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. 2022 Mar 7;12:3678. doi: 10.1038/s41598-022-07313-1

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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ASAP3-R3 accurately reports voltage in motile cells and detects voltage fluctuations during the cell cycle. (a) Top, one segment of membrane in a beating CM was analyzed. Middle, raw traces of green and red signals in the selected region. Red trace indicates significant cell movement in and out of selected region detected by the voltage-independent mCyRFP. Bottom, relative change in green fluorescence alone (− ∆F/F) and the relative change in the green/red ratio (− ∆R/R). While green intensity changes are larger than those observed previously in non-contracting CMs due to these motion artifacts, ratio changes are similar in magnitude to those observed previously. (b) Similar analysis on a different membrane segment of the same cell. Here, the green fluorescence changes are opposite in direction from that expected due to movement, which is detected in the red channel. While green intensity traces do not resemble AP waveforms, ratio traces are indistinguishable from AP waveforms in non-motile CMs and similar in shape to those in (a). Epifluorescence images were acquired using Hamamatsu HCImage software and color channels assigned and overlaid in NIH Fiji 2.1. Graphs were generated in Microsoft Excel for Mac 16. (c) Top, images of the ASAP3-R3 green/red ratio at different time points relative to cytokinesis in a HeLa cell. Middle, individual green/red ratio time courses of three HeLa cells during cell division. The cell in the images is represented by the blue trace. Cytokinesis occurs immediately before the 0-h time point. Below, mean green/red ratios of the three cells. Error bars represent standard deviation. Epifluorescence images were acquired using Hamamatsu HCImage software and pseudocoloring with a ratiometric lookup table was performed in NIH Fiji 2.1. Graphs were generated in Microsoft Excel for Mac 16.