Team Clarity

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

]

Branching organs
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. Images are coming soon.

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. In addition to straight sided pores that possess a zero gaussian curvature, I also performed experiments with non-zero gaussian curvature pores to explore the impact of 3D curvature on tissue production.

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!



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: