Supplementary Figure 1: Diagnostics for assessing excitation input to diSPIM.

a. After projecting the excitation beam through the scanner onto a far screen or wall (approximately three feet from the output of the scanner, without mounting the tube lens), the beam should fill the image of the 2D MEMS mirror (right). An example of a misaligned beam is shown at left: note that the MEMS mirror is underfilled and the beam spills off the MEMS aperture. b. When checking centration and collimation of the beams through the scanner + tube lens assemblies, we mount them together on the bracket (ASI, Cat. # B1013 & B1034) as shown. c. Attaching a circular piece of white paper (2 inches in diameter) to the output of each tube lens is helpful in assessing beam centration. d. When properly collimated, both beams should maintain their size, and appear the same size at a screen placed far from the tube lens face (here a wall 12 feet from the output of each tube lens was used to assess mutual beam size).