Summary

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Published: July 19, 2024
doi:

Summary

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

Abstract

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

Introduction

Ensuring minimal phototoxicity is a major requirement for imaging live embryos with high spatiotemporal resolution for long periods. Over the last decade, light sheet microscopy has become the methodology of choice to meet this requirement1,2,3,4,5,6,7. Briefly, in this technique that was first used in 2004 to capture developmental processes8, two aligned thin sheets of laser pass through the embryo from opposing ends, illuminating only the plane of interest. A detection objective is placed orthogonally, and then the emitted fluorescent light from all illuminated points in the sample is collected simultaneously. A 3D image is then obtained by sequentially moving the embryo through the static light sheet.

In addition, in a specific form of this methodology, termed multiview light sheet microscopy, the samples can be suspended in a polymer tube that can be rotated using a rotor, enabling imaging of the same embryo from multiple angles9,10,11. Following imaging, the images from multiple angles are fused based on registration markers, which are typically globular fluorescent markers within the embryo (e.g., nuclei) or in the tube (e.g., fluorescent beads). Multiview imaging and fusion significantly improve the axial resolution, providing isotropic resolution across all three dimensions12. While this is a big advantage, a major challenge of multiview methodology is sample mounting, where embryos have to be mounted and kept in place in the tubes during the entire time course of imaging.

For performing multiview imaging, to keep the embryos in place and prevent movement while imaging, embryos can be embedded in agarose. However, this often leads to detrimental growth and development, particularly for early-stage zebrafish embryos13, the model system that is discussed here. A second mounting strategy is to use a thin tube that is only slightly bigger than the diameter of the embryo, where the embryo can be pulled into the tube along with the embryo medium, followed by closing the bottom of the tube with an agarose plug14. In this method, because the tube is filled with embryo medium, registration markers such as fluorescent beads cannot be used for the fusion of the different views, and registration is therefore reliant on markers within the embryo. In general, beads act as better registration markers as the signal of the markers within the embryo degrades upon moving deeper into the sample owing to both illumination and detection limitations of any microscope.

Thus, a third approach, which will be detailed here and used previously5,13,14,15,16, is imaging early zebrafish embryos with an intact chorion and filling the tube with a minimal percentage of agarose, which contains beads as registration markers. In this scenario, because manual intervention for positioning embryos within a chorion is not possible, this study provides statistics on the default orientation early zebrafish embryos fall into, particularly focusing on 70% epiboly and bud stages. It then discusses the optimal number of views required for imaging early-stage embryos at cellular-scale resolution and details the process of fusion using BigStitcher, a FIJI-based plugin10,17,18. Together, this protocol, which uses a 20x/1 NA objective, aims to facilitate zebrafish embryologists in using multiview light sheet systems for imaging embryos with nuclei and membrane markers from gastrulation to early somite stages.

Protocol

The zebrafish maintenance and experimental procedures used in this study were approved by the institutional animal ethics committee, vide Reference TIFR/IAEC/2023-1 and TIFR/IAEC/2023-5. Embryos obtained by crossing heterozygous fish expressing Tg(actb2:GFP-Hsa.UTRN)19 were injected with H2A-mCherry mRNA (30 pg) at the one-cell stage. H2A-mCherry mRNA was synthesized using the pCS2+ H2A-mCherry plasmid (a gift from the Oates lab, EPFL) by in vitro transcription. Embryos expr…

Representative Results

Orienting the sample in a precise manner is a vital part of efficiently using a microscopy set-up. However, manually orienting samples is often not possible when using a multiview light sheet system, given the requirement for preparing the samples in a tube. Therefore, to check if there are stereotypical positions that embryos take up within the chorion, zebrafish embryos were imaged at 70% epiboly (about 7 h post-fertilization (hpf)), since time-lapse imaging from gastrulation to early somite stages was the focus of thi…

Discussion

Positioning an embryo in the right orientation to image the region of interest is one of the rate-limiting steps that often results in a failed microscopy session for a user. This is more so in a multiview light sheet microscope where manual manipulation of the orientation is difficult as the samples are embedded within a tube. To aid in this process, this study reports the statistics of various positions a zebrafish embryo takes up between 70% epiboly and early somite stages within a chorion when the polymer tube with e…

Divulgations

The authors have nothing to disclose.

