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      Dinuclear Ruthenium(II) Complexes as Two-Photon, Time-Resolved Emission Microscopy Probes for Cellular DNA**

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          Abstract

          The first transition-metal complex-based two-photon absorbing luminescence lifetime probes for cellular DNA are presented. This allows cell imaging of DNA free from endogenous fluorophores and potentially facilitates deep tissue imaging. In this initial study, ruthenium(II) luminophores are used as phosphorescent lifetime imaging microscopy (PLIM) probes for nuclear DNA in both live and fixed cells. The DNA-bound probes display characteristic emission lifetimes of more than 160 ns, while shorter-lived cytoplasmic emission is also observed. These timescales are orders of magnitude longer than conventional FLIM, leading to previously unattainable levels of sensitivity, and autofluorescence-free imaging.

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          Fluorescence lifetime measurements and biological imaging.

          Mikhail Y Berezin, Samuel Achilefu (2010)
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            Improving FRET dynamic range with bright green and red fluorescent proteins

            Amy Lam, François St-Pierre, Yiyang Gong … (2012)
            A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of cyan and yellow fluorescent proteins (CFP and YFP), but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light, and complex photokinetic events such as reversible photobleaching and photoconversion. Here, we engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date, and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP in reporters of kinase activity, small GTPase activity, and transmembrane voltage significantly improves photostability, FRET dynamic range, and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action potential firing and RhoA activation in growth cones.
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              Ruthenium(II) polypyridyl complexes and DNA--from structural probes to cellular imaging and therapeutics.

              Martin R. Gill, Jim A Thomas (2012)
              In the last few decades, coordination complexes based on d(6) metal centres and polypyridyl ligand architectures been developed as structure- and site-specific reversible DNA binding agents. Due to their attractive photophysical properties, much of this research has focused on complexes based on ruthenium(II) centres and, more recently, attention has turned to the use of these complexes in biological contexts. As the rules that govern the cellular uptake and cellular localisation of such systems are determined they are finding numerous applications ranging from imaging to therapeutics. This review illustrates how the interdisciplinary nature of this research-which takes in synthetic chemistry, biophysical and in cellulo studies-makes this an exciting area in which an array of further applications are likely to emerge. This journal is © The Royal Society of Chemistry 2012
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                Author and article information

                Journal
                Journal ID (nlm-ta): Angew Chem Int Ed Engl
                Journal ID (iso-abbrev): Angew. Chem. Int. Ed. Engl
                Journal ID (publisher-id): anie
                Title: Angewandte Chemie (International Ed. in English)
                Publisher: WILEY-VCH Verlag (Weinheim )
                ISSN (Print): 1433-7851
                ISSN (Electronic): 1521-3773
                Publication date (Print): 24 March 2014
                Publication date (Electronic): 23 January 2014
                Volume: 53
                Issue: 13
                Pages: 3367-3371
                Affiliations
                Department of Chemistry, University of Sheffield Sheffield, S3 7HF (UK)
                Department of Biomedical Science, University of Sheffield Sheffield, S10 2TN (UK)
                The Kroto Institute, University of Sheffield Sheffield, S3 7HQ (UK)
                Central Laser Facility, Research Complex at Harwell, STFC, Rutherford Appleton Laboratory Oxfordshire OX11 0QX (UK)
                Author notes
                [*] Dr. E. Baggaley, Dr. I. V. Sazanovich, Dr. J. A. Weinstein, Dr. J. A. Thomas Department of Chemistry, University of Sheffield Sheffield, S3 7HF (UK) E-mail: julia.weinstein@ 123456sheffield.ac.uk james.thomas@ 123456sheffield.ac.uk
                [[**]]

                We gratefully acknowledge the support of The Wellcome Trust Discipline Hopping Fellowships (MRG), The MRC for a studentship and Centenary Early Career Award (DT), BBSRC “Tools & Resources” program, STFC for programme mode facility access to the LSF, Laserlab Europe, and Becker & Hickl GmbH for fruitful collaboration.

                Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201309427.

                Article
                DOI: 10.1002/anie.201309427
                PMC ID: 4298790
                PubMed ID: 24458590
                SO-VID: dbbf4188-3108-4055-8237-3b5443812ee2
                Copyright © © 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited
                History
                Date received : 29 October 2013
                Date revision received : 11 December 2013
                Categories
                Subject: Communications
                Subject: DNA Imaging

                ScienceOpen disciplines: Chemistry
                Keywords: dna,imaging microscopy,luminescence,ruthenium,two-photon emission imaging
                Data availability:
                ScienceOpen disciplines: Chemistry
                Keywords: dna, imaging microscopy, luminescence, ruthenium, two-photon emission imaging

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