate from scattered light, autofluorescence, and prompt fluorescence it has been possible to resolve double emission images of living amoeba cells containing an intensely stained lucifer yellow in pinocytosed vesicles and membrane surface-bound SBMC-labeled biotinylated concanavalin A. Images of fixed cells represented in terms of the time decay of the sensitized emission show the lifetime of the europium ion emission is sensitive to the environment in which it is found. Through the coupling of SBMC to streptavidin,a plethora of biotin-based tracer molecules are available for immunocytochemical studies.

Polymer beacons for luminescence and magnetic resonance imaging of DNA delivery

“…Complexes housing luminescent Lns, such as europium (Eu 3ϩ ), offer many unique advantages over the aforementioned labeling methods due to their small hydrophilic structures, long luminescence lifetimes, large Stokes shifts, and their stability from quenching and photobleaching (13,14). The chelates of Lns are relatively nontoxic; structures containing gadolinium (Gd 3ϩ ) are FDA-approved as MRI contrast agents due to their paramagnetic nature and slow electronic relaxation time (15).…”

Platinum Porphyrins as Phosphorescent Label for Time-resolved Microscopy

“…At present, the ultimate detection sensitivity in fluorescence microscopy is mainly determined by the nonspecific background signals caused by autofluorescence of the biological objects, fixative-induced fluorescence, and the natural fluorescence of components such as microscope objectives, immersion oil, and optical filters (Aubin 1979;Jovin et al 1989). These fast-decaying types of autofluorescence can be suppressed by application of time-resolved microscopy (Zotikov and Polyakov 1977;Seveus et al 1992;Marriott et al 1991Marriott et al ,1994Verwoerd et al 1994;Hennink et al 1996) using a delayed luminescent label (Hemmilä et al 1982;Seveus et al 1992;Hennink et al 1996) as reporter molecule bound to immunospecific reagents or nucleic acid probes (Diamandis et al 1988;Savitski et al 1989;Beverloo et al 1990;Marriott et al 1991Marriott et al ,1994Seveus et al 1992; de Hennink et al 1996). Most of the applications of time-resolved microscopy reported thus far use europium chelates.…”

“…These requirements have not been combined in one single chelate thus far (Savitski et al 1989;Alpha et al 1990;Mukkala et al 1992). Nevertheless, their potential value has been clearly shown in cases in which some of these problems were overcome (Beverloo et al 1990;Seveus et al 1992;Marriott et al 1994;de Haas et al 1996).…”

Development of Responsive Lanthanide-Based Magnetic Resonance Imaging and Luminescent Probes for Biological Applications

“…12). [66][67][68][69][70][71][72][73][74][75] Thus, this TRLLM imaging has very different characteristics from conventional timeresolved fluorescence imaging. A schematic of the TRLLM system, which was newly developed by us, is shown in Fig.…”

“…unit, xenon flash lamp and timing controller. Applications of mechanical choppers in TRLLM with long-lived luminescent dyes have been reported by some groups, [66][67][68][69][70][71][72][73] and other groups have employed an I.I. unit for the time-resolution.…”