Acknowledgements

We acknowledge Dr. Kalidas Kohale and his team for the maintenance of the fish facility and KV Boby for the maintenance of the light sheet microscope. SRN acknowledges financial support from the Department of Atomic Energy (DAE), Govt. of India (Project Identification no. RTI4003, DAE OM no. 1303/2/2019/R\&D-II/DAE/2079 dated 11.02.2020), the Max Planck Society Partner Group program (M.PG.A MOZG0010) and the Science and Engineering Research Board Start-up Research Grant (SRG/2023/001716).

Materials

Agarose, low gelling temperature Sigma-Aldrich A9414
Calcium Chloride dihydrate Sigma-Aldrich 12022
FIJI Version: ImageJ 1.54f
Latex beads, carboxylate-modified polystyrene, fluorescent red, 0.5 μm mean particle size, aqueous suspension Sigma-Aldrich L3280
Magnesium sulfate heptahydrate Sigma-Aldrich M2773
mMESSAGE mMACHINE SP6 Transcription kit ThermoFischer Scientific AM1340 For in vitro transccription of H2A-mCherry plasmid
Potassium Chhloride Sigma-Aldrich P9541
Potassium phosphate monobasic Sigma-Aldrich P0662
PTFE Sleeving AWG 15L – 1.58 mm ID x 0.15 mm Wall +/-0.05  Adtech Innovations in Fluoroplastics STW15 PTFE tubes
Sodium Chloride Sigma-Aldrich S3014
Sodium phosphate dibasic Sigma-Aldrich 71640
Ultrasonic Cleaner Labman LMUC3 Ultrasonicator
Zeiss LightSheet 7 System Zeiss

