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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

Posted on: 16th February 2018 , updated on: 18th February 2018

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