Supplementary Materials

Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution

Bi-Chang Chen, Wesley R. Legant, Kai Wang, Lin Shao, Daniel E. Milkie, Michael W. Davidson, Chris Janetopoulos, Xufeng S. Wu, John A. Hammer III, Zhe Liu, Brian P. English, Yuko Mimori-Kiyosue, Daniel P. Romero, Alex T. Ritter, Jennifer Lippincott-Schwartz, Lillian Fritz-Laylin, R. Dyche Mullins, Diana M. Mitchell, Joshua N. Bembenek, Anne-Cecile Reymann, Ralph Böhme, Stephan W. Grill, Jennifer T. Wang, Geraldine Seydoux, U. Serdar Tulu, Daniel P. Kiehart, Eric Betzig

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

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  • Supplementary Text
  • Figs. S1 to S26
  • Table S1
  • Full Reference List

Images, Video, and Other Other Media

Movie S1
Transition from an infinite 2D optical lattice to an axially bound lattice light sheet, as seen in views of the illumination pattern in the rear pupil of the excitation objective (left) and the cross-sectional intensity profile of the resulting light sheet at the specimen (right).
Movie S2
Animation of the image acquisition process, showing a lattice light sheet (blue/green) intersecting a cell (gray) to produce fluorescence (orange). As the cell is swept through the light sheet, a series of 2D images of the fluorescence are recorded to build up a 3D image of the cell. The lattice is dithered in its patterned direction to mimic a continuous light sheet.
Movie S3
Top and side view volume renderings of HeLa cells expressing mEmerald-Lifeact, demonstrating the higher speed and reduced phototoxicity of the lattice light (right) compared to Bessel beam plane illumination (left) when each is applied to super-resolution structured illumination microscopy (SR-SIM).
Movie S4
Raw 2D image frames during acquisition of a 3D data volume in the SR-SIM mode for two HeLa cells expressing mEmerald-Lifeact, demonstrating the higher modulation amplitude and lower background obtained with a lattice light sheet (right) as opposed to a stepped Bessel beam (left).
Movie S5
Side view volume rendering of filopodia dynamics at 4.0 sec intervals on the dorsal surface of a HeLa cell expressing mEmerald-Lifeact, acquired in the SR-SIM mode using a three phase hexagonal lattice light sheet.
Movie S6
Rapid muscle contractions in a C. elegans embryo in the three-fold stage, with labeled GFP-PH domains (green) and mCherry-histones (magenta), as recorded in a single 2D optical section at 50 frames/sec. Scale bar, 10 μm.
Movie S7
Volume rendering of GFP-dajumin in a D. discoideum cell, highlighting the rapid dynamics of the osmoregulatory contractile vacuole network over 400 time points at 0.6 sec intervals. A slight rocking is present in the viewer perspective during the time lapse to illustrate 3D depth.
Movie S8
Single molecule tracking of TMR-labeled Sox2 transcription factors in an ~35 μm diameter spheroid of mouse embryonic stem cells, as seen by a dithered lattice light sheet at a fixed plane (top), a single Bessel beam scanned across the same plane (middle), and widefield, epi-illumination across the entire specimen (bottom).
Movie S9
SNR as a function of exposure time when tracking single TMR-labeled Sox2 transcription factors in one plane across a stem cell spheroid illuminated by a dithered lattice light sheet.
Movie S10
Orthoslices and volume renderings of the nuclear envelope of a U2OS cell labeled with Dendra2-lamin A as imaged by lattice-based super-resolution 3D PALM (left) and diffraction-limited lattice light sheet microscopy (right).
Movie S11
Maximum intensity projections and measured GFP-EB1 trajectories for a single HeLa cell in interphase. Growing microtubule endpoints and tracks are color coded by z-position above the substrate. Bounding box, 47 × 50 × 11 μm.
Movie S12
Maximum intensity projections and measured GFP-EB1 trajectories for a single HeLa cell in metaphase. Growing microtubule endpoints and tracks are color coded by growth phase lifetime. Bounding box, 29 × 36 × 26 μm.
Movie S13
Imaging T. thermophila expressing GFP-scramblase in a single 2D plane at 3 ms intervals reveals the motions of individual cilia (cf., fig. S10). Magnified view is shown at right.
Movie S14
Top and oblique view volume rendering of the mutual chemotaxis of starved D. discoideum cells expressing RFP-LimE, marking their transition from unicellular to multicellular morphology (cf., fig. S11).
Movie S15
Volume rendering of GFP-PH domains (green) and mCherry-H2B (orange) in a C. elegans embryo at the three-fold stage, after the onset of muscle contractions. Bounding box, 41 × 39 × 60 μm.
Movie S16
Volume rendering of the amnioserosa and the encroaching epithelium during dorsal closure in a D. melanogaster embryo expressing GFP/DE-cadherin to highlight cell-cell junctions, over 840 time points at 8 sec intervals (cf., Fig. 6C). Bounding box, 86 × 80 × 31 μm.
Movie S17
Various views of dorsal closure in a D. melanogaster embryo expressing sGMCA, a marker of filamentous actin at the plasma membrane, over 639 time points at 12.0 sec intervals. Top left: orthoslice through the center of the amnioserosa. Top right: maximum intensity projection through a thin rectangular slab encompassing the apical surface of the amnioserosa. Bottom right: MIP through a similar slab encompassing the basal surface of the amnioserosa. Bottom left, volume rendering, with the apical surface in blue/green and the basal surface in orange/red (cf., Fig. 6E, F). Bounding box, 73 × 73 × 26 μm.
Movie S18
Rear pupil excitation pattern (left) and cross-sectional intensity at the sample (right) for a linear array of mutually interfering Bessel-Gauss beams, as a function of the period of the array. Top: all beams have the same phase; Bottom: adjacent beams have opposite phase.