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The Effect of Cryo Temperature on Commonly used Fluorophores

Metskas, Lauren Ann and Ho, Samuel and Weaver, Sara J. and Jensen, Grant J. and Tirrell, David A. (2020) The Effect of Cryo Temperature on Commonly used Fluorophores. Biophysical Journal, 118 (3). 150a-151a. ISSN 0006-3495. doi:10.1016/j.bpj.2019.11.942.

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Correlated cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) has become an increasingly popular method for combining the resolving power of cryo-EM with the specificity of fluorescence. Although cryo-fluorescence microscopy suffers from optical limitations, it is a powerful way to target the resolving power of cryo-EM toward proteins of interest in heterogeneous cellular environments. Super-resolution microscopy at cryo temperatures has also been established using several different approaches. While fluorescence-derived localization is a key benefit of cryo-CLEM, fluorescence can also be used to bring orthogonal information into cryo-EM images. We recently developed a fusion assay compatible with cryo-CLEM equipment and conditions (Metskas and Briggs, Microscopy & Microanalysis 2019). This development employs auto-quenching by resonant energy transfer to specifically target a function rather than a protein in cryo-CLEM - in this case, adding information on lipid mixing to morphologies from micrographs of influenza virus fusion. However, further methods developments, particularly those involving FRET, are currently hampered by limited characterization of modern fluorophores at cryo-CLEM temperatures (77-100 K). Here, we present a study of commonly used synthetic fluorophores and fluorescent proteins, characterizing excitation and emission spectra, singlet state lifetime, and quantum yield at 77 K. We note that 10 nm shifts of the modes are common for both excitation and emission spectra, but are fluorophore specific in magnitude and even in direction. Vibronic coupling and spectral narrowing are visible in all cases characterized, and singlet state lifetimes increase or decrease in a fluorophore-specific manner. Taken together, these data suggest guidelines for choosing cryo-CLEM fluorophores and filter sets, and demonstrate promise for techniques such as FRET in carefully-adapted applications.

Item Type:Article
Related URLs:
URLURL TypeDescription
Metskas, Lauren Ann0000-0002-8073-6960
Ho, Samuel0000-0001-7647-0752
Weaver, Sara J.0000-0001-7753-6215
Jensen, Grant J.0000-0003-1556-4864
Tirrell, David A.0000-0003-3175-4596
Additional Information:© 2020 Biophysical Society. Available online 7 February 2020.
Issue or Number:3
Record Number:CaltechAUTHORS:20200210-110813112
Persistent URL:
Official Citation:Lauren Ann Metskas, Samuel Ho, Sara J. Weaver, Grant J. Jensen, David A. Tirrell, The Effect of Cryo Temperature on Commonly used Fluorophores, Biophysical Journal, Volume 118, Issue 3, Supplement 1, 2020, Pages 150a-151a, ISSN 0006-3495, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:101199
Deposited By: Tony Diaz
Deposited On:10 Feb 2020 21:43
Last Modified:16 Nov 2021 18:00

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