Extended Data Fig. 1: INSPR framework, degeneracy illustration, and setup diagram.
From: Three-dimensional nanoscopy of whole cells and tissues with in situ point spread function retrieval
a, INSPR framework and detailed process of in situ model generation. b, Single molecules are localized by a pair of channel-specific models which share the same shape information with the corresponding sub-regions. c, Degeneracy exists in single plane configuration, where PSFs with positive and negative vertical astigmatism aberrations (Ast) are identical at opposite axial positions. d, Degeneracy is broken in biplane configuration, where PSF pairs with positive and negative vertical astigmatism aberrations are different at opposite axial positions. e, Degeneracy is absent in single plane configuration with prior knowledge of astigmatism orientation, where PSFs with additional positive and negative primary spherical aberrations (1st Sph) are different at opposite axial positions. Scale bar in (c–e): 1 μm. f, Setup diagram. M1–M8: mirrors in the excitation path; Di1–Di3: dichroic mirrors; AOTF: acousto-optic tunable filter; L1–L5: lenses in the excitation path; FM: flip mirror; MLS: mercury light source; Obj: objective lens; M1’–M11’: mirrors in the emission path; TL: tube lens; L1’–L6’: lenses in the emission path; DM: deformable mirror; BS: 50/50 non-polarizing beam splitter; SM: 90° specialty mirror; FW: filter wheel. Nominal focal lengths of lenses are, L1: 19 mm, L2: 19 mm, L3: 20 mm, L4: 125 mm, L5: 400 mm, Obj: 1.8 mm, TL: 180 mm, L1’: 88.9 mm, L2’: 250 mm, L3’: 400 mm, L4’: 150 mm, L5’: 500 mm, L6’: 250 mm. g, Definition of biplane distance. The objective lens is moved axially to make plane 1 (case 1) and plane 2 (case 2) in focus successively. The axial movement is defined as biplane distance δ.
