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Coda: Trade-Offs, Cautions, and Limitations of Superresolution Optical Microscopes

  • Barry R. MastersEmail author
Chapter
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Part of the Springer Series in Optical Sciences book series (SSOS, volume 227)

Abstract

My aim in this chapter is to encourage critical skeptical thinking about superresolution microscopy. It is important to note that the precision and accuracy of single-molecule localization are different concepts. Typically, authors report the precision of localization of a single molecule. That is not equivalent to the accuracy of localization. The accuracy of localization is a measure of the fidelity with which single-molecule localization is identical to the actual location within the specimen. To assess the accuracy of superresolution single-molecule locations and therefore the final superresolution image we require calibration specimens whose structures are independently determined. An alternative is to use correlative microscopy (e.g., transmission electron microscopy) to validate the accuracy of information obtained from the superresolution image. Localization techniques achieve high precision in localization of a fluorescent molecule. The size of a fluorescent molecule affects the accuracy of the position of the structure to which the label is attached. This is of increasing importance as the size of the label, including its associated linker molecules, approaches the required resolution of the superresolution image.

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Further Reading

  1. Betzig, E. (2014a). Single Molecules, Cells, and Super-Resolution Optics: Lecture Slides. https://www.nobelprize.org/uploads/2018/06/betzig-lecture-slides.pdf (Accessed April 2, 2019).
  2. Betzig, E. (2014b). Single Molecules, Cells, and Super-Resolution Optics: Nobel Lecture. https://www.nobelprize.org/prizes/chemistry/2014/betzig/lecture/ (Accessed April 2, 2019).
  3. Betzig, E. (2014c). Biographical sketch. https://www.nobelprize.org/prizes/chemistry/2014/betzig/biographical/ (Accessed April 2, 2019).
  4. Shtengel, G., Wang, Y., Zhang, Z., Goh, W. I., Hess, H. F., and Kanchanawong, P. (2014). Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM. Methods in Cell Biology, 123, 273–294.Google Scholar
  5. van de Linde, S., Heilemann, M., and Sauer, M. (2012). Live-cell super-resolution imaging with synthetic fluorophores. Annual Review of Physical Chemistry, 63, 519–540.Google Scholar
  6. Yao, Z., and Carballido-López, R. (2014). Fluorescence imaging for bacterial cell biology: From localization to dynamics, from ensembles to single molecules. Annual Review of Microbiology, 68, 459–476.Google Scholar

Copyright information

© Springer Nature Switzerland AG 2020

Authors and Affiliations

  1. 1.Previously, Visiting Scientist Department of Biological EngineeringMassachusetts Institute of TechnologyCambridgeUSA
  2. 2.Previously, Visiting Scholar Department of the History of ScienceHarvard UniversityCambridgeUSA

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