References

  1. Wan, Y., McDole, K., Keller, P. J. Light sheet microscopy and its potential for understanding developmental processes. Annu Rev Cell Dev Biol. 35, 655-681 (2019).
  2. Keller, P. J., Schmidt, A. D., Wittbrodt, J., Stelzer, E. H. Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science. 322 (5904), 1065-1069 (2008).
  3. Keller, P. J., et al. Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy. Nat Methods. 7 (8), 637-642 (2010).
  4. Keller, P. J. Imaging morphogenesis: Technological advances and biological insights. Science. 340 (6137), 1234168 (2013).
  5. Schmid, B., et al. High-speed panoramic Light sheet microscopy reveals global endodermal cell dynamics. Nat Commun. 4 (1), 2207 (2013).
  6. Strnad, P., et al. Inverted Light sheet microscope for imaging mouse pre-implantation development. Nat Methods. 13 (2), 139-142 (2016).
  7. McDole, K., et al. In toto imaging and reconstruction of post-implantation mouse development at the single-cell level. Cell. 175 (3), 859-876.e33 (2018).
  8. Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J., Stelzer, E. H. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science. 305 (5686), 1007-1009 (2004).
  9. Krzic, U., Gunther, S., Saunders, T. E., Streichan, S. J., Hufnagel, L. Multiview Light sheet microscope for rapid in toto imaging. Nat Methods. 9 (7), 730-733 (2012).
  10. Preibisch, S., Saalfeld, S., Schindelin, J., Tomancak, P. Software for bead-based registration of selective plane illumination microscopy data. Nat Methods. 7 (6), 418-419 (2010).
  11. Swoger, J., Verveer, P., Greger, K., Huisken, J., Stelzer, E. H. K. Multiview image fusion improves resolution in three-dimensional microscopy. Opt Express. 15 (13), 8029-8042 (2007).
  12. Swoger, J., Huisken, J., Stelzer, E. H. Multiple imaging axis microscopy improves resolution for thick-sample applications. Opt Lett. 28 (18), 1654-1656 (2003).
  13. Kaufmann, A., Mickoleit, M., Weber, M., Huisken, J. Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope. Development. 139 (17), 3242-3247 (2012).
  14. Weber, M., Mickoleit, M., Huisken, J. Multilayer mounting for long-term light sheet microscopy of zebrafish. J Vis Exp. (84), e51119 (2014).
  15. Naganathan, S. R., Popović, M., Oates, A. C. Left-right symmetry of zebrafish embryos requires somite surface tension. Nature. 605 (7910), 516-521 (2022).
  16. Shah, G., et al. Multi-scale imaging and analysis identify pan-embryo cell dynamics of germlayer formation in zebrafish. Nat Commun. 10 (1), 5753 (2019).
  17. Schindelin, J., et al. Fiji: An open-source platform for biological-image analysis. Nat Methods. 9 (7), 676-682 (2012).
  18. Hörl, D., et al. BigStitcher: Reconstructing high-resolution image datasets of cleared and expanded samples. Nat Methods. 16 (9), 870-874 (2019).
  19. Behrndt, M., et al. Forces driving epithelial spreading in zebrafish gastrulation. Science. 338 (6104), 257-260 (2012).
  20. Westerfield, M. . The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio Rerio). , (2000).
  21. Icha, J., et al. Using light sheet fluorescence microscopy to image zebrafish eye development. J Vis Exp. 110, e53966 (2016).
  22. Tomer, R., Khairy, K., Amat, F., Keller, P. J. Quantitative high-speed imaging of entire developing embryos with simultaneous multiview Light sheet microscopy. Nat Methods. 9 (7), 755-763 (2012).
  23. Aigouy, B., Prud’homme, B. Segmentation and quantitative analysis of epithelial tissues. Methods Mol Biol. 2540, 387-399 (2022).
  24. Mancini, L., et al. Apical size and deltaA expression predict adult neural stem cell decisions along lineage progression. Science Adv. 9, eadg7519 (2023).
  25. Piscitello-Gomez, R., Mahmoud, A., Dye, N., Eaton, S. Sensitivity of the timing of Drosophila pupal wing morphogenesis to external perturbations. bioRxiv. , (2023).
  26. Tsuboi, A., Fujimoto, K., Kondo, T. Spatiotemporal remodeling of extracellular matrix orients epithelial sheet folding. Science Adv. 9, eadh2154 (2023).
  27. Fu, Q., Martin, B. L., Matus, D. Q., Gao, L. Imaging multicellular specimens with real-time optimized tiling Light sheet selective plane illumination microscopy. Nat Commun. 7 (1), 11088 (2016).
  28. Chen, B. C., et al. Lattice Light sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science. 346 (6208), 1257998 (2014).
  29. York, H. M., et al. Deterministic early endosomal maturations emerge from a stochastic trigger-and-convert mechanism. Nat Commun. 14 (1), 4652 (2023).
  30. Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. Stages of embryonic development of the zebrafish. Dev Dyn. 203 (3), 253-310 (1995).
  31. Melançon, E., Liu, D. W. C., Westerfield, M., Eisen, J. S. Pathfinding by identified zebrafish motoneurons in the absence of muscle pioneers. J Neurosci. 17 (20), 7796 (1997).
  32. Rohde, L. A., et al. Cell-autonomous timing drives the vertebrate segmentation clock’s wave pattern. eLife. 13:RP93764, (2024).
  33. Haynes, E. M., et al. KLC4 shapes axon arbors during development and mediates adult behavior. eLife. (11), e74270 (2022).
This article has been published
Video Coming Soon
Keep me updated:

.

Citer Cet Article
Nagarajan, S., Bardhan, S., Naganathan, S. R. Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos. J. Vis. Exp. (209), e66735, doi:10.3791/66735 (2024).

View Video