TABLE 2.

Troubleshooting table.

Step	Problem	Possible reason(s)	Solution(s)
11, 13, 19	Difficulty anesthetizing the mouse before surgery	Interruption of flow of anesthesia to the animal	Tubing may be disconnected or occluded, preventing flow to the nose cone or the ventilator. Check existing tubing to ensure adequate gas flow. In addition, the anesthesia chamber may be empty. Ensure that sufficient isoflurane is present in the chamber to last through the entire procedure
16, 45, 46B(iv)	Low-quality ECG trace measured through the differential amplifier	There is poor tissue contact with the ECG leads, or interfering noise is present	Minimize interfering electrical noise from other active devices such as the heat plate and the overhead lights. Consider wrapping the length of the ECG leads with insulating electrical tape. If noise affects the quality of the ECG, we recommend the use of a metal mesh Faraday cage placed around the mouse. In addition, a general signal-processing algorithm can be very effective in removing noise spikes and white noise, and in increasing QRS detection accuracy. A protocol describing ECG monitoring in mice can be found in ref. 25
19	Animal is breathing spontaneously despite ventilation	Endotracheal tube may be in the esophagus rather than in the trachea	Check for the rise of the chest with ventilation. If it is not present, remove the endotracheal catheter and reintubate the mouse according to Step 18
37, 39	Inadequate seal of the adhesive stabilizer to the heart surface	Excess liquid on the heart surface or poor-quality adhesive	Gently wick off the excess moisture from the heart surface using a Kimwipe. In addition, open a new bottle of adhesive and repeat the attempt at stabilization
45	Low or absent image signal level	Image is not in focus, or there is inadequate light exposure on the tissue	There are multiple possible causes; verify that the objective’s focal point is set near the surface of the tissue by manually scanning the z axis until the image is visible. Ensure that the laser shutter for the selected illumination wavelength is open. Increase the exposure power level if needed. In two-photon mode, verify that the femtosecond laser is mode-locked by checking the monitoring software for the laser
46	Difficulty pacing the heart	Native heart rate is too high, or there is inadequate contact of the pacing leads with the tissue	The pacing rate must be higher than the native heart rate in order to overdrive the pace of the heart. If necessary, consider using a pharmacologic agent such as a beta blocker to lower the mouse heart rate to allow pacing. Also check that the pacing lead is securely attached to the heart surface with the exposed wire in direct contact with the myocardium. Turn up the amplitude of the stimulus pulse if necessary to achieve capture
47	Retrospective gating software fails to read the ECG correctly	Poor ECG signal quality	Before initiating an imaging session, make sure that the ECG electrode needles are well placed and that the ECG signal is clean