Supplementary Figure 1: Measured Point Spread Functions.

R-Z (lateral-axial) images of 200nm beads obtained with SLAP’s line scan (top left) and raster scan modes (top center). The full-width half-maximum extents of the PSFs are: Raster beam: 415 nm X, 1469 nm Z Line beam: 432 nm X, 1617 nm Z Without correction for the size of the bead. The corresponding diffraction limit for a point source is 377nm X, 1423nm Z (Zipfel et al., 2003). Line excitation results in slightly reduced axial sectioning because light is focused on only one lateral axis. As a result, the total intensity of the line focus decreases as 1/z away from the focal point, compared to 1/z² for the point focus, where z is axial distance from the focal plane. Subtraction of the raster and line PSF highlights the axial side lobes in the line PSF (top right). We incorporate a discrete convolution when using the image obtained with the raster beam to estimate measurements under the line beams, to account for these side lobes. The convolution kernel (example, bottom center) is estimated by deconvolving the line PSF with the raster PSF and constraining the solution (bottom left) to be positive and lie within the plane spacing of the reference image (0.75 µm). The raster PSF convolved with this kernel agrees well with the line PSF. Use of this model PSF improves agreement between expected and measured SLAP recordings (Supplementary Fig. 6j).