Open top dual view light sheet microscope for live imaging of 3D cultures in multi-well format

Session

Poster Session

Authors

Franziska Moos (1, 4), Simon Suppinger (1, 4), Gustavo de Medeiros (1), Davide Gambarotto (3), Andrea Boni (2), Petr Strnad (2), Prisca Liberali (1, 4)

Affiliations

1. Friedrich Miescher Institute for Biomedical Research
2. Viventis Microscopy Sàrl
3. Viventis Microscopy Sarl
4. University of Basel

Keywords

Light-sheet, open top, dual view, dual illumination, multi-well, organoids.

Abstract text

Light sheet fluorescence microscopy (LSFM) is due to its high imaging speed and low phototoxicity a method of choice for imaging of living samples [1]. Open top [2] or inverted [3, 4] LSFM is a further development of the original concept where the sample is mechanically supported from the bottom by a sample holder and it can be accessed through the opened top. Open top or inverted LSFM are compatible with multi-position imaging and suitable to image samples that cannot be embedded in agarose or gel for mechanical stabilization. It was used for medium throughput imaging or sensitive sample such as mouse embryos [3] and organoids [4]. Organoid models such as gastruloids develop for several days and grow to large size making their imaging challenging. Because of image degradation deeper in the sample cells further from the detection objective cannot be identified and tracked. To image large samples SiMView [5], multiview [6] and panoramatic [7] LSFM have been proposed. All three systems use identical concept: the object is viewed from two opposing directions using two detection objectives and illuminated from two opposing directions using two illumination objectives. For an optimally sized sample this increases the penetration depth by a factor of two. The sample however needs to held in a constrained space between the four objectives making multi position and organoid imaging challenging. Here we present a dual view and dual illumination light sheet microscope compatible with multi-well format. We use two opposing detection objectives to image sample from two directions and two illumination objectives which are opposing but slightly rotated to point upwards. This creates an unobstructed linear space just above the two illumination objectives for a custom designed sample holder containing a linear array of wells and samples for multiposition imaging. We demonstrate the capabilities of the system by long term live imaging of murine intestinal organoids, hydra, liver organoids, gastruloids and human colon cancer organoids for more than 5 days. To highlight its potential to achieve single cell resolution, we present single cell tracking and segmentation during regeneration of intestinal organoids and gastruloid development. Our analysis of gastruloids revealed changes in cell shape coupled with increased cell motility, which suggests a partial epithelial to mesenchymal transition in gastruloids induced by a Wnt activation pulse.

References

1. Huisken et al.: Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy, Science, 2014 

2. McGorty et al.: Open-top selective plane illumination microscope for conventionally mounted specimens. Optics Express, 2015 

3. Strnad et al.: Inverted light-sheet microscope for imaging mouse pre-implantation development. Nature Methods, 2015 

4. Serra, Mayr, Boni et al.: Self-organization and symmetry breaking in intestinal organoid development, Nature, 2019