Research Article

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

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Science  24 Oct 2014:
Vol. 346, Issue 6208, 1257998
DOI: 10.1126/science.1257998

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  1. Fig. 1 Methods of light-sheet microscopy.

    (A) The traditional approach, where a Gaussian beam is swept across a plane to create the light sheet. a.u., arbitrary units. (B) A Bessel beam of comparable length produces a swept sheet with a much narrower core but flanked by sidebands arising from concentric side lobes of the beam. (C and D) Bound optical lattices (compare with movie S1) create periodic patterns of high modulation depth across the plane, greatly reducing the peak intensity and, as we have found, the phototoxicity in live cell imaging. The square lattice in (C) optimizes the confinement of the excitation to the central plane, and the hexagonal lattice in (D) optimizes the axial resolution as defined by the overall PSF of the microscope. The columns in (A) to (D) show the intensity pattern at the rear pupil plane of the excitation objective; the cross-sectional intensity of the pattern in the xz plane at the focus of the excitation objective (scale bar, 1.0 μm); the cross-sectional intensity of the light sheet created by dithering the focal pattern along the x axis (scale bar, 1.0 μm); and the xz cross section of the overall PSF of the microscope (scale bar, 200 nm). (E) Model showing the core of our microscope, with orthogonal excitation (left) and detection (right) objectives dipped in a media-filled bath (compare with fig. S4). (F) Higher magnification view, showing the excitation (yellow) and detection (red) light cones, which meet at a common focus within a specimen that is either mounted or cultured onto a cover glass within the media. The x, y, and z directions are indicated. The s-axis defines the direction the specimen moves from image plane to image plane. (G) Representation of a lattice light sheet (blue-green) intersecting a cell (gray) to produce fluorescence (orange) in a single plane. The cell is swept through the light sheet to generate a 3D image (compare with movie S2).

  2. Fig. 2 Experimental comparisons of Bessel beam and lattice light-sheet microscopy.

    (A) Three-dimensional renderings in the xy (top) and xz (bottom) directions of the 300th time point of a 4D data set of a living HeLa cell transfected with mEmerald-Lifeact, taken in the SIM mode with a five-phase hexagonal lattice at 7.5-s intervals. (B) First time point from a different live HeLa cell imaged with a stepped Bessel beam and five-phase SIM at 30-s intervals. Square inset shows the same cell, retracted at the 32nd time point (compare with movie S3). The coherent lattice light sheet generates a pattern of much greater modulation depth (compare with movie S4), requiring less power and generating less damage to produce an image of comparable SNR. It also allows finer, three-phase patterns to be used for SIM mode imaging as fast as 4 s per volume (compare with movie S5). (C) The 300th time point from a third HeLa cell, acquired with a hexagonal lattice in the dithered mode at 1.5 s intervals (compare with Movie 1). Scale bar, 5 μm. The low photoxicity of this mode permits even light-sensitive specimens such as D. discoideum to be imaged for long periods (compare with movie S7 and Movie 3). (D) First time point from a fourth HeLa cell, acquired with a swept Bessel beam at 1.5-s intervals. Circular insets in (A) to (D) show magnified xz views of filopodia. (E) OTFs in log scale showing amplitudes of spatial frequencies for the hexagonal lattice SIM mode used in (A). (F) OTFs for the Bessel SIM mode used in (B). (G) OTFs for the dithered hexagonal lattice used in (C). (H) OTFs for the swept Bessel mode used in (D). Green curves in (E) to (H) represent the diffraction limit of the detection. Blue curves in (E) to (H) represent the diffraction limit of the excitation.

  3. Fig. 3 Single-molecule tracking and superresolution.

    (A) Lattice light sheet 3D rendering of a ∼35-μm-diameter spheroid of mouse embryonic stem cells containing TMR-labeled Sox2 transcription factors. (B) Same spheroid, showing bleached region after single-molecule imaging with the light sheet dithered at a fixed plane (compare with movies S8 and S9). (C) Local apparent diffusion coefficients of Sox2 molecules. Scale bar, 5 μm. (D) MSDs of molecules in the nuclei (7608 trajectories) or cytoplasm (2339 trajectories) with error bars indicating the standard errors of the means. (E) Corresponding cumulative distribution functions (CDFs) representing the fraction of all tracked molecules at a given MSD or below. (F) Diffraction-limited maximum intensity projection (MIP) from a 600-nm-thick slab cut through the bottom of the nuclear membrane of a fixed U2OS cell expressing Dendra2–lamin A, taken in the dithered lattice mode. (G) Superresolution MIP from the same slab, taken with 3D PALM, where molecules in successive planes are excited with a dithered lattice light sheet. Scale bars, 2 μm in upper views of (F) and (G), 1 μm in zoomed boxes below. (H) Exploded view of the full 3D rendering of the nuclear envelope by PALM (compare with movie S10).

