Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 105, Issue 47, Pages 18176-18181Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0809250105
Keywords
fluorescence correlation spectroscopy; live cells; sensitivity
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Funding
- Swedish Research Council, Cancerdegradome
- The Knut and Alice Wallenberg Foundation
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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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