Recent developments in super resolution imaging have led to improvement in both axial and lateral resolution for fluorescence imaging. However, the improved axial resolution has come at the cost of imaging depth, with most techniques limited to a few micrometres from the base of the sample. For conventional imaging techniques, improvements in axial resolution are still sought. The advent of lightsheet microscopy.has improved axial contrast in larger specimens such as zebrafish, embryos and worms. However, due to the optical design constraints, increases in throughput are limited to a few dozen samples per hour and have not reached the speeds necessary for routine screening of compound or mutant libraries.
Improvements in axial resolution and contrast would greatly enhance the image quality obtained from conventional techniques allowing greater accuracy of segmentation of structures during image analysis. This would allow more robust quantitative analysis of the acquired images.
EDGETM confocal is an enhanced confocal method that acquires 2D images or 3D image stacks with improved image contrast in all dimensions. This increase in contrast improves visualization and segmentation of structures in thick samples that would otherwise be obscured by high levels of background fluorescence. Inspired by Poher et al., we have implemented this method on the IN Cell 6500 line scanning confocal high content analysis system to allow fast, robust confocal imaging with a theoretical doubling of axial resolution and an order of magnitude attenuation of the out-of-focus signal. With imaging depths beyond 100 micrometers, EDGE confocal allows 3D imaging of tissue and organoids with minimal signal loss and blur in the centre of the objects. The high quality images resulting from this unique hardware combines with the IN Carta image analysis software to improve segmentation and quantitation. We demonstrate that it is the combination of improvements in both hardware and software that lead to improved analysis outcomes.