Supplementary Figure 3: Membrane localization and voltage sensitivities of Ace-FP fusions.
From: Fast, in vivo voltage imaging using a red fluorescent indicator
(a) Schematic of spectral overlap of the absorption of Ace and emission of indicated fluorophores4–7. (b) Epifluorescence images of HEK cells transfected with the Ace-FP fusion constructs. While Ace-SEP227D, Ace-mNeon and Ace-mRuby3 expressed brightly and showed excellent membrane localization, Ace-mCitrine and Ace-mCardinal2 exhibited dim fluorescence and large intracellular aggregates (n = 4 wells per condition, 500 cells per well). (c) ∆F/∆V curves for Ace-mCitrine, Ace-SEP227D and Ace-mCardinal2 in HEK cells acquired during concurrent optical and electrical recordings in voltage clamp (n = 6 cells for Ace-mCitrine and 4 cells each for Ace-SEP227D and Ace-mCardinal2). Values represent mean ± s.e.m. Illumination intensities were 15–20 mW mm–2 except for Ace-mCardinal2, which was imaged at 40 mW mm–2 at the specimen plane. 4. Bajar, B. T. et al. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. Sci. Rep. 6, 20889 (2016). 5. Chu, J. et al. Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein. Nat. Methods 11, 572–578 (2014). 6. Gong, Y. et al. High-speed recording of neural spikes in awake mice and flies with a fluorescent voltage sensor. Science 350, 1361–1366 (2015). 7. Gong, Y., Wagner, M. J., Zhong Li, J. & Schnitzer, M. J. Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors. Nat. Commun. 5, 3674 (2014).
