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Counting Molecules: Toward Quantitative Imaging

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Far-Field Optical Nanoscopy

Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 14))

Abstract

In this chapter, we describe how single-molecule fluorescence can be used to analyze protein–protein interactions by enabling the direct visualization of protein complexes and the number and species of their constituent subunits. The prerequisites for this visualization are a low protein density, facilitating the discrimination of individual fluorescent complexes, and high labeling efficiency and specificity to allow for quantitative measurements of protein interactions. We describe three experimental realizations of quantitative imaging techniques made possible by the single-molecule approach: counting the photobleaching steps of fluorescent complexes, the analysis of a histogram of measured spot intensities, and the colocalization of fluorescent tags of multiple colors. Applications of this approach range from the determination of a protein’s oligomerization state to the analysis of the subunit content of large protein assemblies containing multiple subunit types. We describe the minimal setup required for these experiments and present several examples of this approach applied to current biological questions.

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Authors and Affiliations

  1. Centre for Biological Signalling Studies (BIOSS) and Institute for Physiology at the Department of Medicine, Albert-Ludwigs-University Freiburg, 79104, Freiburg im Breisgau, Germany

    Maximilian H. Ulbrich

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  1. Maximilian H. Ulbrich
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Correspondence to Maximilian H. Ulbrich .

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Editors and Affiliations

  1. NanoBioSciences Institute of Physical and Theoretical Chemistry, TU Braunschweig, Braunschweig, Germany

    Philip Tinnefeld

  2. MRC Human Immunology Unit and Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom

    Christian Eggeling

  3. Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Stefan W. Hell

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© 2011 Springer-Verlag Berlin Heidelberg

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Ulbrich, M.H. (2011). Counting Molecules: Toward Quantitative Imaging. In: Tinnefeld, P., Eggeling, C., Hell, S. (eds) Far-Field Optical Nanoscopy. Springer Series on Fluorescence, vol 14. Springer, Berlin, Heidelberg. https://doi.org/10.1007/4243_2011_36

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