Imaging biocolonization in opaque materials : a 3D approach using absorption and spectral X-ray Micro-CT

Without preservation, heritage and art deteriorate. They are colonized by microorganisms, forming biofilms on and within the pores. Biofilms can induce biodeterioration but also protect or restore the substrate. Visualizing biofilms is crucial to understand their effect. Absorption microcomputed tomography (µCT), a non-destructive technique popular to study heritage objects reveals their interior in 3D. However, it fails to imaging biofilms due to low contrast. Contrastenhancing staining agents (CESAs) can help but are toxic or impractical, highlighting the need for alternatives. In previous research, we examined five CESAs on cyanobacterial biofilms covering sandstone [1], of which Isotonic Lugol (IL) and Hf-WD 1:2 POM were successful. This study tested them on biofilms of heterotrophic bacteria in between sand. Both CESAs enhanced their contrast, revealing the biofilm's location, size and substrate relationship (Fig. 1A-C), which would not be possible with traditional techniques. IL caused stronger contrast but could cause shrinkage. Moreover, novel spectral CT provided chemical 3D insights and confirmed the CESA's affinity to the biofilms. It distinguished the IL-stained biofilms from the sand (Fig. 1D), but detecting Hf and W was challenging. These results highlight the potential of absorption and spectral CT for studying microorganisms in opaque materials, including art objects. Future research should focus on the CESA-biofilm interactions and apply these CESAs in the field. At last, the added value of spectral-CT should be explored in the whole field of heritage.