Fig. 5: Optimization of excitation laser polarization for single and multi-focal microscopy.

a–c Epifluorescence microscopy. Excitation and detection light travel through the same objective. Horizontally polarized excitation laser (a) vertically polarized excitation laser (b) circularly polarized excitation laser (c). d–f Example image of equatorial plane of a giant unilamellar vesicle (GUV) taken with epifluorescence microscopy. GUV consists of an unsaturated phospholipid (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), is labeled with Fast-DiO and excited with 950 nm. Horizontally polarized (d) vertically polarized (e) circularly polarized, simulated image (f). Scale bar, 10 µm. g, h Multifocal orthogonal LSFM. Excitation and detection light travel through different objectives. Horizontally polarized excitation laser (g) vertically polarized excitation laser (h). i, j Example image of a 7-day post-fertilization zebrafish midbrain expressing pan-neuronal H2B::jGCaMP7f¹⁹⁴ taken with orthogonal LSFM. The sample is illuminated by a single lightsheet from bottom of the image at 920 nm with 175 mW of power. Example image is averaged across 30 s showing a single z-plane of a volumetric time series taken at 1 volume per second. Scale bar, 100 µm. a–c, g, h Green arrows show one possible radial emission direction; Green ellipsoid has the highest fluorescence emission probability perpendicular to the dipole moment, at the ellipsoid’s equator. Circularly polarized light was adapted from “Clockwise circularly polarized light” by Dave3457, Wikimedia Commons, licensed under CC BY-SA 4.0.