Extended Data Fig. 1: Axial calibration of electrically-tunable lens.

a. A change in the current input to the lens generates a curvature change in the lens, which alters the focus. b. Synchronization of ETL focus change with microscope scanning. A TTL pulse is generated at the beginning of each frame from the PCIe board in the computer controlling the microscope (top left). An Arduino Uno was programmed to filter all pulses besides the first of the stack (middle left). This pulse was then sent to the ETL controller to prompt a predetermined set of current steps that were sent to the ETL (bottom left). These current changes created a rapid stack with each depth captured as a single frame (right). c. The point spread function in the X (left) and Z (right) directions of the two-photon microscope created with a 0.17 µm fluorescent microsphere and a 0.8 NA N16XLWD-PF 16x Nikon objective. The ETL obscures part of the back aperture, resulting in a lower effective NA. d. Calibration of the ETL focal range. To provide a fluorescent three-dimensional structure, cotton stands were dipped in a solution of fluorescein isothiocyanate and suspended in optical adhesive within a concave slide. e. Three-dimensional segmentations created using fluorescent cotton strands from three locations (left to right). f. Calibration of the ETL diopter shifts to focal plane shifts. Three locations in the cotton (shown in e) were imaged by shifting the ETL focus and by translating the object in Z and aligned by correlational matching of images (top). These averages are plotted for each location in colored lines with the shaded standard deviation. The linear regression is also plotted as a solid blue line, with zero µm being the focus neutral diopter value (bottom). g. From top to bottom, change in X and Y scaling and laser power as a function of diopter value. Changing the diopter of the ETL had negligible changes in magnification and laser power output in the typical imaging range.