Spatial and temporal profiling of metabolites within and between living systems is vital to understanding how chemical signaling shapes the composition and function of these complex systems. Measurement of metabolites is challenging because they are often not amenable to extrinsic tags, are diverse in nature, and are present with a broad range of concentrations. Moreover, direct imaging by chemically informative tools can significantly compromise viability of the system of interest or lack adequate resolution. The development of technologies to “image” metabolites is essential to provide spatiotemporal resolution that cannot be obtained by previous approaches. Here, we present a nano-enabled and label-free imaging technology using a microfluidic sampling network to track production and distribution of chemical information in the microenvironment of a living organism. We demonstrate the sampling of exometabolites secreted from a growing plant over time, providing spatial and temporal information about development within a model environment. We describe two approaches that integrate a polyester track-etched (PETE) nanofluidic interface to physically confine the biological sample within the model environment, while allowing fluidic access via an underlying microfluidic network. The nanoporous interface enables sampling of the microenvironment above in a time-dependent and spatially-resolved manner. Chemical information was extracted at two different locations, relative to the growing plant root, over time. The flow of metabolites was controlled by tuning the sample aperture size or “pixel size” of the patterned PETE membrane. The diffusional flux through the PETE membrane was characterized to understand membrane performance. Exometabolites of a growing plant root were successfully profiled in a space- and time-resolved manner. This method and device provide a frame-by-frame description of the chemical environment that maps to the physical and biological characteristics of the sample.