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. 2019 Aug 20;9:287. doi: 10.3389/fcimb.2019.00287

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This work is authored by Hillman, Stewart, Strnad, Stone, Starr, Carmody, Evans, Carracoi, Wachter and Rosa on behalf of the U.S. Government and, as regards Hillman, Stewart, Strnad, Stone, Starr, Carmody, Evans, Carracoi, Wachter, Rosa, and the U.S. Government, is not subject to copyright protection in the United States. Foreign and other copyrights may apply

This work is authored by Hillman, Stewart, Strnad, Stone, Starr, Carmody, Evans, Carracoi, Wachter and Rosa on behalf of the U.S. Government and, as regards Hillman, Stewart, Strnad, Stone, Starr, Carmody, Evans, Carracoi, Wachter, Rosa, and the U.S. Government, is not subject to copyright protection in the United States. Foreign and other copyrights may apply. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Spirochetes making tetracysteine-tagged OspD can be labeled with FlAsH and relabeled with ReAsH over time. All samples were imaged by dark field and fluorescence microscopy using FITC and Texas Red channels. (A) FlAsH-labeled ΔOspD-SV-2xTC fluorescence monitored in the FITC channel over time. No fluorescence was observed in the Texas Red channel (TxRed). (B) Fluorescence of FlAsH-labeled and heat-killed ΔOspD-SV-2xTC (control) visualized over time, no fluorescence was observed in the Texas Red channel. (C) Fluorescence of ΔOspD-SV-2xTC spirochetes labeled with FlAsH (monitored in the FITC channel) on Day 0 and relabeled with ReAsH (monitored in the TxRed channel) at subsequent time points as indicated. All cultures were incubated at 22°C and analyzed over 8 days. All images were acquired using a 1 s exposure for FlAsH and a 2 s exposure for ReAsH. Images have been cropped, are not inverted, and representative of what was observed. This experiment was repeated 3 times.