title: Dendrimer probes for enhanced photostability and localization in fluorescence imaging.
abstract: Recent advances in fluorescence microscopy have enabled high-resolution imaging and tracking of single proteins and biomolecules in cells. To achieve high spatial resolutions in the nanometer range, bright and photostable fluorescent probes are critically required. From this view, there is a strong need for development of advanced fluorescent probes with molecular-scale dimensions for fluorescence imaging. Polymer-based dendrimer nanoconjugates hold strong potential to serve as versatile fluorescent probes due to an intrinsic capacity for tailored spectral properties such as brightness and emission wavelength. In this work, we report a new, to our knowledge, class of molecular probes based on dye-conjugated dendrimers for fluorescence imaging and single-molecule fluorescence microscopy. We engineered fluorescent dendritic nanoprobes (FDNs) to contain multiple organic dyes and reactive groups for target-specific biomolecule labeling. The photophysical properties of dye-conjugated FDNs (Cy5-FDNs and Cy3-FDNs) were characterized using single-molecule fluorescence microscopy, which revealed greatly enhanced photostability, increased probe brightness, and improved localization precision in high-resolution fluorescence imaging compared to single organic dyes. As proof-of-principle demonstration, Cy5-FDNs were used to assay single-molecule nucleic acid hybridization and for immunofluorescence imaging of microtubules in cytoskeletal networks. In addition, Cy5-FDNs were used as reporter probes in a single-molecule protein pull-down assay to characterize antibody binding and target protein capture. In all cases, the photophysical properties of FDNs resulted in enhanced fluorescence imaging via improved brightness and/or photostability.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: Younghoon
authors.initials: Y
authors.lastname: Kim
authors.forename: Sung Hoon
authors.initials: SH
authors.lastname: Kim
authors.forename: Melikhan
authors.initials: M
authors.lastname: Tanyeri
authors.forename: John A
authors.initials: JA
authors.lastname: Katzenellenbogen
authors.forename: Charles M
authors.initials: CM
authors.lastname: Schroeder
mesh_terms: D001692:Biological Transport; D002232:Carbocyanines / Q000737:chemistry; D050091:Dendrimers / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008027:Light; D008856:Microscopy, Fluorescence / Q000379:methods*; D049329:Nanostructures / Q000737:chemistry; D009693:Nucleic Acid Hybridization
chemical_list: D002232:Carbocyanines; D050091:Dendrimers; D005456:Fluorescent Dyes; C089187:cyanine dye 3; C085321:cyanine dye 5
references.citation: J Am Chem Soc. 2001 Aug 22;123(33):8101-8
references.pmid: 11506567
references.citation: Cell. 2010 Dec 23;143(7):1047-58
references.pmid: 21168201
references.citation: Nature. 2009 Jun 4;459(7247):686-9
references.pmid: 19430463
references.citation: Nat Methods. 2011 Jun;8(6):499-508
references.pmid: 21552254
references.citation: Science. 2005 Jun 3;308(5727):1469-72
references.pmid: 15817813
references.citation: Science. 1998 Sep 25;281(5385):2016-8
references.pmid: 9748158
references.citation: Science. 2002 Sep 13;297(5588):1873-7
references.pmid: 12228718
references.citation: Chem Soc Rev. 2004 Jan 10;33(1):43-63
references.pmid: 14737508
references.citation: Annu Rev Biophys. 2008;37:417-44
references.pmid: 18573089
references.citation: Angew Chem Int Ed Engl. 2001 Dec 17;40(24):4643-4648
references.pmid: 12404372
references.citation: Science. 1999 Jul 16;285(5426):400-2
references.pmid: 10411501
references.citation: Science. 2003 Jun 27;300(5628):2061-5
references.pmid: 12791999
references.citation: Science. 2006 Sep 15;313(5793):1642-5
references.pmid: 16902090
references.citation: Nat Methods. 2006 Oct;3(10):793-5
references.pmid: 16896339
references.citation: J Am Chem Soc. 2003 May 14;125(19):5832-8
references.pmid: 12733924
references.citation: Nanomedicine. 2005 Sep;1(3):193-212
references.pmid: 17292079
references.citation: Nature. 2011 May 26;473(7348):484-8
references.pmid: 21614075
references.citation: Science. 2005 Jan 28;307(5709):538-44
references.pmid: 15681376
references.citation: Nat Methods. 2008 Nov;5(11):943-5
references.pmid: 18794861
references.citation: Angew Chem Int Ed Engl. 2008;47(29):5465-9
references.pmid: 18601270
references.citation: Nat Methods. 2010 May;7(5):377-81
references.pmid: 20364147
references.citation: Biophys J. 2002 May;82(5):2775-83
references.pmid: 11964263
references.citation: Annu Rev Phys Chem. 2012;63:595-617
references.pmid: 22404588
references.citation: J Phys Chem B. 2007 Jul 19;111(28):7929-31
references.pmid: 17583944
references.citation: Nat Chem. 2010 Mar;2(3):179-86
references.pmid: 20625479
references.citation: Nano Lett. 2011 Nov 9;11(11):4656-60
references.pmid: 21919444
references.citation: Opt Lett. 1994 Jun 1;19(11):780-2
references.pmid: 19844443
references.citation: J Am Chem Soc. 2007 Apr 18;129(15):4595-605
references.pmid: 17378559
references.citation: Science. 2008 Feb 8;319(5864):810-3
references.pmid: 18174397
references.citation: Science. 1953 Oct 16;118(3068):447-8
references.pmid: 13101775
references.citation: Anal Chem. 2010 Mar 15;82(6):2192-203
references.pmid: 20163145
references.citation: Proc Natl Acad Sci U S A. 2005 Sep 13;102(37):13081-6
references.pmid: 16141335
references.citation: Adv Drug Deliv Rev. 2005 Dec 14;57(15):2106-29
references.pmid: 16305813
references.citation: Chem Soc Rev. 2010 Apr;39(4):1272-9
references.pmid: 20349533
references.citation: Nat Methods. 2008 Feb;5(2):155-7
references.pmid: 18193054
references.citation: J Am Chem Soc. 2010 Dec 29;132(51):18054-6
references.pmid: 21133363
references.citation: Chemistry. 2009 Sep 21;15(37):9270-85
references.pmid: 19718727
references.citation: Nat Methods. 2006 Nov;3(11):891-3
references.pmid: 17013382
references.citation: Chem Rev. 2001 May;101(5):1267-300
references.pmid: 11710221
references.citation: Science. 2007 May 25;316(5828):1153-8
references.pmid: 17525330
references.citation: Biomacromolecules. 2006 Feb;7(2):572-9
references.pmid: 16471932
references.citation: Nano Lett. 2006 Jul;6(7):1459-63
references.pmid: 16834429
references.citation: Nat Biotechnol. 2004 Dec;22(12):1567-72
references.pmid: 15558047
references.citation: Nat Methods. 2008 Jun;5(6):507-16
references.pmid: 18511918
references.citation: Science. 1998 Sep 25;281(5385):2013-6
references.pmid: 9748157