Supplementary Materials

Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics

Dong Li, Lin Shao, Bi-Chang Chen, Xi Zhang, Mingshu Zhang, Brian Moses, Daniel E. Milkie, Jordan R. Beach, John A. Hammer III, Mithun Pasham, Tomas Kirchhausen, Michelle A. Baird, Michael W. Davidson, Pingyong Xu, Eric Betzig

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Supplementary Text
  • Figs. S1 to S55
  • Table S1 and S2

Images, Video, and Other Other Media

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Movie S1
Phototoxicity of WF-RESOLFT vs. PA NL-SIM. Left: Behavior over time of a U2OS cell expressing Skylan-NS-Lifeact, at the excitation intensity and exposure time used by WF-RESOLFT (11). Right: Behavior of a similar cell under excitation and exposure conditions typical of PA NL-SIM.
Movie S2
Cytoskeletal attachment at focal adhesions in a live U2OS cell at 37°C. Dynamics of filamentous actin (mApple-F-tractin, magenta) and its interaction with mEmerald-paxillin (green) at focal adhesions, at 20 sec intervals for 81 time points, as seen by high NA TIRF-SIM.
Movie S3
Dynamics of clathrin-medited endocytosis of transferrin receptors at 37°C. High NA TIRF-SIM for 28 frames at 5 sec intervals of the interaction of clathrin coated pits (green) and transferrin cargo (magenta) in MEF cells labeled with mEmerald-CLTB and transferrin conjugated to Alex568.
Movie S4
Dynamics of clathrin coated pits at 37°C. Formation, maturation, and internalization of clathrin coated pits in a live BSC-1 cell expressing EGFP-CLTA as seen by high NA TIRF-SIM for 150 time points at 0.8 sec intervals.
Movie S5
Actin-clathrin interactions in a second cell. High NA TIRF-SIM for 181 frames at 5 sec intervals in another COS-7 cell at 37°C expressing mEmerald-CLTA (green) and mCherry-Lifeact (orange-red).
Movie S6
Molecular states during the phases of nonlinear SIM. Illustration of the sequential steps of activation, depletion, and read-out in three forms of nonlinear SIM: patterned activation, saturated patterned activation, and saturated depletion.
Movie S7
Actin dynamics as seen by SD NL-SIM. Dynamics of the actin cytoskeleton over 12 time points at 20 sec intervals by nonlinear SIM with saturated depletion, in a COS-7 cell at 23°C expressing Skylan-NS-Lifeact. The rapid decline in SNR compared to movie S11 is indicative of the relative photon inefficiency of SD NL-SIM compared to PA NL-SIM.
Movie S8
Actin rings and cytoskeletal dynamics as seen by PA NL-SIM. A COS-7 cell at 23°C expressing Skylan-NS-Lifeact as seen over 34 frames at 18 sec intervals by PA NL-SIM.
Movie S9
Actin dynamics as seen by high NA PA NL-SIM. Dynamics of the actin cytoskeleton over 26 time points at 15 sec intervals as seen by patterned activation nonlinear SIM, in a COS-7 cell at 37°C expressing Skylan-NS-Lifeact.
Movie S10
Actin dynamics as seen by high NA PA NL-SIM in a second cell. Dynamics of the actin cytoskeleton over 40 time points at 15 sec intervals as seen by patterned activation nonlinear SIM, in a COS-7 cell at 37°C expressing Skylan-NS-Lifeact.
Movie S11
Dynamics of keratin as seen by PA NL-SIM. Progressive resolution gain in a live COS-7 cell at 23°C expressing Skylan-NS-keratin from diffraction-limited TIRF, to TIRF with deconvolution, linear TIRF-SIM, and nonlinear SIM with patterned activation (PA NL-SIM), followed by the dynamics of keratin for 11 frames at 20 sec intervals by PA NL-SIM.
Movie S12
Dynamics of caveolae as seen by different methods. Caveolae in a COS-7 cell at 37°C expressing Skylan-NS-caveolin for 21 time points at 3 sec intervals as seen by diffraction-limited TIRF, TIRF with deconvolution, linear TIRF-SIM and PA NL-SIM.
Movie S13
Caveolae dynamics as seen by saturated PA NL-SIM. Caveolae in a COS-7 cell at 23°C expressing Skylan-NS-caveolin for 12 time points at 3 sec intervals as seen by TIRF with deconvolution, linear TIRF-SIM, PA NL-SIM, and saturated PA NL-SIM.
Movie S14
Cortical actin and early endosomes by TIRF-SIM and PA NL-SIM in a second cell. A COS-7 cell at 23°C expressing expressing Skylan-NS-Lifeact (orange-red) as imaged by PA NL-SIM and mCherry-Rab5a (green) as imaged by TIRF-SIM, both over 28 frames at 15 sec intervals.
Movie S15
Dynamic interactions of f-actin and ??-actinin by two-color combined TIRF-SIM and PA NL-SIM. A COS-7 cell at 23°C expressing expressing Skylan-NS-Lifeact (green) as imaged by PA NL-SIM and mCherry-??-actinin (magenta) as imaged by TIRF-SIM, both over 24 frames at 20 sec intervals.
Movie S16
3D dynamics of actin by lattice light sheet PA NL-SIM. 2D xy and xz scrolling orthoslice views at one time point, followed by the 3D evolution of the actin cytoskeleton over 18 time points at 2 min intervals, in a U2OS cell at 23°C expressing Skylan-NS-Lifeact.
Movie S17
3D dynamics of the Golgi apparatus by lattice light sheet PA NL-SIM. 3D evolution of a volume-rendered representation of the Golgi apparatus over 42 time points at 95 sec intervals, in a U2OS cell at 23°C expressing Skylan-NS-MannII, showing nuclear-facing hollowing structure and surrounding vesicles.
Movie S18
3D color-coded representation of Golgi dynamics by lattice light sheet PA NL-SIM. 3D evolution of a the Golgi apparatus, color coded by height above the cover slip, over 42 time points at 95 sec intervals, in a U2OS cell at 23°C expressing Skylan-NS-MannII.