Fig. 2

a Schematic (top) and photograph (bottom) of the setup used to directly film excitation volumes. IR, infrared. b effective scan-planes directly visualised as indicated in (a) for all optical configurations as indicated, in each case with scan-points spaced to facilitate inspection of individual PSFs. c–e PSF measurements using fluorescent beads (cf. Fig. 1m) across the full FOV for all optical configurations as indicated in (c). Shown are exemplary scan profiles (pairs of xz and yz projections) from (d) and (e) quantification of full width half maxima (FWHM) In lateral (xy) and axial dimensions (z). For statistical evaluation we compared each “lateral” PSF estimates to the respective centre estimates using 1-way ANOVA. No significant differences were detected, except in two top-right (TR) xy values for nTC₂ conditions, as indicated. These small differences are likely explained by slightly imperfect laser centring. Error bars in mean ± s.d., n = 20 experimentally independent samples per measurement. TL, top left, TR, top right, BL, bottom left, BR, bottom right. f–h, example scan under nTC₂ 3.5 mm configuration of a mouse brain slice labelled with SMI31 for phosphorylated Neurofilament-H (NFH). Full usable FOV (f) and two zoomed in regions (g, h) as indicated in (f), without moving the objective, to illustrate image quality at the FOV edges (f–h, 1024 × 1024 px, 0.49 Hz). Data leading to e in Source data file.