Combined Ion Conductance and Atomic Force Microscope for Fast Simultaneous Topographical and Surface Charge Imaging.

We report here an advanced approach for simultaneous and independent submicroscale imaging of local surface charge and topography using microchanneled cantilevers, also known as FluidFM nanopipette probes. These hollow cantilevers with a 300 nm opening are employed for ion current measurements that provide access to the local properties of the electrical double layer using the phenomenon of ion current rectification, while also taking advantage of the force sensing capabilities for accurate probe vertical positioning and topography imaging. The independent nature of this atomic force microscope (AFM) feedback opens up a possibility to significantly increase the sensitivity for probing local surface charges in a wider range of salt concentrations, especially in electrolytes of low ionic strength (below 10 mM), where classical local ion conductance measurements with glass nanopipettes would suffer from inaccuracies and instabilities, but where the electrical double layer extends further into the liquid medium and has stronger effect on the measured ion currents for charge imaging. We demonstrate that the measurements with FluidFM do not compromise the positioning accuracy and enable accurate and simultaneous topographical and charge imaging in contact mode (similar to AFM) at high scanning rates, approaching thousands of pixels per second, therefore overtaking state-of-the-art techniques for charge mapping by at least 2 orders of magnitude (the probes reach translation rates of 120 µm s-1 equating to 2 ms per image pixel). We also reveal experimentally the physical limit of this high speed scanning, constrained by the rate of ion redistribution in surface-induced rectification required for double layer sensing and charge mapping.

Local Chemical Stimulation of Neurons with the Fluidic Force Microscope (FluidFM).

Physiological communication between neurons is dependent on the exchange of neurotransmitters at the synapses. Although this chemical signal transmission targets specific receptors and allows for subtle adaptation of the action potential, in vitro neuroscience typically relies on electrical currents and potentials to stimulate neurons. The electric stimulus is unspecific and the confinement of the stimuli within the media is technically difficult to control and introduces large artifacts in electric recordings of the activity. Here, we present a local chemical stimulation platform that resembles in vivo physiological conditions and can be used to target specific receptors of synapses. Neurotransmitters were dispensed using the force-controlled fluidic force microscope (FluidFM) nanopipette, which provides exact positioning and precise liquid delivery. We show that controlled release of the excitatory neurotransmitter glutamate induces spiking activity in primary rat hippocampal neurons, as measured by concurrent electrical and optical recordings using a microelectrode array and a calcium-sensitive dye, respectively. Furthermore, we characterized the glutamate dose response of neurons by applying stimulation pulses of glutamate with concentrations from 0 to 0.5 mm. This new stimulation approach, which combines FluidFM for gentle and precise positioning with a microelectrode array read-out, makes it possible to modulate the activity of individual neurons chemically and simultaneously record their induced activity across the entire neuronal network. The presented platform not only offers a more physiological alternative compared with electrical stimulation, but also provides the possibility to study the effects of the local application of neuromodulators and other drugs.

Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks.

Bottom-up neuroscience aims to engineer well-defined networks of neurons to investigate the functions of the brain. By reducing the complexity of the brain to achievable target questions, such in vitro bioassays better control experimental variables and can serve as a versatile tool for fundamental and pharmacological research. Astrocytes are a cell type critical to neuronal function, and the addition of astrocytes to neuron cultures can improve the quality of in vitro assays. Here, we present cellulose as an astrocyte culture substrate. Astrocytes cultured on the cellulose fiber matrix thrived and formed a dense 3D network. We devised a novel co-culture platform by suspending the easy-to-handle astrocytic paper cultures above neuronal networks of low densities typically needed for bottom-up neuroscience. There was significant improvement in neuronal viability after 5 days in vitro at densities ranging from 50,000 cells/cm2 down to isolated cells at 1,000 cells/cm2. Cultures exhibited spontaneous spiking even at the very low densities, with a significantly greater spike frequency per cell compared to control mono-cultures. Applying the co-culture platform to an engineered network of neurons on a patterned substrate resulted in significantly improved viability and almost doubled the density of live cells. Lastly, the shape of the cellulose substrate can easily be customized to a wide range of culture vessels, making the platform versatile for different applications that will further enable research in bottom-up neuroscience and drug development.

Collaborative effects of electric field and fluid shear stress on fibroblast migration.

Cells are inherently exposed to a number of different biophysical stimuli such as electric fields, shear stress, and tensile or compressive stress from the extracellular environment in vivo. Each of these biophysical cues can work simultaneously or independently to regulate cellular functions and tissue integrity in both physiological and pathological conditions. Thus, it is vital to understand the interaction of multiple stimuli on cells by decoupling and coupling the stimuli in simple combinations and by investigating cellular behaviors in response to these cues. Here, we report a novel microfluidic platform to apply the combinatorial stimulation of an electric field and fluid shear stress by controlling two directional cues independently. An integrated microfluidic platform was developed using soft lithography to monitor the cellular migration in real-time in response to an electric field and fluid shear stress in single, simultaneous, and sequential modes. When each of these stimulations is applied separately, normal human dermal fibroblasts migrate toward the anode and in the direction of fluid flow in a dose-dependent manner. Simultaneous stimulation with an electric field and shear stress, which mimics a wound in vivo, enhances the directional migration of fibroblasts by increasing both directedness and trajectory speed, suggesting the plausible scenario of cooperation between two physical cues to promote wound healing. When an electric field and shear stress are applied sequentially, migration behavior is affected by the applied stimulation as well as pre-existing stimulating conditions. This microfluidic platform can be utilized to understand other microenvironments such as embryogenesis, angiogenesis and tumor metastasis.