Objective

The drug discovery pipeline needs accurate information on the toxicity profile of potential drugs. Bulk (non-imaging) and image-based fluorescent readouts of fluorescent probes in 2D cell cultures can provide quantitative information in an assay format, but do not capture the complexity of biological tissues, which are inherently 3D structures. Multi-cellular spheroids (MCS)represent the next level of model complexity, as they incorporate inter-cellular interactions and gradients of the drug and microenvironmental parameters such as oxygen, nutrients, pH, etc, and are therefore used increasingly in the pharmaceutical industry. High-content 2D imaging of fluorescent probes in MCS is performed routinely using commercially available instrumentation, but fast 3D imaging of MCS in 96 and 384-well plates is more challenging. Light sheet fluorescence microscopy (LSFM) has the potential to provide high speed 3D imaging with low out-of-plane photobleaching and phototoxicity¹, but the requirement in conventional LSFM for the excitation and collection lenses to be perpendicular to each other and close to the sample restricts the ability to use conventional sample preparation approaches, including multi-well plates. Oblique plane microscopy (OPM)² is an alternative light sheet approach that is compatible with multi-well plate for 3D imaging and provides subcellular resolution, and uses a single high numerical aperture microscope objective for both fluorescence excitation and collection whilst maintaining the advantages of LSFM. The OPM system has been developed previously and demonstrated for rapid volumetric imaging with subcellular resolution^2-5. Here we report the application of OPM to drug toxicity screening using live MCS mounted in commercially available glass-bottomed 384-well plates. A first challenge in implementing OPM for imaging MCS in 384-well plates is to automatically determine the x-y-z location of the spheroid in each well prior to 3D imaging. We have developed a solution to this problem in x-y using an automated pre-find procedure based on an initial low magnification epi-fluorescence image of each well acquired using a 4× objective lens followed by automatic spheroid detection in software. Using this approach, the x-y location of ~235 out of 240 spheroids in a 384-well plate can be found automatically in ~25 minutes. Currently, we then employ a manual focus step in each well to determine the z position, but we are in the process of automating this step too. Multi-cellular HepG2 C3a-clone and HepaRG spheroids consisting of approximately 500 cells with diameters in the range of 200-300 mm were grown in low attachment U-bottomed dishes and then transferred to glass-bottomed 384-well plates for imaging by stage scanning OPM (ssOPM)⁵. Spheroids were pre-dosed with 12 clinically-relevant compounds at a range of concentrations and loaded with fluorescent probes reporting cytotoxicity (Sytox Green) and mitochondrial function (tetramethylrhodamine, methyl ester). 3D volumetric images in two spectral channels were acquired by scanning the sample through the tilted light sheet at a constant velocity (0.1 µm.ms^-1). For each spheroid, a volume of 250 images was acquired in each channel in 5 s. Overall, the image acquisition, data storage and stage movement for 240 wells took 80 minutes. We present preliminary image data acquired using stage-scanning OPM system.