Team Clarity

Projects
Our first try: 3D Reconstraction of Drosophila Embryo.

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Mice development
Work plan during the course: 1) Samples of different sizes: 500(L)x300(R) um to 1(L)x0.7(R) cm including- Mouse embryos at different developmental stages (E10.5, E11.5, E12.5 & E13.5) & Brain, Lung & Heart of E18.5 embryo 2) A variety of signal intensity (expression levels, stability, etc…) – Different “labeling” patterns: membrane GFP in all tissues in the context of an intact embryo or dissected embryonic organs, Cytoplasmic GFP in scattered cells  at different regions of the embryo & Cytoplasmic GFP expressed exclusively in PGCs 3) Samples of different morphology: opaque, transparent, branched, auto-fluorescent, etc…)



Imaging Results during the course:

Ultra microscope – Cleared E13.5 mouse embryo:

Branching organs
"Goal": 3D Life imaging of the thick, uncleared embryonic tissue with the light sheet microscopy.

Mouse embryonic kidneys at E11.5, E12 and E12.5 were imaged with Zeiss Lightsheet Z.1. Light sheet microscopy provides a cellular resolution at the organ scale. Green and red depict renal epithelium and mesenchyme.



Optical sectioning of embryonic mouse kidney with OpenSPIM. Greyscale depicts e-cadherin staining in the epithelium.



Take home message: commercial and home build light sheet microscopes can be used to image embryonic organ explants, however two photon and active optics might be necessary to fight scattering.

Investigation of 3d tissue growth using Light Sheet Microscopy
My aim was to use Light Sheet Microscopy to resolve the three-dimensional structure of fixed tissues grown in transparent straight sided pores of controlled shape. The transparent scaffolds have two different circular pore sizes and are fabricated with the aid of rapid-prototyping. Pre-osteoblast (MC3T3-E1) Cells were then seeded on top of the scaffolds and cultured in a cell culture media for up to 28 days. The grown tissue has been fixed at certain time-points and, in order to quantify the volumetric distribution of the tissue, the scaffolds were mounted in a sample holder for subsequent visualization with a Light Sheet Microscope.

Unfortunately, due to the scaffold design and limitations of the objective lens, the conventional mounting technique using FEP-tubes or agarose was not applicable. Therefor the sample has been mounted on a double-hook and imaged from multiple views. The obtained multiview images were then reconstructed in Fiji.

Due to the large amount of data (approx. 100GB for 6 views) and the fact that the scaffolds contain no beeds, reconstruction of the multiview images in Fiji is complicated and still in progress!



Sketch of the mounting technique to image scaffolds in a Zeiss Lightsheet.Z1



Tissue grown in a circular pore.



Maximum projection of tissue grown in a circular pore obtained with Zeiss Lightsheet.Z1 and a 5x objective lens. One can clearly see cell-nuclei and actin-bundles. Actin fibres are depicted in green and cell-nuclei in red. The pore size is 2mm.



Images were obtained using a Zeiss Lightsheet.Z1 Microscope with a 5x objective lens and reconstructed in Fiji.

Live samples
Colonies of Membranipora membranacea were shipped twice from Bergen, Norway. In both cases colonies arrived dead or mostly-dead. However, I managed to scrape better-looking embryos from inside the zooids. Some embryos got activated and started cleaving. I tried to image them in the Scanned Lightsheet SPIM (Meyers SPIM) since it has temperature control in the chamber and is adapted to small samples. Embryos were stained with a membrane marker (FM 4-64). Unfortunately, we only manage to acquire a small time-lapse from an embryo that was not healthy: