Vortex phase plate alignment

The vortex phase plate or spiral phase plate is the same phase profile used to create high-quality donut-shaped depletion and excitation profiles from Gaussian laser beams for STED³⁷ and MINFLUX⁷¹. Coincidentally this is also the lowest order component of a Double-helix PSF⁷², and a discretized version of a spiral phase plate with only 3-4 phase-steps could be used to generate a rotating PSF⁷³. Here we use a vortex phase plate with a single spiral from 0 to 2π in 64 steps (V-593-10-1, vortex photonics).

A 1:1 optical relay is built on the emission path of the microscope to place the vortex phase plate in a plane conjugate to the back focal plane of the objective as illustrated in Supplementary Fig. ¹⁷. The phase plate is placed roughly halfway between the two relay lenses. To align the vortex phase plate, defocused images are taken of 1 μm beads (TetraSpeck Fluorescent Microspheres Size Kit, ThermoFisher) and diagonally opposing regions are recorded (Supplementary Fig. ^18a). If the vortex phase plate is aligned properly, all the PSFs should have the same shape throughout the FOV as shown in Supplementary Fig. ^18b. When the vortex phase plate is not in the correct axial position the PSF will vary over the field of view. This is because light from different areas in the sample does not pass through the vortex phase plate in the same place as it is not conjugate to the back focal plane of the objective. The main parts of the PSF to observe for alignment is the peak in the center that does not move when the phase plate is moved and a ring that moves with the phase plate. The process of aligning the vortex phase plate involves moving the rings to be centered over the peaks over the entire FOV. The vortex phase plate should be translated along the optical axis until all the beads look the same throughout the field of view (same offset between the peak and the ring throughout the FOV) (Supplementary Fig. ^{18c, d}). Thereafter the vortex phase plate can be shifted horizontally and vertically so the dark spot overlaps with the center of the bead (Supplementary Fig. ^{18e, f}). Lastly the beads are translated along the optical axis and defocused in the opposite direction to verify that the rings also overlap with the center of the bead there. If the rotation direction of the vortex phase plate is unknown (K(ρ) = β/(2π) or K(ρ) = −β/(2π)), then fixed single molecules can be fitted with both orientations and the correct setting will have a visually better fit (should be especially evident on molecules with θ = ±45°).