Simultaneous real-time imaging of oxygen gradients and microbial community spatial organization in confined environments

The ecological functioning of subsurface environments—including soils, lake and marine sediments, and aquifers—is largely governed by redox processes mediated by complex microbial communities inhabiting porous media. The composition and spatial organization of these communities emerge from the interplay between porous geometry, porewater flow, microbial interactions within and across populations, and microscale geochemical heterogeneities. Identifying the biological and environmental controls on microbial community structure is therefore crucial for understanding and predicting the functions of subsurface ecosystems. However, progress remains limited by the difficulty of observing microbial communities and characterizing their cell-scale geochemical environment within opaque porous matrices.

Recent advances in microscale imaging technologies offer new opportunities to overcome these challenges. Microfluidic devices—transparent platforms that reproduce the pore structure and flow conditions of soils and sediments under controlled laboratory settings—have emerged as powerful tools for investigating subsurface biogeochemical dynamics. When combined with fluorescently tagged bacteria, microfluidics enables non-invasive, real-time visualization of microbial populations and their self-organization in response to physicochemical gradients or microbial interactions. 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 challenging. Existing luminescent sensors typically emit in the visible range of the spectrum, overlapping with the emission wavelengths of commonly used fluorescent protein tags. This spectral interference has so far prevented the concurrent detection of fluorescently tagged microorganisms and sensor signals within the same microfluidic platform.

Here, we present a novel microfluidic platform integrating a transparent oxygen optode emitting in the near-infrared (NIR) region of the light spectrum. Spectrofluorometric characterization demonstrates that this NIR-emitting optode eliminates spectral interference with most used fluorescent tags, enabling simultaneous imaging of microbial populations and oxygen dynamics at the microscale.

We demonstrate the potential of this approach by investigating the progressive colonization of sandy sediment under flowing conditions by an aerobic microbial community and the associated formation of microscale oxygen gradients. The community consists of two bacterial strains representing distinct phenotypes—elongated and rounded cell shapes—engineered to express mScarletI and GFP, respectively. The results reveal clear differences in spatial organization and cluster morphology between the two strains, consistent with previous observations of shape-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 application, the proposed methodology is highly versatile. Spectral analyses indicate that up to four microbial populations could potentially be imaged simultaneously alongside oxygen dynamics. Furthermore, rapid advances in luminescent sensor chemistry are expanding the range of physicochemical parameters that can be mapped in the NIR. Finally, 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 mineralogical transformations in subsurface environments.