One of the patterns present on a PSFcheck calibration slide consists of a 3D array of small diffraction limited shell features separated 10 µm from each other. The fluorescent thickness of a shell is small compared to the PSF so that the average FWHM across several features was calculated to be of 208 nm in a SIM microscope. We imaged this shell pattern using widefield fluorescence excitation on a NanoImager-S (Oxford Nanoimaging Ltd), equipped with a 100X, 1.4 NA, oil-immersion objective (Olympus). The PSFcheck sample was excited with a 561 nm laser and the emitted fluorescence acquired in the Emission Filter 2: Band 1 575-616.5 and recorded on an sCMOS Hamamatsu Orca Flash 4.0 V3. Acquisition time = 33 ms, effective pixel size at object plane = 117 nm. Imaging of the PSFcheck pattern was done once.

{"references": ["Alexander D. Corbett, Michael Shaw, Andrew Yacoot, Andrew Jefferson, Lothar Schermelleh, Tony Wilson, Martin Booth, and Patrick S. Salter, \"Microscope calibration using laser written fluorescence,\" Opt. Express 26, 21887-21899 (2018)"]}