(A) Simplified optical diagram of W-4PiSMSN.

(B) Overview and cross-sections of the ER network in an immunolabeled COS-7 cell. Cross-sections of the W-4PiSMSN reconstruction show clearly separated membranes of the tubular structures, which cannot be resolved with conventional astigmatism-based nanoscopy (light blue frame).

(C) x-y slice through the mid-section of the ER network shown in (B) highlights the distinct membrane contour of ER tubules (arrowheads).

(D) Overview of immunolabeled microtubules in a COS-7 cell.

(E and F) 20-nm-thin x-y slices of the red (E) and green (F) segments shown in (D) demonstrate that microtubules can be easily resolved as hollow cylinders in W-4PiSMSN.

(G) A look through a 120-nm-long segment of the microtubule of (F).

(H) A histogram showing the number of localizations in a cross-section of the microtubule, white dotted box in (G).

(I) A bacteriophage reconstructed from 115 averaged viral particles rendered in 3D.

(J and K) 5-nm-thin vertical (J) and horizontal (K) slices through the averaged dataset corresponding to the planes shown in (I).

(L) The internal angle measurements of the hexagon shape identified from the viral capsid shown in (J).

OBJ, objective; QWP, quarter-wave plate; DM, dichroic mirror; QBF, quad-band band-pass filter; Def. Mirror, deformable mirror; Cam, camera; 50/50, beam splitter cube.