Original paper

Biofilms fluorescence and image analysis in hypogean monuments research

Roldán, Mónica; Clavero, Ester; Castel, Susanna; Hernández-Mariné, Mariona


Confocal Laser Scanning Microscopy (CLSM) was used to investigate aerophytic phototrophic biofilms thriving on artificially illuminated surfaces in Roman hypogean monuments, particularly the St. Callistus and Domitilla Catacombs (Rome, Italy). Phototrophic organisms were visualized by their in vivo pigment fluorescence, whereas extrapolymeric substances (EPS) and DNA structures of both photo- and heterotrophic microorganisms were revealed respectively with the carbohydrate recognising lectin Concanavalin-A conjugated with the fluorophore Alexa Fluor 488 and the fluorochrome Hoechst 33258 labels. Inherent fluorescences other than pigment fluorescence such as CaCO3 fluorescence of the substratum were also detected. Distribution of microorganisms and EPS in biofilms and their relationship with substratum was evidenced with the application of diverse kinds of image analyses. Detailed localization of certain structures or taxa was achieved with the examination of gallery images. On the other hand, particular image analyses enlightened one or several partial aspects of the biofilm heterogeneity. Extended focus images built with the maximum intensity projections of the x-y, x-z, and y-z planes were best to compare among biofilms and to reveal their porosity and stratification. 3D-reconstructions of biofilms as perspective images gave an account of the surface characteristics such as roughness or coverage of the substratum surface. Topographic reconstruction was used to measure geometrical and biological heterogeneity parameters like sample thickness or surface roughness. What makes CLSM so powerful is the ability to analyse samples with minimal preparation and non-disturbing architectural observation, the use of multiple excitation and detection wavelengths at different depths of focus, and the large pool of digital image processing algorithms with which one can effectively target specific elements in the biofilm sample, such as molecules (e.g. DNA and pigments), structure (e.g. surfaces, matrix, sheaths and filaments) and properties (e.g. stage of cell division, growth and senescence), with the capability to fully visualise them all in 3D image.


biofilmsroman catacombsconfocal laser scanning microscopy (clsm)cyanobacteriacon-aepshypogean environmentshoechst 332583d-image structure of biofilms