Menu

Close

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

Tsung-li Liu, Srigokul Upadhyayula, Daniel E Milkie, Ved Singh, Kai Wang, Ian A Swinburne, Kishore R Mosaliganti, Zach M Collin, Tom W Hiscock, Jamien Shea, Abraham Q Kohrman, Taylor N Medwig, Daphne Dambournet, Ryan Forster, Brian Cunniff, Yuan Ruan, Hanako Yashiro, Steffen Scholpp, Elliot M Meyerowitz, Dirk Hockemeyer, David G Drubin, Benjamin L Martin, David Q Matus, Minoru Koyama, Sean G Megason, Tom Kirchhausen, Eric Betzig

Preprint posted on January 08, 2018 https://www.biorxiv.org/content/early/2018/01/07/243352

Pushing the boundaries of light sheet microscopy to image subcellular processes in intact living organisms.

Selected by Arnaud Monnard, Gautam Dey

Context

The recent development of live cell super-resolution microscopy1 and, subsequently, of light sheet microscopy2, have dramatically increased our ability to probe living cells and organisms. Light sheet microscopy has rapidly gained popularity within the scientific community, in particular for its low phototoxicity and ability to image large samples. It thereby enables long-term imaging of developing multicellular organisms3. However, the ideal combination of low phototoxicity, speed, super-resolution, and high signal-to-noise ratio has proved elusive4.

 

Key findings and technical advances 

In the preprint, the authors characterize a lattice light sheet microscope fitted with adaptive optics (AO-LLSM) to correct sample-induced aberrations. The optics of a lattice light sheet microscope involve different excitation and detection light paths. The authors’ strategy thus involved scanning a guide star produced by two-photon excited fluorescence across the sample volume, and collecting the de-scanned light with two Shack-Hartmann wavefront sensors, one linked to the excitation objective, and one to the detection objective.

The authors go on to demonstrate elegantly the power of their system in vivo. They first track the dynamics of clathrin-coated vesicles in the dorsal developing muscle of zebrafish larvae and then they image organelle dynamics through the cell cycle in zebrafish brain progenitor cells. In a third case study, they also used tiled acquisitions (each with individual adaptive corrections) to image large volumes in the tail and eye of the developing zebrafish. Finally, they imaged growth cone dynamics and cell migration at other specific sites in the zebrafish embryo. In an attempt to generalize their findings beyond the zebrafish models, the supplementary information also includes additional data from C. elegans and Arabidopsis.

 

Why we chose it

Labs around the world are trying to shift experimental cell biology from culture models to native multicellular environments; developing and refining imaging approaches that increase resolution in three dimensions – while reducing phototoxicity – are critical components of this effort. In highlighting the ability of adaptive optics to solve some of the challenges presented by optically complex, heterogeneous multicellular environments, the Betzig lab and collaborators provide a powerful add-on to lattice light sheet microscopy. In doing so, they produce a set of beautiful time-lapse images of subcellular processes in a living vertebrate.

 

Challenges for the future

The authors themselves are quick to point out two caveats of the current work. First, the lion’s share of experiments was carried out using zebrafish embryos; other larger, less transparent systems will present additional imaging challenges. Second, the AO-LLSM approach, like many other sophisticated imaging approaches, generates enormous quantities of raw data. The hardware and software described in this preprint would require significant additional investment across the board. In addition, it is worth noting that there are currently only a handful of operational lattice light sheet systems around the world. These systems represent a significant financial investment and require skilled maintenance, as well as highly trained users. For these reasons, it will likely be a while before you spot an AO-LLSM microscope in your local imaging facility.

 

References

  1. Beyond the diffraction limit. Nat. Photonics 3, 361–361 (2009).
  2. Huisken, J. & Stainier, D. Y. R. Selective plane illumination microscopy techniques in developmental biology. Development 136, 1963–1975 (2009).
  3. Keller, P. J. et al. Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy. Nat. Methods 7, 637–42 (2010).
  4. Laissue, P. P., Alghamdi, R. A., Tomancak, P., Reynaud, E. G. & Shroff, H. Assessing phototoxicity in live fluorescence imaging. Nat. Methods 14, 657–661 (2017).

Tags: imaging

Read preprint (2 votes)




  • Have your say

    Your email address will not be published. Required fields are marked *

    Sign up to customise the site to your preferences and to receive alerts

    Register here

    Also in the cell biology category:

    Spatiotemporally controlled Myosin relocalization and internal pressure cause biased cortical extension to generate sibling cell size asymmetry

    Tri Thanh Pham, Arnaud Monnard, Jonne Helenius, et al.



    Selected by Giuliana Clemente

    Nuclear decoupling is part of a rapid protein-level cellular response to high-intensity mechanical loading

    Hamish T J Gilbert, Venkatesh Mallikarjun, Oana Dobre, et al.



    Selected by Rebecca Quelch

    1

    A robust method for transfection in choanoflagellates illuminates their cell biology and the ancestry of animal septins

    David Booth, Heather Middleton, Nicole King



    Selected by Maya Emmons-Bell

    SWI/SNF remains localized to chromatin in the presence of SCHLAP1

    Jesse R Raab, Keriayn N Smith, Camarie C Spear, et al.



    Selected by Carmen Adriaens

    1

    Clathrin plaques form mechanotransducing platforms

    Agathe Franck, Jeanne Laine, Gilles Moulay, et al.



    Selected by Amanda Haage

    Cellular Crowding Influences Extrusion and Proliferation to Facilitate Epithelial Tissue Repair

    Jovany Jeomar Franco, Youmna Maryline Atieh, Chase Dallas Bryan, et al.



