Description
The ecological functioning of subsurface environments—including soils, lake and marine sediments, and aquifers—is strongly controlled by redox processes mediated by diverse microbial communities inhabiting porous media. The composition and spatial organization of these communities arise from the coupled effects of pore-scale geometry, fluid flow, microbial interactions, and microscale geochemical heterogeneity. Understanding how these factors jointly shape microbial community structure is essential for predicting subsurface ecosystem functioning and its role in biogeochemical cycling. However, progress is hindered by the limited accessibility of opaque porous matrices, which complicates direct observation of microbial dynamics and their local physicochemical environment.
Microfluidic approaches provide a great opportunity to address these challenges. Specifically, microfluidic devices that mimic the pore structure and flow conditions of natural soils and sediments are being used as powerful tools to investigate the dynamics of microbial colony formation. When combined with fluorescently tagged microorganisms, microfluidics enables non-invasive, real-time visualization of microbial colonization, self-organization, and interactions under well-defined physicochemical conditions. In parallel, microfluidic integration with transparent optical sensors, such as optodes and luminescent nanoparticles, has been shown to allow mapping of microscale physicochemical gradients, e.g., oxygen concentrations, driven by microbial activity coupled with advection and diffusion processes.
Despite these advances, the simultaneous imaging of microbial community dynamics and geochemical gradients remains technically challenging. Most existing luminescent sensors emit in the visible part of the light spectrum, overlapping spectrally with commonly used fluorescent protein tags, thereby preventing concurrent detection within a single microfluidic platform.
Here, we present a sensing microfluidic platform integrating a transparent oxygen optode emitting in the near-infrared (NIR) region of the spectrum. Spectrofluorometric characterization shows that this NIR-emitting optode avoids spectral overlap with widely used fluorescent reporters, enabling simultaneous, high-resolution imaging of different microbial populations and oxygen dynamics at the microscale.
We demonstrate the capabilities of this platform by examining the colonization of sandy sediment under flow by an aerobic microbial community and the resulting development of microscale oxygen gradients. The community comprises two bacterial strains with contrasting cell morphologies—elongated and rounded—engineered to express mScarlet and GFP, respectively. Our results reveal distinct spatial organization patterns and cluster morphologies between the two strains, consistent with morphology-dependent colonization under flow. Moreover, the data suggest that distinct cell morphologies differentially influence local oxygen gradients, highlighting a direct link between microbial physical traits and microscale redox dynamics.
Beyond this proof of concept, the proposed approach is broadly applicable to a wide range of microbial ecological applications. Spectral analyses suggest that up to four microbial populations could be imaged simultaneously alongside oxygen dynamics, and ongoing advances in NIR luminescent sensor chemistry are expanding the range of measurable physicochemical parameters. Moreover, the platform is compatible with complementary microscale analytical techniques, such as SIMS or synchrotron-based methods, enabling integrated investigations of microbial activity, geochemical gradients, and mineral transformations relevant to subsurface ecosystem functioning.