3D Visualization of cyanobacterial biofilms using micro-computed tomography with contrast-enhancing staining agents

Currently, biofilms colonizing surfaces are mainly imaged in 2D by conventional techniques, such as optical or scanning electron microscopy. Confocal laser scanning microscopy or optical coherence tomography can visualize biofilms in 3D, but they suffer from a limited penetration depth and cannot visualize biofilms in opaque materials. Micro-computed tomography (µCT) can overcome these issues, but µCT cannot easily distinguish biofilm structures from water due to a lack of contrast difference. Within this research, five contrast-enhancing staining agents (CESAs) were evaluated for their staining potential of cyanobacterial biofilms, aiming to visualize these biofilms in 3D. Isotonic Lugol and 1:2 hafnium(IV)-substituted Wells-Dawson polyoxometalate (Hf-WD 1:2 POM) were the most promising, as they allowed visualization of the biofilms and revealed structures in the stained biofilms. Staining with isotonic Lugol could clearly visualize bundles of filaments within the biofilm, while Hf-WD 1:2 POM revealed a smooth biofilm. It is assumed that both CESAs have a different affinity towards the biofilms and could thus be used complementary. Monolacunary Wells-Dawson polyoxometalate (Mono-WD POM) showed moderate discrimination while staining with cationic iodinated CA4+ and Hexabrix® (Guerbet) containing anionic ioxaglate did not allow to distinctly visualize the biofilms. These results indicate that µCT, together with CESAs such as isotonic Lugol and Hf-WD 1:2 POM, can be used as a tool to image extensive biofilms or microbial mats in 3D. Further research will determine whether these CESAs are suitable for visualizing biofilms within opaque porous media.