Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution without inducing undue stress on either. We combined lattice light sheet microscopy with two-channel adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages, and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments. One Sentence Summary Combining lattice light sheet microscopy with adaptive optics enables high speed, high resolution in vivo 3D imaging of dynamic processes inside cells under physiological conditions within their parent organisms.

[1]  Stephan Saalfeld,et al.  Globally optimal stitching of tiled 3D microscopic image acquisitions , 2009, Bioinform..

[2]  M. Mione,et al.  Novel transgenic lines to fluorescently label clathrin and caveolin endosomes in live zebrafish. , 2015, Zebrafish.

[3]  P. Strzyz,et al.  Interkinetic nuclear migration is centrosome independent and ensures apical cell division to maintain tissue integrity. , 2015, Developmental cell.

[4]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[5]  L. A. Lowery,et al.  The trip of the tip: understanding the growth cone machinery , 2009, Nature Reviews Molecular Cell Biology.

[6]  Anna Huttenlocher,et al.  Neutrophil migration in infection and wound repair: going forward in reverse , 2016, Nature Reviews Immunology.

[7]  Jing Yang,et al.  Visualizing extravasation dynamics of metastatic tumor cells , 2010, Journal of Cell Science.

[8]  J. Sanes,et al.  Improved tools for the Brainbow toolbox. , 2013, Nature methods.

[9]  T. Kirchhausen,et al.  Cisternal organization of the endoplasmic reticulum during mitosis. , 2009, Molecular biology of the cell.

[10]  Omar E. Olarte,et al.  Measurement and correction of in vivo sample aberrations employing a nonlinear guide-star in two-photon excited fluorescence microscopy , 2011, Biomedical optics express.

[11]  A. Kinkhabwala,et al.  A structural and functional ground plan for neurons in the hindbrain of zebrafish , 2011, Proceedings of the National Academy of Sciences.

[12]  Ian A. Swinburne,et al.  Improved Long-Term Imaging of Embryos with Genetically Encoded α-Bungarotoxin , 2015, PloS one.

[13]  Michael Sixt,et al.  Breaching multiple barriers: leukocyte motility through venular walls and the interstitium , 2010, Nature Reviews Molecular Cell Biology.

[14]  F. C. Sauer.  Mitosis in the neural tube , 1935 .

[15]  Martin J. Booth,et al.  Modelling of multi-conjugate adaptive optics for spatially variant aberrations in microscopy , 2013 .

[16]  Michael M. Kazhdan,et al.  Distributed gradient-domain processing of planar and spherical images , 2010, TOGS.

[17]  Jonathan M. Taylor,et al.  3D adaptive optics in a light sheet microscope. , 2012, Optics express.

[18]  Alexandra Bokinsky,et al.  Dual-view plane illumination microscopy for rapid and spatially isotropic imaging , 2014, Nature Protocols.

[19]  Randal C. Burns,et al.  Gradient-Domain Fusion for Color Correction in Large EM Image Stacks , 2015, ArXiv.

[20]  M. Poo,et al.  Essential role of filopodia in chemotropic turning of nerve growth cone induced by a glutamate gradient , 1996, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[21]  Wesley R. Legant,et al.  Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution , 2014, Science.

[22]  Na Ji.  Adaptive optical fluorescence microscopy , 2017, Nature Methods.

[23]  D. Bentley,et al.  Disoriented pathfinding by pioneer neurone growth cones deprived of filopodia by cytochalasin treatment , 1986, Nature.

[24]  Wesley R. Legant,et al.  Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy , 2016, Molecular biology of the cell.

[25]  D. Milkie,et al.  Rapid Adaptive Optical Recovery of Optimal Resolution over LargeVolumes , 2014, Nature Methods.

[26]  Philipp J. Keller,et al.  Whole-brain functional imaging at cellular resolution using light-sheet microscopy , 2013, Nature Methods.

[27]  Michel Verhaegen,et al.  Adaptive illumination based on direct wavefront sensing in a light-sheet fluorescence microscope. , 2016, Optics express.

[28]  C. Holt,et al.  Navigational errors made by growth cones without filopodia in the embryonic xenopus brain , 1993, Neuron.

[29]  Kishore Mosaliganti,et al.  ACME: Automated Cell Morphology Extractor for Comprehensive Reconstruction of Cell Membranes , 2012, PLoS Comput. Biol..

[30]  Philipp J. Keller,et al.  Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy , 2012, Nature Methods.

[31]  F. Brodsky,et al.  Actin scaffolding by clathrin heavy chain is required for skeletal muscle sarcomere organization , 2014, The Journal of cell biology.

[32]  W. Southwell.  Wave-front estimation from wave-front slope measurements , 1980 .

[33]  Jan Huisken,et al.  Multi-view image fusion improves resolution in three-dimensional microscopy. , 2007, Optics express.

[34]  Kartik Chandran,et al.  Endocytosis by Random Initiation and Stabilization of Clathrin-Coated Pits , 2004, Cell.

[35]  M. Davidson,et al.  Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics , 2015, Science.

[36]  A. Ridley,et al.  Crossing the endothelial barrier during metastasis , 2013, Nature Reviews Cancer.

[37]  Philipp J. Keller,et al.  Whole-animal functional and developmental imaging with isotropic spatial resolution , 2015, Nature Methods.

[38]  Stephanie J. Spielman,et al.  Regulation of clathrin-mediated endocytosis by hierarchical allosteric activation of AP2 , 2017, The Journal of cell biology.