Lateral alignment of image panels. To further analyze the image data, the nine z-planes recorded for each time point need to be aligned along the optical axis. On the left we show the initial alignment of the nine z-planes. White represents full overlap and green and violet indicate mismatches. On the right we show the alignment of the nine planes after lateral alignment as described in Fig. 2. This alignment is done for each time point in the movies we acquired. The alignment signal is a DNA stain with Life cycle dye Vybrant DyeCycle Violet (Life Technologies). This dye, if excited at 405 nm, emits in both the green and red channel. Images were recorded on a custom-built MFM using a 60×, 1.3 NA, 1.405 RI silicone immersion objective (Olympus); a total magnification of 80× after the MFM unit; and a 1k, back-illuminated EMCCD with 8-µm-pixel size (model 888, built in 2011; Andor Technology). Alignment was done using Fiji and custom-written code as available here.
