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Caveat fluorophore: an insiders’ guide to small-molecule fluorescent labels

Nature Methods volume 19, pages 149–158 (2022)Cite this article

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Abstract

The last three decades have brought a revolution in fluorescence microscopy. The development of new microscopes, fluorescent labels and analysis techniques has pushed the frontiers of biological imaging forward, moving from fixed to live cells, from diffraction-limited to super-resolution imaging and from simple cell culture systems to experiments in vivo. The large and ever-evolving collection of tools can be daunting for biologists, who must invest substantial time and effort in adopting new technologies to answer their specific questions. This is particularly relevant when working with small-molecule fluorescent labels, where users must navigate the jargon, idiosyncrasies and caveats of chemistry. Here, we present an overview of chemical dyes used in biology and provide frank advice from a chemist’s perspective.

From its inception, fluorescence microscopy has been driven by advances in dyes. Early protein labels excited by ultraviolet (UV) light proved difficult to use for cellular imaging, and so Coons—a trained physician—developed the amine-reactive, blue-excited fluorescein isothiocyanate (FITC) in the 1940s1. This longer wavelength label established the use of small-molecule dyes for the nascent field of fluorescence microscopy. Although the investigation of fluorophores continued apace in the subsequent decades, the last 30 years have seen exponential growth in imaging agents thanks to the discovery and extension of genetically encoded fluorescent proteins, the ongoing application of new chemistry to small-molecule dye synthesis, the advent of super-resolution and single-molecule microscopies and the creation of new ‘hybrid’ labeling strategies useful in live cells and tissue2,3,4. The development of small-molecule fluorophores is an intra- and interdisciplinary effort, leveraging the marvelous flexibility of organic synthesis, the predictive power of physical organic chemistry, the innovations of optical physics and the creative questions of biology. Here, we review classic and modern small-molecule fluorophores across the electromagnetic spectrum and highlight the advantages and caveats of this type of label.

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Fig. 1: Fluorescence basics.
Fig. 2: Labeling strategies.
Fig. 3: Fluorophores across the visible spectrum.
Fig. 4: Caveats of small-molecule fluorescent dyes.
Fig. 5: Photobleaching of fluorescent dyes.
Fig. 6: Synthesis of Alexa Fluor 488.

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Acknowledgements

We thank E. Schreiter for assistance with protein structures and E. Betzig, U. Boehm, X. Darzacq, B. English, H. Farrants, A. Hansen, Z. Liu, T. Lionnet, J. Lippincott-Schwartz, J. Schmidt and S.-H. Shu for contributive discussions. Related work in our laboratory is supported by the Howard Hughes Medical Institute. Chemical structures and spectral data were taken from references or obtained from vendor websites.

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    Jonathan B. Grimm & Luke D. Lavis

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Grimm, J.B., Lavis, L.D. Caveat fluorophore: an insiders’ guide to small-molecule fluorescent labels. Nat Methods 19, 149–158 (2022). https://doi.org/10.1038/s41592-021-01338-6

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