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 inProceedings of the National Academy of Sciences - PNAS Vol. 105; no. 47; pp. 18176 - 18181
Main Authors Vukojević, Vladana, Heidkamp, Marcus, Ming, Yu, Johansson, Björn, Terenius, Lars, Rigler, Rudolf
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 25.11.2008
National Acad Sciences
Subjects
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
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Summary: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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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
ISSN:0027-8424
1091-6490
1091-6490
DOI:10.1073/pnas.0809250105