Furthermore, spectroscopic analyses elucidated that during formation of biofilms, sulfated polysaccharides initially form linear structures followed by entrapment of cyanobacterial cells. This showed that cyanobacterial cells were aligned along acidic/sulfated polysaccharides in the extracellular environment. By leveraging the advantages of MIP microscopy, such as its sub-micrometer spatial resolution, autofluorescence-free features, and high detection sensitivity, the distribution of cyanobacteria and their extracellular polysaccharides in the biofilm matrix were successfully visualized. Herein, we visualized photosynthetic microbial biofilms in a label-free manner using a super-resolution optical infrared absorption imaging technique, called mid-infrared photothermal (MIP) microscopy.

However, interference resulting from the bright autofluorescence of photosynthetic molecules and the low detection efficiency of Raman scattering make visualization a challenge. Raman spectroscopy is useful for label-free visualization of biofilm constituents based on chemical contrast. Visualization of photosynthetic microbial biofilms requires label-free methods, because labelling EPSs results in structural changes or aggregation. Although there are numerous studies on photosynthetic microbial biofilms, the spatial distribution of EPS components that are vital for microbial biofilm formation, such as exopolysaccharides and proteins, is not well understood. The formation of photosynthetic microbial biofilms comprising multispecies biomolecules, such as extracellular polymeric substances (EPSs), and microbial cells play pivotal roles in maintaining or stimulating their biological functions.

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