Fungi and bacteria are found living together in a wide variety of environments. Their interactions are significant drivers of many ecosystem functions and are important for the health of plants and animals. A large number of fungal and bacterial families are engaged in complex interactions that lead to critical behavioural shifts of the microorganisms ranging from mutualism to pathogenicity. The importance of bacterial-fungal interactions (BFI) in environmental science, medicine and biotechnology has led to the emergence of a dynamic and multidisciplinary research field that combines highly diverse approaches including molecular biology, genomics, geochemistry, chemical and microbial ecology, biophysics and ecological modelling. In this review, we discuss most recent advances that underscore the roles of BFI across relevant habitats and ecosystems. A particular focus is placed on the understanding of BFI within complex microbial communities and in regards of the metaorganism concept. We also discuss recent discoveries that clarify the (molecular) mechanisms involved in bacterial-fungal relationships, and the contribution of new technologies to decipher generic principles of BFI in terms of physical associations and molecular dialogues. Finally, we discuss future directions for researches in order to catalyse a synergy within the BFI research area and to resolve outstanding questions.

We present spatial light interference microscopy (SLIM) as a new optical microscopy technique, capable of measuring nanoscale structures and dynamics in live cells via interferometry. SLIM combines two classic ideas in light imaging: Zernike’s phase contrast microscopy, which renders high contrast intensity images of transparent specimens, and Gabor’s holography, where the phase information from the object is recorded. Thus, SLIM reveals the intrinsic contrast of cell structures and, in addition, renders quantitative optical path-length maps across the sample. The resulting topographic accuracy is comparable to that of atomic force microscopy, while the acquisition speed is 1,000 times higher. We illustrate the novel insight into cell dynamics via SLIM by experiments on primary cell cultures from the rat brain. SLIM is implemented as an add-on module to an existing phase contrast microscope, which may prove instrumental in impacting the light microscopy field at a large scale.

Herein, we show that the geometrical cues of the substrate influence the differentiation process of C2C12 myoblasts.

The characterization of physical properties of cells such as their mass and stiffness has been of great interest and can have profound implications in cell biology, tissue engineering, cancer, and disease research. For example, the direct dependence of cell growth rate on cell mass for individual adherent human cells can elucidate the mechanisms underlying cell cycle progression. Here we develop an array of micro-electro-mechanical systems (MEMS) resonant mass sensors that can be used to directly measure the biophysical properties, mass, and growth rate of single adherent cells. Unlike conventional cantilever mass sensors, our sensors retain a uniform mass sensitivity over the cell attachment surface. By measuring the frequency shift of the mass sensors with growing (soft) cells and fixed (stiff) cells, and through analytical modeling, we derive the Young’s modulus of the unfixed cell and unravel the dependence of the cell mass measurement on cell stiffness. Finally, we grew individual cells on the mass sensors and measured their mass for 50+ hours. Our results demonstrate that adherent human colon epithelial cells have increased growth rates with a larger cell mass, and the average growth rate increases linearly with the cell mass, at 3.25%/hr. Our sensitive mass sensors with a position-independent mass sensitivity can be coupled with microscopy for simultaneous monitoring of cell growth and status, and provide an ideal method to study cell growth, cell cycle progression, differentiation, and apoptosis.

Microfluidic devices have been used to study high-density cultures of many cell types. Because cell-to-cell signaling is local, however, there exists a need to develop culture systems that sustain small numbers of neurons and enable analyses of the microenvironments. Such cultures are hard to maintain in stable form, and it is difficult to prevent cell death when using primary mammalian neurons. We demonstrate that postnatal primary hippocampal neurons from rat can be cultured at low densities within nanoliter-volume microdevices fabricated using polydimethylsiloxane (PDMS). Doing so requires an additional fabrication step, serial extractions/washes of PDMS with several solvents, which removes uncrosslinked oligomers, solvent and residues of the platinum catalyst used to cure the polymer. We found this step improves the biocompatibility of the PDMS devices significantly. Whereas neurons survive for ≥ 7 days in open channel microdevices, the ability to culture neurons in closed-channel devices made of untreated, native PDMS is limited to ≤ 2 days. When the closed-channel PDMS devices are extracted, biocompatibility improves allowing for reliable neuron cultures at low densities for ≥ 7 days. Comparisons made to autoclaved PDMS and native, untreated PDMS reveal that the solvent-treated polymer is superior in sustaining low densities of primary neurons in culture. When neuronal affinity for local substrates is observed directly, we find that axons localize to channel corners and prefer PDMS surfaces to glass in hybrid devices. When perfusing the channels with media by gravity flow, cultured hippocampal neurons survive for ≥ 11 days. Extracting PDMS improves biocompatibility of microfluidic devices and thus enables the study of differentiation of identifiable neurons and the characterization of local extracellular signals.

Wiring the nervous system relies on the interplay of intrinsic and extrinsic signaling molecules that control neurite extension, neuronal polarity, process maturation and experience-dependent refinement. Extrinsic signals establish and enrich neuron–neuron interactions during development. Understanding how such extrinsic cues direct neurons to establish neural connections in vitro will facilitate the development of organized neural networks for investigating the development and function of nervous system networks. Producing ordered networks of neurons with defined connectivity in vitro presents special technical challenges because the results must be compliant with the biological requirements of rewiring neural networks. Here we demonstrate the ability to form stable, instructive surface-bound gradients of laminin that guide postnatal hippocampal neuron development in vitro. Our work uses a three-channel, interconnected microfluidic device that permits the production of adlayers of planar substrates through the combination of laminar flow, diffusion and physisorption. Through simple flow modifications, a variety of patterns and gradients of laminin (LN) and fluorescein isothiocyanate-conjugated poly-L-lysine (FITC–PLL) were deposited to present neurons with an instructive substratum to guide neuronal development. We present three variations in substrate design that produce distinct growth regimens for postnatal neurons in dispersed cell cultures. In the first approach, diffusion-mediated gradients of LN were formed on cover slips to guide neurons toward increasing LN concentrations. In the second approach, a combined gradient of LN and FITC–PLL was produced using aspiration-driven laminar flow to restrict neuronal growth to a 15 µm wide growth zone at the center of the two superimposed gradients. The last approach demonstrates the capacity to combine binary lines of FITC–PLL in conjunction with surface gradients of LN and bovine serum albumin (BSA) to produce substrate adlayers that provide additional levels of control over growth. This work demonstrates the advantages of spatio-temporal fluid control for patterning surface-bound gradients using a simple microfluidics-based substrate deposition procedure. We anticipate that this microfluidics-based patterning approach will provide instructive patterns and surface-bound gradients to enable a new level of control in guiding neuron development and network formation.