# Methods for Studying Bacterial-Fungal Interactions in the Microenvironments of Soil
## 3 Get Your Hands Dirty: Methods for Studying BFI
### What Are They Doing? Investigating the Molecular Interactions in BFI  
The ability to confine small numbers of microorganisms and determine their location in space is an important step towards understanding the impact of spatial structure on microbial behaviour. However, the development of techniques for the in situ measurement of small molecule signals, and for the detection of other factors responsible for behavioural modulation, are equally important. These techniques are applicable to simple bacterial and fungal communities and ultimately allow cell-to-cell resolution. Scanning electrochemical microscopy (SECM) can be considered as the most straightforward technique to quantify and spatially map the concentration of specific redox-active molecules (i.e., one compound at the time) proximal to populations of cells [175], such as the small molecules that are involved in the interaction in multispecies biofilms [176]. For more comprehensive studies, with imaging mass spectrometry (IMS), in all its variations, one can detect and describe multiple molecules at the same time, and superimpose their spatial distribution onto optical or fluorescence images of the sample. To put these advantages in perspective, a key difference between IMS and standard fluorescence microscopy is the capability of IMS to detect up to thousands of unique signals from one biological substrate, compared to only up to eight in the average fluorescence microscopes [177]. The interplay between competing organisms, the metabolic exchange in complex intra- and inter- species signaling, or the detection of new and uncharacterized molecules can be studied with this IMS technique. Furthermore, the direct link between the distribution of detected compounds and the observed phenotypes of the biological sample may give essential information on the function of the molecules themselves. Finally, the endogenous molecules can directly be analysed in their microenvironment, without the indirect biases that artificial substrates may cause (e.g., isotope feeding experiments) [177; 178; 179]. Matrix-assisted laser desorption-ionization (MALDI) [180] and desorption electrospray ionization (DESI) [181] are two types of IMS techniques that suit particularly well for studying the production of natural products by microorganisms in the laboratory [178]. Whereas the soft and absorbing nature of agar medium limits DESI-IMS analysis, with MALDI-IMS microorganisms can be grown on a 0.5-1.5-mm layer of agar for a defined period of time before being covered with a matrix and subjected to the analyses. Albeit certain media that have a high salt or sugar content may prove difficult to analyse, owing to ion suppression or uneven matrix crystallization, the use of agar-based IMS approach can provide important time-dependent correlations [180]. MALDI- and DESI-IMS have been widely applied for the characterization of microbial monocultures, such as cyanobacteria [182], bacterial colonies [183], or fungal-fungal interaction [184], and their application in BFI studies is slowly increasing, especially for the detection of antifungal compounds in co-culturing experiments [185; 186; 187]. Some of the limitations of these techniques derive from biases due to sample preparation, the simplification of otherwise complex environments, and signal distortion from background noise [179]. Secondary ion mass spectrometry (SIMS) [188] has the highest resolution (order of nanometres) of all the IMS techniques due to its extremely narrow ion beam. It can be applied to have either a focus on the analytical mass range (static) or to have a greater spatial resolution (dynamic). SIMS platforms can also perform very fine depth-profiling experiments by etching away the sample surface with the ion beam [178]. When an ultra-sensitive characterization of the molecular microenvironment between interacting cells is needed (down to 50 nm of resolution), nanoSIMS can be used. Obviously, with this level of resolution, the sampling area that can be analysed decreases to a few mm\({}^{2}\).