Figure 3.
Temporal fidelity of DiO/DPA FRET pair.
A
, Fluorescence quenching responses to brief depolarizations from a HEK-293 cell bath labeled with DiO and incubated with 1 μm DPA. Membrane potential was stepped to +100 mV from an hp of −70 mV for durations of 0.1, 0.3, 1, 2, and 5 ms, yielding peak ΔF/F amplitudes of −14, −25, −28, −28, and −29%, respectively. Gray trace (τ rise) is a single-exponential fit to the initial quenching response of the 5 ms pulse. Black dashed traces (τ decay) are double-exponential fits of 0.17, 0.29, and 0.38 to the recovery of the quenched fluorescence response following termination of the 0.3, 1, and 5 ms step pulses, respectively. Each trace is an average of 10 sweeps and was filtered offline to 2 kHz.
B
, Correlation plot of the τ rise of fluorescence (+100 mV pulse for 5 ms) versus the weighted time constant of a double-exponential fit of the capacitance transients in response to a 5 mV step. Dashed line is a linear fit to all the data (Pearson correlation). The y-intercept is 0.12 ms and approximates the intrinsic time constant of the voltage-dependent quenching of the DiO fluorescence.
C
, Fluorescence quenching responses to trains of 1 ms, 100 mV depolarizations (hp = −70 mV) delivered at various frequencies (1000 Hz stimuli used 0.5 ms pulses). Traces were filtered offline to 3.5 kHz, and are averages of 10 and 15 trials for the 100 Hz and 300 Hz trains, respectively, and 17 trials for both 666 and 1000 Hz. DPA concentration was 1 μm. Inset is an expanded time scale of the 1000 Hz train (calibration: 4 ms, 12% ΔF/F).