In soil, microbial communities survive, reproduce and die in a complex 3-D physical framework. This 3-D framework has variable geometry, composition and stability across scales spanning several orders of magnitude, from the molecular to the landscape. Consequently, external environmental conditions do not have a uniform effect throughout the soil enabling a large diversity of microhabitats to develop. This allows microbial processes with contrasting requirements for substrate and environmental conditions, such as e.g. nitrification and denitrification, to occur simultaneously. For the most part, studies have been done at experimentally convenient scales far larger than the scales at which microbial communities exist and function. Recent research has indicated that the distribution of micro-organisms at the micro-scale and their interactions with the physical micro-habitat are non-random and has suggested that the micro-scale distribution of bacteria has a direct effect on activity. Although there is an increasing acceptance of the need to study spatio-temporal patterns at micro- and nano-scales in soils in order to fully understand macro-scale behaviour (at field and catchment scales), few studies have attempted to address the spatial 3-D complexity of the soil with respect to soil biotic interactions and process rates. A major obstacle to progress is the lack of techniques of adequate sensitivity for data collection at the appropriate scales needed to integrate an understanding of structural heterogeneity of the soil physical and chemical environment with the ecological function of microbial communities. Nano-Secondary Ion Mass Spectrometry (NanoSIMS) is a cutting edge technology that links high resolution microscopy with isotopic analysis. NanoSIMS involves bombardment of a sample with an energetic primary beam (usually ¹³³Cs⁺ or ¹⁶O^-). This results in sputtering of the sample surface and the liberation of secondary ions that are dispersed in a mass spectrometer according to their energies and their mass to charge ratios. An image ('map') can be formed for any selected mass and a number of ionic species can be recorded simultaneously. The power of NanoSIMS lies in the ability of the instrument to distinguish stable isotopes of elements with a high sensitivity, i.e. concentrations in parts per million can be detected. The level of spatial resolution achievable is better than 50 nm (¹³³Cs⁺ primary beam) with NanoSIMS, a significant improvement on other SIMS instruments and on X-ray micro-analytical techniques. The high resolution of NanoSIMS allows the tracing of isotopes into individual bacteria and the simultaneous exploration of factors that determine micro-site heterogeneity in soil. NanoSIMS has therefore the potential to improve our understanding of links between the 3-D architecture of the soil matrix, the location of key microbial communities and the regulation of the fate and cycling of nutrients within soils. Our objective is to explore the potential of NanoSIMS for the study of the physical-chemical-biological interface in soils. Currently, we are focusing upon soil sample preparatory techniques and soil application development. We will report the use of NanoSIMS to measure, in parallel, the ²⁸Si^-, ¹²C^-, ¹³C^-, ¹²C¹⁴N^-, and ¹²C¹⁵N^- ion currents in soils inoculated with Pseudomonas fluorescens cultures that had been cultured in mineral medium containing glucose and either natural abundance (NH₄)₂SO₄ or as 54 atom% (¹⁵NH₄)₂SO₄. Practical considerations for NanoSIMS analysis and soil sample preparation will be presented; and implications for integrating the physical-chemical-biological interface in soils using NanoSIMS as a new analytical tool will be discussed.