Label-free visualization of photosynthetic microbial biofilms using mid-infrared photothermal and autofluorescence imaging

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. 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. Visualization of photosynthetic microbial biofilms requires label-free methods, because labelling EPSs results in structural changes or aggregation. Raman spectroscopy is useful for label-free visualization of biofilm constituents based on chemical contrast. However, interference resulting from the bright autofluorescence of photosynthetic molecules and the low detection efficiency of Raman scattering make visualization a challenge. 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. 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. This showed that cyanobacterial cells were aligned along acidic/sulfated polysaccharides in the extracellular environment. Furthermore, spectroscopic analyses elucidated that during formation of biofilms, sulfated polysaccharides initially form linear structures followed by entrapment of cyanobacterial cells. The present study provides the foundation for further studies on the formation, structure, and biological functions of microbial biofilms. Photosynthetic microbial biofilms were studied using mid-infrared photothermal (MIP) microscopy in a label-free manner. The distribution of cyanobacterial cells and their extracellular polysaccharides in the biofilm matrix were successfully visualized.