  4. Fig. 4 Intracellular dynamics in three dimensions.

    (A) Cells in prophase (left) and anaphase (right), showing histones and 3D tracks of growing microtubule ends, color-coded by velocity. Color-coding of each track by height (movie S11) or growth-phase lifetime (movie S12) is also possible. Each image in (A) represents a distillation of a few time points from a 4D, two-color data set typically covering hundreds of time points per cell (compare with (Movie 5). Graph shows the distribution of growth rates at different stages of mitosis, averaged across 9 to 12 cells (compare with figs. S8 and S9). (B) The 3D spatial relationship of histones (green), mitochondria (yellow), and ER (magenta) at four time points during mitosis in a slab extracted from a larger 4D, three-color data set of HeLa cells imaged for 300 time points (compare with Movie 6). (C) Volume renderings at eight consecutive time points of a single specimen of the protozoan T. thermophila taken from a 4D data set spanning 1250 time points (compare with Movie 7). Imaging at 3 ms per frame in a single plane (compare with movie S13) reveals the motions of individual cilia.

  5. Fig. 5 Imaging cell-cell and cell-matrix interactions.

    (A) T cell expressing mEmerald-Lifeact (orange) approaching a target cell expressing a plasma membrane marker fused to TagRFP (blue), as seen from the side. (B) Initial contact. (C) The two cells, after immunological synapse formation. These data represent three time points from a 4D, two-color data set spanning 400 time points. Scale bars, 4.0 μm. Other cell-cell interactions, such as the aggregation-starved D. discoideum cells, can likewise be studied for extended periods (compare with movie S14). (D) Neutrophil-like human HL-60 cell expressing mCherry-utrophin in a fluorescently labeled collagen matrix. (E) Volume renderings of the cell at three time points extracted from a 4D, two-color data set of the cell and the matrix covering 250 time points (compare with Movie 9). Scale bar, 5.0 μm. (F) Overlaid MIPs in three different colors of the collagen at the same three time points. Colored regions highlight local displacements of the matrix by the cell. Scale bar, 5.0 μm.

  6. Fig. 6 Embryogenesis in three dimensions.

    (A) Distribution of chromosomal passenger protein GFP–AIR-2 (green) relative to plasma membranes and histones (red) in C. elegans embryos at the two-cell (left, compare with fig. S12 and Movie 11) through six-cell developmental stages. Scale bar, 5 μm. Even late-stage development of C. elegans can be studied (compare with movie S15), although muscle contractions make it difficult to follow specific features continuously. (B) xy view MIPs of the distribution of Lifeact in a C. elegans embryo during pseudocleavage ingression (left), in maintenance phase just before the first division (center), and during the first division (right), extracted from a 4D data set covering 368 time points (compare with fig. S13 and Movie 12). Scale bar, 5 μm. (C) Volume rendering of DE-cadherin during dorsal closure in Drosophila development, highlighting boundaries between amnioserosa cells and the formation of spiracles, from a 4D data set containing 840 time points (compare with movie S16); bounding box, 86 μm by 80 μm by 31 μm. (D) Myosin II during dorsal closure, highlighting the myosin-rich purse string at the interface between the outer epithelium and the amnioserosa, from a 4D data set covering 1000 time points (compare with Movie 13); bounding box, 80 μm by 80 μm by 26 μm. (E) sGMCA during dorsal closure, from a 4D data set covering 639 time points (compare with movie S17); bounding box, 73 μm by 73 μm by 26 μm. (F) MIPs highlighting cortical actin at the apical and basal surfaces of the amnioserosa, extracted from (E). Scale bar, 10 μm.