# Methods for Studying Bacterial-Fungal Interactions in the Microenvironments of Soil
## 3 Get Your Hands Dirty: Methods for Studying BFI
### Playing Hide and Seek with Bacteria and Fungi: Unravel Their Physical Interactions
In addition, bleaching caused during image acquisition and the impossibility to view \"the whole picture\" in a single ocular field can be an additional problem. Visualization techniques do generally require long processing times, a lot of handling skills, and result interpretation experience. This probably explains why there is still a relatively low number of articles published on BFI, especially given that these tools have been around for already few decades. Lastly, but most importantly, a fundamental problem remains: when samples are physically altered and chemically fixed (i.e., killing), it is impossible to monitor microbial cell dynamics in situ and in vivo. With regard to non-invasively studies on soil microbes in their undisturbed, structured environment, several new technical approaches have been developed and proposed during recent years: X-ray computed tomography (CT) [3, 111] and its variations (e.g., optical coherence tomography (OCT) [60], synchroton tomography (SR-CT) [163], and X-ray nanotography [164]) have been used, in combination with other methods, to study the reciprocal interaction between microbes and their surrounding environment, in both native and artificial soils [41, 96, 97, 98, 102, 165]. With X-ray CT, for example, the effect of the soil environment on fungal exploration behaviour and gas release was investigated by repeated scanning of microcosm systems over several weeks [90]. This type of approach was also adopted for direct observation of plant-pathogen interactions [166], and to detect the migratory capabilities of _Pseudomonas fluorescens_[167, 168] in situ at high spatial resolution for the first time. However, studies on BFI implementing these techniques have lacked knowledge up to now. A fundamental problem of radiation-based imaging is that it is ionising and penetrative, meaning that it could possibly damage both structures and microorganisms within the substrate [164]. On the other hand, low irradiation doses applied to plant-soil interfaces were reported to neither influence root growth [169], nor microbial cell number, microbial community structure, or their potential activity [165, 170]. Nevertheless, even if not lethal, care should be taken for the insurgence of mutations with unpredictable consequences. Lastly, the imaging resolution chosen can limit either the range or the feature of detection [111]. When concerned with the in vivo study of dynamically interacting organisms, simple and reliable applications for the live-cell screening of microbial organisms is the use of fluorescent protein-expressing bacteria (e.g., GFP, [85, 142, 171]) or fungi (e.g., tdTomato, [172]). A combination of red and green fluorescent proteins allows for a simultaneous detection of multiple organisms, and can be used to follow BFI both in situ and in vivo [172, 173]. Although these modern molecular tools only permits to work with isolates or model organisms, if combined with a relevant experimental set-up, such as microfluidics, video microscopy, or other techniques, described in the next section, they can give a great deal of advantages [174].