Alberta has one of the largest oil reserves in the world. Large-scale commercial oil production from oil sands in Alberta for the past 40 years has led to accumulation of tailings water in tailings ponds covering areas ranging over 150 km2. Less than 1% of this area has been certified as reclaimed leading to both economic and environmental consequences. Research is underway to reduce tailings ponds reclamation time from decades to weeks by developing new polymer flocculants, better tailings treatment methods and recovering bitumen from tailings. Information about impact of residual bitumen on the shear strength, trafficability, densification, hydraulic conductivity, consolidation, post-reclamation settlement for oil sands tailings is insufficient. Outstanding challenges exist in understanding bitumen and clay interaction in tailings to help with the development of techniques which accelerate clay sedimentation and enhance bitumen recovery. To shed light on the bitumen-clay interactions, here we develop advanced three-dimensional optical tomography approaches approaching sub-micron resolution. In this paper, we report, the first ever Total Internal Reflection Fluorescence (TIRF) microscope tomography for Mature Fine Tailings (MFT) samples to reveal bitumen distribution on clay in MFT. We employ a unique evanescent wave illumination approach as opposed to conventional fluorescence microscopy with enhanced axial resolution and high signal-to-noise ratio. The resolution of TIRF is further improved by using an Axial Super-Resolution Evanescent-wave Tomography (AxSET) technique. The information obtained from this study not only gives evidence of the presence of hydrophilic and oleophilic clays but with aid of 3D reconstruction using advance image processing also validates that bitumen is partially coating some of clay surfaces, thus verifying the presence of biwettable clays in oil sands MFT. The advances from our imaging work can aid the development of bitumen recovery techniques for environmental and economic impact.