    Selected by Helen Weavers

    A non-cell autonomous actin redistribution enables isotropic retinal growth

    Marija Matejcic, Guillaume Salbreux, Caren Norden



    Selected by Yara E. Sánchez Corrales

    1

    Rearing temperature and fatty acid supplementation jointly affect membrane fluidity and heat tolerance in Daphnia

    Dominik Martin-Creuzburg, Bret L. Coggins, Dieter Ebert, et al.



    Selected by Alexander Little

    Live-cell imaging of marked chromosome regions reveals dynamics of mitotic chromosome resolution and compaction

    John K Eykelenboom, Marek Gierlinski, Zuojun Yue, et al.

    AND

    Quantitative imaging of chromatin decompaction in living cells

    Elisa Dultz, Roberta Mancini, Guido Polles, et al.



    Selected by Carmen Adriaens, Gautam Dey

    Optogenetic reconstitution reveals that Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble

    Masako Okumura, Toyoaki Natsume, Masato T Kanemaki, et al.



    Selected by Arnaud Monnard

    1

    Optogenetic reconstitution reveals that Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble

    Masako Okumura, Toyoaki Natsume, Masato T Kanemaki, et al.



    Selected by Ben Craske, Thibault Legal and Toni McHugh

    A novel microtubule nucleation pathway for meiotic spindle assembly in oocytes

    Pierre ROME, Hiroyuki OHKURA



    Selected by Binyam Mogessie

    ERM proteins: The missing actin linkers in clathrin-mediated endocytosis

    Audun Sverre Kvalvaag, Kay Oliver Schink, Andreas Brech, et al.



    Selected by Nicola Stevenson

    Cell type-specific interchromosomal interactions as a mechanism for transcriptional diversity

    Adan Horta, Kevin Monahan, Lisa Bashkirova, et al.



    Selected by Boyan Bonev

    A non-canonical role for dynamin-1 in regulating early stages of clathrin-mediated endocytosis in non-neuronal cells

    Saipraveen Srinivasan, Christoph J. Burckhardt, Madhura Bhave, et al.



    Selected by Penelope La-Borde

    Atomic model of microtubule-bound tau

    Elizabeth H Kellogg, Nisreen M.A. Hejab, Simon Poepsel, et al.



    Selected by Satish Bodakuntla

    1

    Also in the developmental biology category:

    Human macrophages survive and adopt activated genotypes in living zebrafish

    Colin D. Paul, Alexus Devine, Kevin Bishop, et al.



    Selected by Giuliana Clemente

    Altering the temporal regulation of one transcription factor drives sensory trade-offs

    Ariane Ramaekers, Simon Weinberger, Annelies Claeys, et al.



    Selected by Mariana R.P. Alves

    Presence of midline cilia supersedes the expression of Lefty1 in forming the midline barrier during the establishment of left-right asymmetry

    Natalia A Shylo, Dylan A Ramrattan, Scott D Weatherbee



    Selected by Hannah Brunsdon

    Genetic compensation is triggered by mutant mRNA degradation

    Mohamed El-Brolosy, Andrea Rossi, Zacharias Kontarakis, et al.



    Selected by Andreas van Impel

    1

    Cellular Crowding Influences Extrusion and Proliferation to Facilitate Epithelial Tissue Repair

    Jovany Jeomar Franco, Youmna Maryline Atieh, Chase Dallas Bryan, et al.



    Selected by Helen Weavers

    Photoperiod sensing of the circadian clock is controlled by ELF3 and GI

    Usman Anwer, Amanda Davis, Seth Jon Davis, et al.



    Selected by Annika Weimer

    Limb- and tendon-specific Adamtsl2 deletion identifies a soft tissue mechanism modulating bone length

    Dirk Hubmacher, Stetson Thacker, Sheila M Adams, et al.



    Selected by Alberto Rosello-Diez

    A non-cell autonomous actin redistribution enables isotropic retinal growth

    Marija Matejcic, Guillaume Salbreux, Caren Norden



    Selected by Yara E. Sánchez Corrales

    1

    Rearing temperature and fatty acid supplementation jointly affect membrane fluidity and heat tolerance in Daphnia

    Dominik Martin-Creuzburg, Bret L. Coggins, Dieter Ebert, et al.



    Selected by Alexander Little

    A SoxB gene acts as an anterior gap gene and regulates posterior segment addition in the spider Parasteatoda tepidariorum

    Christian L. B. Paese, Anna Schoenauer, Daniel J. Leite, et al.



    Selected by Erik Clark

    1

    Cell type-specific interchromosomal interactions as a mechanism for transcriptional diversity

    Adan Horta, Kevin Monahan, Lisa Bashkirova, et al.



    Selected by Boyan Bonev

    Germ layer specific regulation of cell polarity and adhesion gives insight into the evolution of mesoderm.

    Miguel Salinas-Saavedra, Amber Q. Rock, Mark Q. Martindale



    Selected by ClaireS & SophieM

    1

    Wnt/β-catenin regulates an ancient signaling network during zebrafish scale development

    Andrew J Aman, Alexis N Fulbright, David M Parichy



    Selected by Andreas van Impel

    Tissue flow induces cell shape changes during organogenesis

    Gonca Erdemci-Tandogan, Madeline J.Clark, Jeffrey D. Amack, et al.



    Selected by Jacky G. Goetz

    Temporal Control of Transcription by Zelda in living Drosophila embryos

    Jeremy Dufourt, Antonio Trullo, Jennifer Hunter, et al.



    Selected by Teresa Rayon

    1

    An atlas of silencer elements for the human and mouse genomes

    Naresh Doni Jayavelu, Ajay Jajodia, Arpit Mishra, et al.



    Selected by Rafael Galupa

    1

    Close

    We want to make our website, and the services we provide, useful and reliable. This sometimes involves placing small amounts of information called cookies on the device you used to access the internet. If you continue to use this website we will assume you are happy to accept our cookies.

    Accept