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Tuesday, October 22 • 10:30am - 11:50am
Advances in Imaging and Analysis- Light Sheet Fluorescence Microscopy for High-Content Analysis in 3D

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Light sheet fluorescence microscopy (LSFM) provides low out-of-plane photobleaching and phototoxicity, but usually requires two microscope objective lenses orientated at 90° to one another, making high content imaging in conventional well plates more challenging. Oblique plane microscopy (OPM) uses a single high numerical aperture microscope objective to provide both fluorescence excitation and detection whilst maintaining the advantages of LSFM. We present the development of a stage scanning OPM approach for light sheet fluorescence imaging in commercially available 96 and 384-well plates, including plastic-bottomed plates.
In a first application, the system was used to measure cell shape parameters in fixed and live cells in grown in 3D in a collagen matrix. We are developing a MATLAB 3D image analysis pipeline for automated segmentation and morphological quantification of the image data. This work aims to enable a better understanding of which genes are responsible for cancer cell size determination and invasion in 3D cultures.
In a second application, the system is being used to develop a 3D assay for spheroid-based compound safety screening. Multi-cellular HepG2/C3a spheroids with diameters in the range 100-200 micron were grown in low attachment u-bottomed dishes and then transferred to 384-well plates for ssOPM imaging. Following initial tests, SYTOX green and TMRM were chosen to read out spheroid viability and mitochondrial function as an indicator for drug toxicity. The assay Z’, signal to noise ratio and coefficient of variation have been measured using max-min plating protocols.

Speakers
avatar for Chris Dunsby

Chris Dunsby

Senior Lecturer, Imperial College London
Dr Chris Dunsby works on the development of quantitative fluorescence imaging techniques for applications in biomedicine, including Förster resonant energy transfer microscopy, automated multiwell plate imaging and fluorescence lifetime imaging (FLIM). He has also worked on super-resolution... Read More →

Chair

Tuesday October 22, 2019 10:30am - 11:50am
Wellcome Auditorium