%0 Journal Article %1 Heinlein2005 %A Heinlein, T %A Biebricher, A %A Schluter, P %A Roth, CM %A Herten, DP %A Wolfrum, J %A Heilemann, M %A Muller, C %A Tinnefeld, P %A Sauer, M %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2005 %I WILEY-V C H VERLAG GMBH %J CHEMPHYSCHEM %K sauer mike %N 5 %P 949-955 %T High-resolution colocalization of single molecules within the resolution gap of far-field microscopy %U http://dx.doi.org/10.1002/cphc.200400622 %V 6 %X To obtain detailed information about the three-dimensional (30) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 6301650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 6301650) that can be efficiently excited by a single pulsed diode loser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.

@article{Heinlein2005, abstract = {To obtain detailed information about the three-dimensional (30) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 6301650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 6301650) that can be efficiently excited by a single pulsed diode loser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.}, added-at = {2011-03-01T10:57:13.000+0100}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, affiliation = {Herten, DP (Reprint Author), Univ Bielefeld, Fac Phys Appl Laser Phys \& Laser Spect, Univ Str 25, D-33615 Bielefeld, Germany. Univ Bielefeld, Fac Phys Appl Laser Phys \& Laser Spect, D-33615 Bielefeld, Germany. Univ Heidelberg, Inst Phys Chem, D-69120 Heidelberg, Germany.}, author = {Heinlein, T and Biebricher, A and Schluter, P and Roth, CM and Herten, DP and Wolfrum, J and Heilemann, M and Muller, C and Tinnefeld, P and Sauer, M}, author-email = {tinnefeld@physik.uni-bielefeld.de sauer@physik.uni-bielefeld.de}, biburl = {https://www.bibsonomy.org/bibtex/20c7af075281b7474203f4f8add18dbfb/reichert}, cited-references = {BUSTAMANTE C, 1994, SCIENCE, V265, P1599. CARDOSO MC, 1993, CELL, V74, P979. CHURCHMAN LS, 2005, P NATL ACAD SCI USA, V102, P1419, DOI 10.1073/pnas.0409487102. CLEGG RM, 1996, FLUORESCENCE IMAGING, P179. COOK PR, 1999, SCIENCE, V284, P1790. DICKSON RM, 1996, SCIENCE, V274, P966. DIETRICH A, 2002, REV MOL BIOTECHNOL, V82, P211. DYBA M, 2002, PHYS REV LETT, V88. ESA A, 2000, J MICROSC-OXFORD 2, V199, P96. GORDON MP, 2004, P NATL ACAD SCI USA, V101, P6462, DOI 10.1073/pnas.0401638101. HA T, 1996, PHYS REV LETT, V77, P3979. HEILEMANN M, 2002, ANAL CHEM, V74, P3511. 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YILDIZ A, 2004, SCIENCE, V303, P676, DOI 10.1126/science.1093753.}, doc-delivery-number = {926OU}, groups = {public}, interhash = {8319ff2f3bea2a47e948563dd04d2519}, intrahash = {0c7af075281b7474203f4f8add18dbfb}, issn = {1439-4235}, journal = {CHEMPHYSCHEM}, journal-iso = {ChemPhysChem}, keywords = {sauer mike}, keywords-plus = {LIFETIME IMAGING MICROSCOPY; HAND-OVER-HAND; FLUORESCENCE MICROSCOPY; STIMULATED-EMISSION; DYE MOLECULES; DYNAMICS; DNA; TRANSCRIPTION; LOCALIZATION; SPECTROSCOPY}, language = {English}, month = MAY, number = 5, number-of-cited-references = {42}, pages = {949-955}, publisher = {WILEY-V C H VERLAG GMBH}, subject-category = {Chemistry, Physical; Physics, Atomic, Molecular \& Chemical}, times-cited = {13}, timestamp = {2011-03-09T14:16:23.000+0100}, title = {High-resolution colocalization of single molecules within the resolution gap of far-field microscopy}, type = {Article}, unique-id = {ISI:000229133300023}, url = {http://dx.doi.org/10.1002/cphc.200400622}, username = {reichert}, volume = 6, year = 2005 }