Ionwizard sarcomere length acquisition software

Cells were stimulated at 1 Hz by carbon fiber electrodes placed at the side of the superfusion bath, and the contractile properties were studied using the IonOptix system (IonOptix Corp.) to measure sarcomere length. Cells were visualized via a ×40 oil objective using an IonOptix MyoCam (IonOptix Corp.) that sampled images at a frequency of 240 Hz. Sinusoidal optical density traces arising from the alternating light and dark bands of the contractile machinery were then transformed into a signal of sarcomere length by application of a fast Fourier transform by the IonWizard sarcomere length acquisition software (IonOptix Corp.). Length measurements were calibrated using a stage micrometer with 2-μm graduations so that the number of pixels per micrometer recorded by the MyoCam could be entered into the software as a fixed value. Amplitude of sarcomere shortening was calculated by deduction of systolic from diastolic sarcomere length. Analysis was performed using IonWizard 5 software (IonOptix Corp.). All values represent an average of 10 contractions. Cells for both Ca²⁺ imaging experiments and contraction studies were superfused with medium containing 130 mm NaCl, 5.4 mm KCl, 3.5 mm MgCl₂, 1.8 mm CaCl₂, 10 mm glucose, 5 mm HEPES, and 0.4 mm NaH₂PO₄ (pH 7.4) at 36 °C.

Cells were stimulated to contract via a patch electrode (permeabilized configuration), and contractile properties were studied using the IonOptix system (IonOptix Corp.) to measure sarcomere length. Cells were visualized via a ×40 oil objective using an IonOptix MyoCam (IonOptix Corp.) that sampled images at a frequency of 240 Hz. Sinusoidal optical density traces arising from the alternating light and dark bands of the contractile machinery were then transformed into a signal of sarcomere length by application of a fast Fourier transform by the IonWizard sarcomere length acquisition software (IonOptix Corp.). Length measurements were calibrated using a stage micrometer with 2-μm graduations such that the number of pixels/μm recorded by the MyoCam could be entered into the software as a fixed value. Amplitude of sarcomere shortening was calculated by deduction of systolic from diastolic sarcomere length and expressed as a percentage of resting sarcomere length. Analysis was performed using IonWizard 5 software (IonOptix Corp.). All values represent an average of 10 contractions.