Fig. 3.

Change of bead intersection length as a measure of three-dimensional scan instability. (A) Three-dimensional projection of z-stack data (maximum intensity projections of z-stack intensities in XY, XZ and YZ plane) and spiral scan trajectory (white dashed line). Positions of intersected fluorescent beads, shown in B, are indicated by numbers (1 – 5). Cube dimensions: 500 × 500 × 200 µm³ (XYZ). (B) Examples of bead intensity profiles at different positions in spiral trajectory, scanned over 10 minutes. 10 pixels ≙ 10 µm. (C) Change of intersection length plotted versus time. The dotted line indicates no detected change. Note that no change in length not necessarily indicates position stability as the intersection of a sphere (bead) is not defined uniquely.