Volume 106, Issue 1, Pages (January 2014)

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Volume 106, Issue 1, Pages 272-278 (January 2014) The Covalent Trimethoprim Chemical Tag Facilitates Single Molecule Imaging with Organic Fluorophores Tracy Y. Wang, Larry J. Friedman, Jeff Gelles, Wei Min, Aaron A. Hoskins, Virginia W. Cornish Biophysical Journal Volume 106, Issue 1, Pages 272-278 (January 2014) DOI: 10.1016/j.bpj.2013.11.4488 Copyright © 2014 Biophysical Society Terms and Conditions

Figure 1 (A) Schematic cartoon of the covalent trimethoprim chemical tag (A-TMP-tag). A target protein is tagged with an E. coli dihydrofolate reductase cysteine mutant (eDHFR:L28C) and covalently bound to a cell-permeable acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore). (B) Schematic of fluorophores and their A-TMP-tag conjugates examined in this investigation. Biophysical Journal 2014 106, 272-278DOI: (10.1016/j.bpj.2013.11.4488) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 2 SM photon fluxes and survival lifetimes of fluorophores (Fl) and tagged-fluorophores (Tagged-Fl) for Atto655 and Alexa647 in PBS buffer, 10 mM MEA, and 40% YE with 250 μW 633 nm laser illumination. (A) SM photon flux, which is determined by the number of detected photons. (B) Average SM survival lifetime before photobleaching. Biophysical Journal 2014 106, 272-278DOI: (10.1016/j.bpj.2013.11.4488) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 3 Total photon output of fluorophores (Fl) and tagged-fluorophores (Tagged-Fl) for Atto655 and Alexa647 in PBS buffer, 10 mM MEA, and 40% YE with 250 μW 633 nm laser illumination. Total photon output is determined by multiplying photon flux by average survival lifetime. Biophysical Journal 2014 106, 272-278DOI: (10.1016/j.bpj.2013.11.4488) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 4 Quantum yield and photostability lifetime for fluorophores (Fl), A-TMP-fluorophore (A-TMP-Fl) and tagged-fluorophores (Tagged-Fl) for Atto655 and Alexa647 in PBS buffer. (A) Quantum yield. (B) Photostability lifetime is the half-life of fluorescence signal due to photobleaching. Biophysical Journal 2014 106, 272-278DOI: (10.1016/j.bpj.2013.11.4488) Copyright © 2014 Biophysical Society Terms and Conditions