High-speed 3-D fluorescence imaging by oblique plane microscopy: multi-well plate-reader development, biological applications and image analysis

Light-sheet microscopy has enabled high-speed 3D fluorescence microscopy with lower levels of phototoxicity and photobleaching compared to more established fluorescence microscopy techniques. However, conventional light sheet configurations prevent the use of many common sample mounting techniques including multi-well plates. The oblique plane microscope (OPM) is a light-sheet microscope implemented on a standard microscope frame. This thesis presents the development of a high-speed OPM plate-reader and analysis of high speed 2D and 3D OPM data of isolated cardiomyocytes. First, the modifications made to the OPM system to achieve the plate-reading mode are presented. In particular, the electrical triggering system and the software developed for image acquisition control are discussed, as well as modifications including an immersion-fluid replenishing cap and to the microscope enclosure system. Finally, the algorithm and software used to reconstruct the OPM data are presented, together with a discussion of their influence on the final image. An experimental characterisation of the OPM system optical performance is then presented. Optical modelling of the different parts of the OPM is detailed together with experimental measurements of the transmission efficiency of light through the OPM system, the light sheet thickness, the aberrations introduced when refocusing away from the designed focal plane, and the influence of the refocusing distance and scanning speed on the size of the system point spread function. Image analysis of high speed 2D and 3D OPM data acquired of calcium dynamics in isolated cardiomyocytes is then presented. The software developed is presented and applied to study the relationships between cardiomyocyte transverse tubule structure and dynamic calcium spark and wave events. Finally, the applications of the OPM plate-reader to ratiometric imaging of FRET biosensors in 3D tissue cultures and a range of biological samples will be detailed. These results demonstrate the ability of the system to acquire data over an extended field of view, in particular to study cell invasion into collagen in multi-well plates. The OPM plate-reader is then applied to the imaging of spheroids, and in particular of spatio-temporal glucose dynamics with ratiometric imaging of a FRET biosensor, along with the image processing to quantity the FRET ratio as a function of the distance from the outside of the spheroid.Open Acces