Depth-dependent resolution quantification in 3D fluorescence microscopy

A method is presented to quantify resolution as a function of depth in features of morphologically complex 3D samples. Applying the method to the brain of Drosophila, resolution is measured at increasing depth throughout the central brain region. The results quantify improvements in image quality when using two-photon microscopy compared to confocal. It is also demonstrated how resolution improvements through tuning a single parameter, laser power, can be measured objectively. Since the metric is interpretable as the average resolution within a feature, it is suitable for comparing results across optical systems, and can be used to inform the design of biological experiments requiring resolution of structures at a specific scale.