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. Author manuscript; available in PMC: 2013 Apr 1.
Published in final edited form as: Nat Methods. 2012 Sep 9;9(10):1005–1012. doi: 10.1038/nmeth.2171

Figure 3.

Figure 3

Clover-mRuby2 FRET in voltage sensing. (a) Organization of VSFP-CR, based on VSFP2.3. (b) Ratiometric images of hippocampal neurons expressing VSFP-CR before and after depolarization with 50 mM KCl. Increased acceptor/donor emission was observed at the cell membranes and in neurites (arrows), but not inside the cell body (asterisk). Scale bar, 10 μm. (c) For voltage-emission ratio relationships, hippocampal neurons expressing VSFP-CR were subjected to various voltage steps from a resting potential of –70mV by patch-clamping (top panel), and emission ratio changes at each voltage were measured (bottom panel). (d) Mean emission ratio changes in response to voltage steps for VSFP2.3 (n = 39 cells) or VSFP-CR (n = 47 cells). Error bars represent s.e.m. Differences at potentials ≤ –100 mV and ≥ –40 mV are statistically significant by two-tailed t-test with Bonferroni correction for 10 repeated measures (P = 6.9e–4, 1.9e–5, 1.9e–5, 3.3e–5, 1.8e–4, 1.2e–3, 4.7e–3, and 1.5e–3 for –120, –100, –40, – 20, 0, 20, 40, and 60 mV respectively, versus required P < 0.005 for α < 0.05). (e) Ratio changes in response to a single AP were 1.03 ± 0.1% (mean ± s.e.m., n = 22 cells) for VSFP-CR and 0.82 ± 0.05% (n = 32 cells) for VSFP2.3 (P = 0.04 by two-tailed t-test). (f) VSFP-CR ratio changes (bottom) reliably detected APs (top) in a single unfiltered trace with a measured peak/noise ratio of 8.0. Measured power at the specimen plane was 1 W cm-2. At this power, the baseline ratio changed by 5% over 25 s.