Multimodal Hyperspectral Imaging for the study of cyanobacterial sub- aerial biofilm on carbonatic stones

The interactions between microorganisms and masonry surfaces in built heritage are the result of the coordinated activity of complex microbial communities attached to the surfaces known as sub-aerial biofilms (SABs). These SABs cause aesthetic alterations and discoloration of the surface. However, recent empirical evidence has highlighted that SABs do not always have a negative impact on the chemical and mechanical properties of the substrate. Hence, the ongoing debate regarding the biofilm’s role, whether it is protective, neutral, or deteriorative, remains unresolved and the research on the topic calls for new methods able to map the presence of traces of biological materials on colonized surfaces and investigate their biological activity. In this context, we suggest using a multimodal hyperspectral imaging (HSI) approach to investigate carbonatic stone samples intentionally colonized in laboratory with a cyanobacterial SAB, which served as a model system for investigating colonized stone surfaces. HSI is a method that combines optical imaging with spectroscopy to obtain spatially resolved spectral information of a sample. This is done by creating the so-called data-cubes, in which each element (pixel) at the image plane carries the information about the spectrum of the light diffusely reflected and/or emitted at that specific location of the sample surface. The approach is implemented here using an HSI camera made of a compact birefringent interferometer - called TWINS - coupled with a monochrome camera. The method provides high robustness and flexibility, as well as high collection throughput thanks to the absence of any type of filter. Indeed, the acquisition time is also strongly reduced compared to standard HSI imaging, and the optical dose delivered to biological samples is considerably lower, even when performing HSI in fluorescence modality. In the present experiments, the HSI camera has been coupled to a collecting optical system to achieve a field of view of 1.5 cm with a spatial resolution <10 μm. For reflectivity studies, the sample is illuminated with 2 halogen lamps, while for fluorescence experiments, a LED source emitting at 630 nm is used. Diffuse reflectance and fluorescence HSI data were sequentially collected for each colonised stone sample. The obtained hyperspectral data were compared with high-definition 3D microscopic maps. The results highlighted those areas appearing as non-colonized under microscopy showed emission peaks at wavelengths indicative of the presence of the cyanobacterial SAB. Indeed, obtaining information on the real distribution of the SABs on porous substrate could help the conservators to deal with biocolonization also in the perspective of preserving it.