High-resolution colocalization of single molecules within the resolution gap of far-field microscopy
Heinlein T, Biebricher A, Schluter P, Roth CM, Herten D-P, Wolfrum J, Heilemann M, Müller C, Tinnefeld P, Sauer M (2005)
ChemPhysChem 6(5): 949-955.
Zeitschriftenaufsatz
| Veröffentlicht | Englisch
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Autor*in
Heinlein, Thomas;
Biebricher, Andreas;
Schluter, Pia;
Roth, Christian Michael;
Herten, Dirk-Peter;
Wolfrum, Jürgen;
Heilemann, MikeUniBi;
Müller, Christian;
Tinnefeld, Philip;
Sauer, MarkusUniBi
Einrichtung
Abstract / Bemerkung
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.
Stichworte
single-molecule spectroscopy;
fluorescence;
photochemistry;
DNA;
high-precision distance microscopy
Erscheinungsjahr
2005
Zeitschriftentitel
ChemPhysChem
Band
6
Ausgabe
5
Seite(n)
949-955
ISSN
1439-4235
eISSN
1439-7641
Page URI
https://pub.uni-bielefeld.de/record/1603877
Zitieren
Heinlein T, Biebricher A, Schluter P, et al. High-resolution colocalization of single molecules within the resolution gap of far-field microscopy. ChemPhysChem. 2005;6(5):949-955.
Heinlein, T., Biebricher, A., Schluter, P., Roth, C. M., Herten, D. - P., Wolfrum, J., Heilemann, M., et al. (2005). High-resolution colocalization of single molecules within the resolution gap of far-field microscopy. ChemPhysChem, 6(5), 949-955. https://doi.org/10.1002/cphc.200400622
Heinlein, Thomas, Biebricher, Andreas, Schluter, Pia, Roth, Christian Michael, Herten, Dirk-Peter, Wolfrum, Jürgen, Heilemann, Mike, Müller, Christian, Tinnefeld, Philip, and Sauer, Markus. 2005. “High-resolution colocalization of single molecules within the resolution gap of far-field microscopy”. ChemPhysChem 6 (5): 949-955.
Heinlein, T., Biebricher, A., Schluter, P., Roth, C. M., Herten, D. - P., Wolfrum, J., Heilemann, M., Müller, C., Tinnefeld, P., and Sauer, M. (2005). High-resolution colocalization of single molecules within the resolution gap of far-field microscopy. ChemPhysChem 6, 949-955.
Heinlein, T., et al., 2005. High-resolution colocalization of single molecules within the resolution gap of far-field microscopy. ChemPhysChem, 6(5), p 949-955.
T. Heinlein, et al., “High-resolution colocalization of single molecules within the resolution gap of far-field microscopy”, ChemPhysChem, vol. 6, 2005, pp. 949-955.
Heinlein, T., Biebricher, A., Schluter, P., Roth, C.M., Herten, D.-P., Wolfrum, J., Heilemann, M., Müller, C., Tinnefeld, P., Sauer, M.: High-resolution colocalization of single molecules within the resolution gap of far-field microscopy. ChemPhysChem. 6, 949-955 (2005).
Heinlein, Thomas, Biebricher, Andreas, Schluter, Pia, Roth, Christian Michael, Herten, Dirk-Peter, Wolfrum, Jürgen, Heilemann, Mike, Müller, Christian, Tinnefeld, Philip, and Sauer, Markus. “High-resolution colocalization of single molecules within the resolution gap of far-field microscopy”. ChemPhysChem 6.5 (2005): 949-955.
Daten bereitgestellt von European Bioinformatics Institute (EBI)
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