Soil microbial processes exert a major influence on many of our planet's ecosystems, yet our ability to directly observe the enzymatic and metabolic activities of microbes within soil is currently critically limited. This in part due to the complexity of soil microbial communities, but another significant limitation is the lack of experimental tools to study these communities and their molecular interactions in situ. Thus, we propose here to develop a novel microscope that combines complementary imaging modalities to overcome these challenges and gain in situ insights into metabolic cycling in soil. Ultimately, this proposed 'high-resolution isotopic microscope' has the potential to be a transformational technology that will enable us to simultaneously visualize the microbial and the molecular fate of environmentally-relevant substrates. This capability will enhance our understanding of the microbial and metabolic interactions occurring within soil communities that are relevant to carbon degradation, among other soil processes. We will develop this instrument at EMSL, where it will then be available to the entire EMSL User Base, enabling a variety of related DOE-relevant systems to be interrogated in the future. Specifically, our goal is to create an integrated platform that combines fluorescence microscopy, Raman microspectroscopy, and nanospray desorption electrospray ionization (21 Tesla) Fourier transform ion cyclotron resonance mass spectrometry (nanoDESI-FTICR-MS) that can exploit the use of both fluorescent- and stable isotope-labels to directly investigate microbial activities and molecular transformations occurring in soil. Explicitly, our integrated instrument will use: (a) fluorescence detection to map microbial gene activity and uptake of polysaccharides by bacteria, (b) Raman detection to visualize incorporation of these carbon species into microbial cells, and (c) ultrahigh-resolution mass spectrometry imaging (MSI) to detect liberated carbon and other metabolites. We will apply this multimodal imaging platform to visualize, in situ, the interactions between bacteria, fungal and plant biomass, and soil carbon in a series of mesocosms of increasing complexity.