Fig. 1: 4Pi-SIM principle and resolution characterization.
From: Elucidating subcellular architecture and dynamics at isotropic 100-nm resolution with 4Pi-SIM
a, Simplified schematic of 4Pi-SIM (top). The illumination beams are shown in blue, and the fluorescence emission is shown in green. Six partially coherent illumination beams interfere at the sample plane. Two fluorescence emission beams are coherently combined at the NPBS to generate two phase-complementary images (the xy and xz cross-sections are shown). In the frequency domain, the illumination pattern of one lateral SIM orientation consists of 19 illumination components (bottom left), and the detection OTF extends axially (bottom middle). Convolution of the two components yields an effective 4Pi-SIM OTF for one lateral SIM orientation (bottom right). OBJ1 and OBJ2, objectives 1 and 2; L1–L4, lens 1 through lens 4; M1 and M2, mirrors 1 and 2; initial phase, φ0; π, constant phase difference between the two phase-complementary interference images. b, A lateral cross-section image of a field of 100-nm beads vertically divided into three sub-fields, showing the difference in lateral resolution of WF (top), 3D-SIM (middle) and 4Pi-SIM (bottom). c, Axial cross-sections along the vertical dashed line in b, showing the difference in axial resolution. d, Experimentally measured OTFs of WF (top), 3D-SIM (middle) and 4Pi-SIM (bottom). e, Axial intensity profiles of the bead highlighted by arrowheads in c, demonstrating the optical sectioning capability of 3D-SIM and 4Pi-SIM and the improved axial resolution. a.u., arbitrary units. f, Resolution quantification with 100-nm fluorescent beads. The FWHMs were measured on 150 beads in 3 independent experiments for each imaging mode. WF: 246.7 ± 4.3 nm laterally, 561.6 ± 10.9 nm axially; 3D-SIM: 107.7 ± 4.7 nm laterally, 322.9 ± 16.9 nm axially; 4Pi-SIM: 108.1 ± 4.0 nm laterally, 102.2 ± 4.6 nm axially. g, Resolution quantification with immunolabeled microtubules in fixed COS-7 cells. The FWHMs were measured at 75 locations in 3 independent experiments for each imaging mode. WF: 254.5 ± 18.7 nm laterally, 584.5 ± 29.1 nm axially; 3D-SIM: 105.4 ± 7.5 nm laterally, 320.7 ± 17.6 nm axially; 4Pi-SIM: 104.4 ± 5.0 nm laterally, 101.7 ± 6.2 nm axially. In f and g, statistical data are presented as mean ± s.d. Scale bars, 500 nm (a,c); 1 µm (b); 1/200 nm–1 (d). Whiskers: 75% and 25% percentiles; centerlines: medians. All cross-section slices are 30 nm thick.
