Analysis of ER tubules, sheets, and nanoholes with superresolution microscopy. (A) Plot of calculated PSF FWHM of STED microscope. Measurements taken from two cells per day; n = 4 imaging days; each point is from n = 50 profiles. FWHM, mean ± SD. (B and C) Live STED images of ER tubules labeled with SNAP-Sec61β. Arrowheads mark ER lumen. Dashed rectangle represents region used to generate fluorescence intensity line profiles to calculate tubule diameters in D. (D) Histogram of ER tubule diameters in living cells. n = 300 tubule profiles; n = 3 imaging days. 50 SNAP-Sec61β-labeled tubule profile measurements taken from each cell; two cells per day; diameter = mean ± SD. (E–G) Overview and cross sections of ER network in fixed COS-7 cell expressing GFP-Sec61β imaged with 4Pi-SMSN. Regions marked by dashed lines in xy view in F are shown as xz views of 20-nm-thick y slices shown in G and I. (H) Histogram of ER tubule diameters labeled with SNAP-Sec61β in fixed cell. n = 45 tubule profiles. Mean ± SD is shown. (I) xz view of 20-nm thick y slices of regions marked by blue dashed line in F. (J–N) Overview and magnified STED images of ER network. Orange line in K and M represents 5-pixel-wide fluorescence intensity line profile shown in L and N. Magenta arrows mark ER tubules, green bars mark uniform ER sheets, and cyan arrows mark ER regions flanking nanoholes. Line profiles in L and N are from raw image (black dots), and image was smoothed with a uniform 3 × 3 filter (orange line). Traces are from one cell.