Supplementary Figure 11: Multi-spot holographic illumination and diffraction efficiency correction

(a) Max projection of the fluorescence generated by a sequence of 15 µm diameter holographic spots placed on a square grid. The generation of a variable size (from 0 to 15 µm) correction holographic spot (not shown) is applied to correct for the position-dependent diffraction efficiency (see Methods). Scale bar: 30 µm. (b) Vertical and horizontal intensity profiles corresponding to panel a with and without the correction of diffraction efficiency, averaged over the 5 columns and lines of the matrix, respectively. (c) Fluorescence generated by the simultaneous projection of 7 randomly placed holographic spots with equal intensity, corrected for diffraction efficiency. Scale bar: 30 µm. (d) Graded input pattern used to calculate the hologram generating the illumination pattern in panel c (see Methods). Scale bar: 30 µm. (e) Axial intensity profile of the fluorescence induced by a 10 µm holographic spot displaced over different axial positions relative to the microscope objective focal plane without diffraction efficiency correction (black curves) and with the correction (red curve) using an external correction spot (not shown). (f) Superposition of the axial intensity profiles of panel e (with correction of diffraction efficiency), after being centered at z = 0. The black curve represents the average profile with FWHM = 18.5 µm. (g) Axial intensity profile of the multispot holographic pattern reported in Figure 5b. The black curve represents the average profile with FWHM = 17.5 µm. (h) Lateral view of the 3D fluorescence pattern generated by 3 holographic spots (diameter 10 µm) of equal intensity at axial positions of z = -120 µm; 0; + 120µm. Scale bar: 30 µm. (i) Graded input pattern used to calculate the hologram generating the illumination pattern in panel h. As in panel d, different input intensities were assigned to each spot to compensate for the diffraction efficiency dependence on the vertical position and to obtain the same output power density on each spot. (j) Superposition of the axial intensity profiles of the spots of panel h. The black curve represents the average profile with FWHM = 18 µm.