Live cell imaging of lysosomal pH changes with pH responsive ratiometric lanthanide probes

Smith, David Geoffrey; McMahon, Brian; Pál, Róbert; Parker, David

doi:10.1039/c2cc34267g

Cited by 125 publications

(85 citation statements)

References 31 publications

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“…Much of the current research in the area of lanthanide complexation chemistry is focussed on the development of emissive europium complexes as ratiometric probes for biological analytes. 31,131,160,161 The sensitivity of the europium emission spectral form to the metal coordination environment is key to these advances and terbium is often overlooked. This example illustrates nicely how the use of CPL spectroscopy may be able to facilitate the use of terbium complexes as emissive probes, where changes in g em at a given wavelength could be used to indicate the presence of a given analyte.…”

Section: Studies Using Circular Dichroismmentioning

confidence: 99%

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Lanthanide complexes as chiral probes exploiting circularly polarized luminescence

2012

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The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that:• a full bibliographic reference is made to the original source• a link is made to the metadata record in Durham E-Theses• the full-text is not changed in any wayThe full-text must not be sold in any format or medium without the formal permission of the copyright holders. Statement of CopyrightThe copyright of this thesis rests with the author. No quotations should be published without prior consent and information derived from it must be acknowledged. ABSTRACT -ii - ABSTRACTA series of studies has been undertaken to facilitate the identification and development of chiral lanthanide complexes that are able to report on changes in their local environment through modulation of the circular polarization of their emission. Reports of such systems remain relatively rare in the literature, notwithstanding the prevalence and importance of chirality in biological systems.The work described herein is separated into five chapters, the first of which comprises a discussion of relevant background information, along with a comprehensive review of responsive lanthanide-based CPL probes reported to date. A classification of these probes is built up, which informs the content of the following three chapters.Chapter 2 describes work undertaken in the pursuit of a novel lanthanide-based system for use as a CPL probe for the detection of proteins. The synthesis of an enantiopure lanthanide complex was undertaken and characterisation of this system carried out with reference to a structurally related racemic complex. A series of comparative investigations designed to probe the relative protein binding capability of these complexes was subsequently performed, which revealed that the observation of induced CPL from racemic lanthanide systems may be brought about by a change in complex constitution. This is the first example of such an effect from a well-defined racemic lanthanide complex in solution. Chapter 3 goes on to detail studies undertaken to demonstrate the utility of this racemic lanthanide system as a probe for chiral detection.Chapter 4 describes investigations carried out in an attempt to identify new systems exhibiting chiral quenching effects in solution. Initially, two pairs of enantiomeric electron-rich quenching species were assessed for their ability to quench the emission from an enantiopure DOTA-derived lanthanide complex differentially. Subsequently, investigations were focussed on examining the quenching of emission from novel enantiopure lanthanide complexes based on a 1,4,7-triazacyclononane framework, using cobalt complexes as the quenching species.Finally, Chapter 5 contains experimental procedures for each compound synthesised, as well as general experimental procedures.

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Section: Studies Using Circular Dichroismmentioning

confidence: 99%

“…Changes in the to an altered CPL output. 131 In this system, a sulphonamide moiety is incorporated into the ligand framework, which is deprotonated at high pH and able to bind to the lanthanide centre. However, at low pH this is not the case and the coordination sphere is instead completed by a bound water molecule.…”

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Lanthanide complexes as chiral probes exploiting circularly polarized luminescence

2012

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“…Time-resolved confocal microscopy has also been explored (19,20). Numerous studies have reported both time-gated and steady-state imaging of responsive lanthanide sensors in living cells, including sensors for Zn 2þ , bicarbonate, pH, and singlet oxygen (21)(22)(23)(24). Lanthanide biosensors may be designed as responsive probes that exhibit an analyte-dependent change in emission intensity or lifetime (6), or as FRET-based systems that use Tb(III) or Eu(III) complexes as donors and FPs, organic dyes, or even quantum dots as acceptors (8,25).…”

Section: Introductionmentioning

confidence: 99%

Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

Rajendran

Miller

2015

Biophysical Journal

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Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts. This work describes the performance of a wide-field, time-gated microscope with respect to its ability to image Tb(III) luminescence and Tb(III)-mediated FRET in cultured mammalian cells. The system employed a UV-emitting LED for low-power, pulsed excitation and an intensified CCD camera for gated detection. Exposure times of ∼1 s were needed to collect 5-25 photons per pixel from cells that contained micromolar concentrations of a Tb(III) complex. The observed photon counts matched those predicted by a theoretical model that incorporated the photophysical properties of the Tb(III) probe and the instrument's light-collection characteristics. Despite low photon counts, images of Tb(III)/green fluorescent protein FRET with a signal/noise ratio ≥ 7 were acquired, and a 90% change in the ratiometric FRET signal was measured. This study shows that the sensitivity and precision of lanthanide-based cellular microscopy can approach that of conventional FRET microscopy with fluorescent proteins. The results should encourage further development of lanthanide biosensors that can measure analyte concentration, enzyme activation, and protein-protein interactions in live cells.

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“…Dysfunction of lysosomal structure or function is associated with multiple pathologies, including inflammation, cancer, neurodegenerative disease, and specific lysosomal storage diseases such as Tay-Sachs12345678910111213141516. Currently, there are limited numbers of fluorescent probes that efficiently enable a comprehensive evaluation of the structure/function correlates of lysosomes17181920212223242526272829303132. Cellular compartments with a low internal pH (pH 4.0-6.0), including the lysosome, enable basic amines of low ionic strength to selectively target and thereby explore the synthesis and actions of lysosomes.…”

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confidence: 99%

“…However, when LysoTracker probes accumulate intracellularly for prolonged periods, the intracellular pH increases which may result in enhanced quenching of the fluorescent dye. Most commercially available lysosome probes require a short excitation wavelength, which considerably restricts the use of these probes in tissue imaging associated with low penetration depth, decreased solubility, and poor photostability due to wavelength restrictions17181920212223242526272829303132. To address this problem, Belfield et al recently developed a novel, two-photon absorbing fluorescence derivatives exhibiting selectivity for the lysosomes of HCT 116 colon cancer cells26.…”

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Lysosomal Targeting with Stable and Sensitive Fluorescent Probes (Superior LysoProbes): Applications for Lysosome Labeling and Tracking during Apoptosis

Chen

Wang

et al. 2015

Sci Rep

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Intracellular pH plays an important role in the response to cancer invasion. We have designed and synthesized a series of new fluorescent probes (Superior LysoProbes) with the capacity to label acidic organelles and monitor lysosomal pH. Unlike commercially available fluorescent dyes, Superior LysoProbes are lysosome-specific and are highly stable. The use of Superior LysoProbes facilitates the direct visualization of the lysosomal response to lobaplatin elicited in human chloangiocarcinoma (CCA) RBE cells, using confocal laser scanning microscopy. Additionally, we have characterized the role of lysosomes in autophagy, the correlation between lysosome function and microtubule strength, and the alteration of lysosomal morphology during apoptosis. Our findings indicate that Superior LysoProbes offer numerous advantages over previous reagents to examine the intracellular activities of lysosomes.

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Live cell imaging of lysosomal pH changes with pH responsive ratiometric lanthanide probes

Cited by 125 publications

References 31 publications

Lanthanide complexes as chiral probes exploiting circularly polarized luminescence

Lanthanide complexes as chiral probes exploiting circularly polarized luminescence

Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

Lysosomal Targeting with Stable and Sensitive Fluorescent Probes (Superior LysoProbes): Applications for Lysosome Labeling and Tracking during Apoptosis

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