TABLE 1.
Troubleshooting table.
| Problem | Possible reason | Solution |
|---|---|---|
| Preparation drifting | Explant is not properly pinned into the dish | Pin the explant stably and flat into the dish; make sure that no parts touch the objective |
| Superfusion or suction lines touch the rim of the dish | Place the superfusion lines in a way that their tips are in the fluid, but do not touch the dish | |
| Temperature has not yet stabilized | Wait until the temperature is stable; avoid temperature spikes; turn off auto-feedback control | |
| Preparation twitching | Muscle fibers have been damaged during the preparation | Discard preparation |
| Tissue is suffering from phototoxicity | Reduce excitation light | |
| Blurry images | Air bubble is trapped in front of the objective | Flush with physiological solution, taking care to avoid a temperature spike due to increased flow rate during flush. Have oxygenated room temperature solution ready for ‘rescue operations’ |
| Consider heating solution in reservoir to avoid ‘degassing’ as temperature rises in in-line heater | ||
| Superfusion or suction lines touch the rim of the dish and transmit vibrations—or air table is not floating | Place the superfusion lines in a way that their tips are in the fluid but do not touch the dish. Check that all four corners of table are floating | |
| Absence of cellular dynamics | Tissue has been mechanically damaged, suffered hypoxia or has been in chamber for too long | Discard the preparation |
| Organelles look abnormal (e.g., rounded mitochondria) | Tissue has been damaged or suffered hypoxia | Discard the preparation |
| Slow warping of imaged synapse | ‘Contraction clots’ in muscle fibers are forming as tissue has been mechanically damaged, suffered hypoxia or was exposed to phototoxicity | Discard the preparation. Reduce light exposure for new sample |
| Bleaching and phototoxicity | Light levels too high or exposure time too long | Reduce excitation light to lowest possible level, especially during search for suitable axons (dark-adapt your eyes before starting the experiment) |
| Undesired wavelengths (UV, infrared) present in excitation light | Check the presence of heat filters. Consider inserting extra UV-filters |