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Measuring Nanometer Distances Between Fluorescent Labels Step-by-Step

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Book cover Super-Resolution Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1663))

Abstract

Super-resolution fluorescence microscopy methods are increasingly applied to study the structure of biological molecules within their natural context or at biomaterial interfaces. We here provide a protocol for Single-molecule High-Resolution Imaging with Photobleaching (SHRImP) that can be used to obtain information about the conformation of large proteins or other macromolecules at the single-molecule level. This procedure requires site-specific protein labeling with fluorescent dyes, immobilization and sample preparation, optimization of imaging buffer composition and microscope settings, and acquisition of short time-lapse movies that capture the stepwise bleaching behavior of individual molecules. We then describe a method for reliably determining the relative positions of labels from bleaching movies using the free image processing package Fiji (ImageJ) with the help of auxiliary macros that are provided as Supplementary Material. The presented approach allows for measuring intramolecular distance distributions in the range of a few to hundreds of nanometers and can be applied to a wide variety of biological systems.

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Author information

Authors and Affiliations

  1. Laboratory of Applied Mechanobiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093, Zurich, Switzerland

    Susanna Maria Früh & Ingmar Schoen

Authors
  1. Susanna Maria Früh
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  2. Ingmar Schoen
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Corresponding author

Correspondence to Ingmar Schoen .

Editor information

Editors and Affiliations

  1. High-Content Analysis of the Cell (HiCell) and Advanced Biological Screening Facility, BioQuant, Heidelberg University, Heidelberg, Germany

    Holger Erfle

1 Electronic Supplementary Material

Data 16.1

SHRImP_Macros (AS 140,674 kb)

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Früh, S.M., Schoen, I. (2017). Measuring Nanometer Distances Between Fluorescent Labels Step-by-Step. In: Erfle, H. (eds) Super-Resolution Microscopy. Methods in Molecular Biology, vol 1663. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7265-4_16

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  • DOI: https://doi.org/10.1007/978-1-4939-7265-4_16

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7264-7

  • Online ISBN: 978-1-4939-7265-4

  • eBook Packages: Springer Protocols

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