Live imaging

Happy holidays, the preprint is out there!

This is a guest post by Marie Held. Credits are given to Andrew Plested for the title/timing of this post (see here).

It had been a long time coming but the preprint of our manuscript Ex vivo live cell tracking in kidney organoids using light sheet fluorescence microscopy is now available on BioRxiv. Together with the preprint, the associated large body of imaging data has been made publicly available in the online Image Data Resource (IDR) repository. Through the power of OMERO you can have a play with the data or even download it and then re-analyse it.

The imaging has been conducted on the Zeiss Z.1 Lightsheet microscope at the Centre for Cell Imaging at the University of Liverpool, whose ever helpful staff enabled us generating lots and lots of image data and providing an efficient infrastructure for data storage as well as support for image and subsequent data analysis. Of course this is only half the story because the imaging would not have been possible without generating the samples first: A big thank you also goes to the students and academic staff in the Institute of Translational Medicine at the University of Liverpool.

In this study we have generated organoids from dissociated and re-aggregated mouse embryonic kidney tissue and imaged them with a light sheet fluorescence microscope. The microscope optically sections the samples, therefore preserving the three-dimensional context of the sample throughout imaging. We have found organotypic kidney structures in the organoids and evidence for the maturation of cells to the point of forming glomeruli, the basic filtration unit of the kidney. A functional assay showed that the developed tubules display secretory function.

Most importantly though, we have also performed live imaging of organoids made from genetically tagged fluorescing cells. The light sheet microscopy setup combines an illumination that is perpendicular to the detection. Therefore, full frame images can be recorded rapidly and only the section of the sample that is recorded is illuminated, thus vastly reducing photobleaching and phototoxic effects that limit long-term live fluorescence imaging in wide field and in particular confocal scanning fluorescence microscopy. We have then tracked the fluorescing cells with the help of an automated algorithm and subsequently analysed the generated tracking data. We have {started to} analyse the tracking data and can now quantitatively compare between experiments.

Yet, there is so much more that can be done with the images and data and we would love to see which ideas and approaches others might have, so please do not be shy to dig in and have a play. Be sure to let us know though.

Featured image caption: Organoid of mouse embryonic kidney cells formed following dissociation and re-aggregation of embryonic kidney rudiments. Yellow: Pax2+ cells indicating the metanephric mesenchyme, a prerequisite for nephron development, Red: Peanut-agglutinin staining basement membranes of the ureteric tree and developed nephrons.