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Choosing the Right Fluorescent Probe

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Fluorescence Spectroscopy and Microscopy in Biology

Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 20))

Abstract

Fluorescence microscopy and spectroscopy are by now used routinely in any laboratory working on the field of basic and applied biological sciences. A wide and expanding library of small organic fluorophores with radically different properties has been made available, offering great flexibility to the user of fluorescence-based methods. Beyond small organic fluorophores, the development of fluorescent proteins allowed for the introduction of genetically encoded fluorescence tagging, a novel tool that quickly revolutionized cellular imaging and cell biology.

Still, a considerable fraction of casual users of fluorescence tools do not follow rational considerations when selecting a fluorescent probe for their intended application, often relying on trial and error alone, which inevitably leads to a decrease in data quality and limits the potential of fluorescence-based methods. This chapter aims to present an overview of the most important considerations to be made when selecting a fluorescent reporter. Fluorophore properties and their importance for fluorescence assays will be discussed. A list of different fluorophores and a summary of their properties will also be presented as a tool to assist in the process of choosing the right fluorescence probe. Finally, specific applications such as super-resolution microscopy require very specific fluorophores, and these will be discussed separately.

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Authors and Affiliations

  1. Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal

    Maria J. Sarmento

  2. iBB – Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal

    Fábio Fernandes

  3. Associate Laboratory i4HB – Institute for Health and Bioeconomy, IST, Universidade de Lisboa, Lisbon, Portugal

    Fábio Fernandes

  4. Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal

    Fábio Fernandes

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  1. Maria J. Sarmento
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  1. J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic

    Radek Šachl

  2. J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic

    Mariana Amaro

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Sarmento, M.J., Fernandes, F. (2022). Choosing the Right Fluorescent Probe. In: Šachl, R., Amaro, M. (eds) Fluorescence Spectroscopy and Microscopy in Biology. Springer Series on Fluorescence, vol 20. Springer, Cham. https://doi.org/10.1007/4243_2022_30

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