Figure 10.

Troubleshooting – examples of drifting in either the Z-axis or Y-axis of the field of view

Drifting can happen due to slow or fast bath flow or due to tissue bulging from cortex breakdown. Examples are provided for both types of drift.

Arrows are placed either at the 60-minute mark (Z-axis example) or at the injection timepoint (Y-axis examples), and are not moved in subsequent timepoints, demonstrating the change in cell soma locations that drift away from arrowhead points.

In the Z-axis, the tissue is drifting out of the plane of view, so the focal plane is diving deeper into the tissue, causing the loss of visibility of imaged processes.

This also explains the cell somas disappearing over the course of one hour.

The Y-axis drift example has the tissue drifting down within the frame, meaning the cell somas are slowly moving down compared to the arrowheads.

Scale bars represent 50 μm.