Data description KnifeEdgeTraces: Each folder contains the knife edge traces for one objective at a specific wavelength. This is the oscilloscope output, that is photodiode signal versus time. Data is text format, comma delimeted, 2 columns. Also files with extension .isf are text format. Each file is named a numeric value, (e.g. 3480.isf) which is the z-position of the knife edge in microns. First column time in seconds Second column Photodiode signal in Volts Each trace form a plot similar to fig. 1c in the paper. From this the 10/90 points can be retrieved and the beam diameter calculated as described in the experimental section in paper. These beam diameters can then be plotted versus z-position from the file name to obtain figure 3. The conversion factors from time to microns in the knife edge traces are: 5X 0.15NA 488 nm 23.12 mm/s 10x 0.4NA 488 nm 2.063 mm/s 20x 0.7NA 488 nm 1.947 mm/s 20x 0.7NA 800 nm 2.390 mm/s 40x 0.75NA 488 nm 21.62 mm/s 40x 1.3 NA 488 nm 2.036 mm/s 40x 1.3 NA 800 nm 2.316 mm/s Z-stacks: Each folder contains a stack of fluorescence images, corresponding to the supplementary movies 1, 2 and 3. Each image is marked on the image with the scalebar and z-position. The format of the images is RGB tiff. These are exported to tiff using LAS AF, Leica microsystems (control software for Leica microscopes) An .xlm file in the folder contains the information on microscope settings.