| Zugriffsnummer | 24821 |
| Dokumenttyp | Zeitschriftenartikel |
| Sprache | Englisch |
| Titel | Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy |
| Autor(in); Institution |
Rüttinger, Steffen; 8.3, Biomedizinische Optik, PTB-Berlin
Buschmann, V.; PicoQuant GmbH, Berlin, GERMANY
Krämer, B.; PicoQuant GmbH, Berlin, GERMANY
Erdmann, R.; PicoQuant GmbH, Berlin, GERMANY
Macdonald, Rainer; 8.3, Biomedizinische Optik, PTB-Berlin
Koberling, F.; PicoQuant GmbH, Berlin, GERMANY
|
| Quelle/Jahr | Journal of Microscopy: 232 (2008), 2, 343 - 352 |
| ISSN | 0022-2720 (PRINT) ; 1365-2818 (ONLINE) |
| DOI | |
| Verlag | Oxford [u.a.]: Wiley-Blackwell |
| Freie Schlagworte | Confocal microscopy ; confocal volume ; FCS |
| Zusammenfassung | Single molecule detection based on fluorescent labels offers the possibility to gain not only qualitative but also quantitative insight into specific functions of complex biological systems. Fluorescence correlation spectroscopy is one of the favourite techniques to determine concentrations and diffusion constants as well as molecular brightness of molecules in the pico- to nano-molar concentration range, with broad applications in biology and chemistry. Although fluorescence correlation spectroscopy in principle has the potential to measure absolute concentrations and diffusion coefficients, the ecessity to know the exact size and shape of the confocal volume very often hampers the possibility to obtain quantitative results and restricts fluorescence correlation spectroscopy to relative measurements mainly. The determination of the confocal volume in situ is difficult because it is sensitive to optical alignment and aberrations, optical saturation and variations of the index of refraction as observed in biological specimen. In the present contribution, we compare different techniques to characterize the confocal volume and to obtain the confocal parameters by fluorescence correlation pectroscopy curve fitting, a fluorescence correlation spectroscopy dilution series and confocal scanning of fluorescent beads. The results are compared in the view of quantitative fluorescence correlation spectroscopy measurement and analysis. We investigate how unavoidable artefacts caused by a non-ideal confocal volume can be experimentally determined and validated. |
Zitierung
Rüttinger, S., Buschmann, V., Krämer, B., Erdmann, R., Macdonald, R., & Koberling, F. (2008). Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy. Journal of Microscopy, 232(2), 343–352. https://doi.org/10.1111/j.1365-2818.2008.02105.x