In microbiological studies, a common goal is to link environmental factors to microbial activities. Both environmental factors and microbial activities are typically derived from bulk samples. It is becoming increasingly clear that such bulk environmental parameters poorly represent the microscale environments microorganisms experience. Using infrared (IR) microspectroscopy, the spatial distribution of chemical compound classes can be visualized, making it a useful tool for studying the interactions between microbial cells and their microenvironments. The spatial resolution of conventional IR microspectroscopy has been limited by the diffraction limit of IR light. The recent development of optical photothermal infrared (O-PTIR) microspectroscopy has pushed the spatial resolution of IR microspectroscopy beyond this diffraction limit, allowing the distribution of chemical compound classes to be visualized at sub-micrometer spatial scales. To examine the potential and limitations of O-PTIR microspectroscopy to probe the interactions between fungal cells and their immediate environments, we imaged the decomposition of cellulose films by cells of the ectomycorrhizal fungus Paxillus involutus and compared O-PTIR results using conventional IR microspectroscopy. Whereas the data collected with conventional IR microspectroscopy indicated that P. involutus has only a very limited ability to decompose cellulose films, O-PTIR data suggested that the ability of P. involutus to decompose cellulose was substantial. Moreover, the O-PTIR method enabled the identification of a zone located outside the fungal hyphae where the cellulose was decomposed by oxidation. We conclude that O-PTIR can provide valuable new insights into the abilities and mechanisms by which microorganisms interact with their surrounding environments.IMPORTANCEInfrared (IR) microspectroscopy allows the spatial distribution of chemical compound classes to be visualized. The use of conventional IR microspectroscopy in microbiological studies has been restricted by limited spatial resolution. Recent developments in laser technology have enabled a new class of IR microspectroscopy instruments to be developed, pushing the spatial resolution beyond the diffraction limit of IR light to approximately 500 nm. This improved spatial resolution now allows microscopic observations of changes in chemical compounds to be made, making IR microspectroscopy a useful tool to investigate microscale changes in chemistry that are caused by microbial activity. We show these new possibilities using optical photothermal infrared microspectroscopy to visualize the changes in cellulose substrates caused by oxidation by the ectomycorrhizal fungus Paxillus involutus at the interface between individual fungal hyphae and cellulose substrates.
Keywords: cellulose; decomposition; infrared microspectroscopy; microenvironment; microorganism; organic matter.