Quantitative single-molecule imaging by confocal laser scanning microscopy
A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent...
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| Published in | Proceedings of the National Academy of Sciences - PNAS Vol. 105; no. 47; pp. 18176 - 18181 |
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| Main Authors | , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
United States
National Academy of Sciences
25.11.2008
National Acad Sciences |
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| Online Access | Get full text |
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| Abstract | A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed. |
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| AbstractList | A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed. A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed. fluorescence correlation spectroscopy live cells sensitivity A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed. [PUBLICATION ABSTRACT] |
| Author | Ming, Yu Vukojević, Vladana Johansson, Björn Rigler, Rudolf Terenius, Lars Heidkamp, Marcus |
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| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/19011092$$D View this record in MEDLINE/PubMed http://kipublications.ki.se/Default.aspx?queryparsed=id:118018740$$DView record from Swedish Publication Index |
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| Notes | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Author contributions: R.R. designed research; V.V. performed research; M.H., Y.M., B.J., and L.T. contributed new reagents/analytic tools; V.V. and R.R. analyzed data; and V.V. and R.R. wrote the paper. Parts of this work were presented at the 9th Carl Zeiss-sponsored workshop on FCS and related methods, December 4–6, 2006, AlbaNova University Center, Stockholm Sweden. Communicated by Walter J. Gehring, University of Basel, Basel, Switzerland, October 7, 2008 |
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| SubjectTerms | Cells Correlations Deoxyribonucleic acid DNA DNA - chemistry Fluorescence Fluorescent Dyes Image analysis Imaging Lasers Medicin och hälsovetenskap Microscopy Microscopy, Confocal - methods Molecular imaging Molecular structure Molecules Photons Physical Sciences Pixels Proteins Spectroscopy |
| Title | Quantitative single-molecule imaging by confocal laser scanning microscopy |
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