Visualization and quantification of cell monolayer integrity and single cell activity using metal clad waveguide imaging based on a high numerical aperture microscope objective

Surface plasmon resonance (SPR) was recently demonstrated by our group and others as a valuable label-free approach for real-time monitoring of changes in cell activity induced by hormones, pharmacological agents, and toxins. For a cell-based biosensing application, cells are cultured on a substrate acting as the sensing layer. Receptors present at the cell's surface recognize biomolecules in the media surrounding the cell, triggering distinct intracellular events such as cell contraction and detachment. Such changes results in refractive index changes on the surface, detectable by SPR. Like most label-free cell-based biosensing approaches, the SPR signal arises from a complex cascade of molecular events within the cell. Additional complexity originates from a heterogeneous cell response within the population. In this context, delineating the cellular events contributing to the measured SPR signal can be challenging. To address this problem, we used metal clad waveguide (MCWG) imaging as an alternative to SPR. MCWGs allow for a higher sensitivity and deeper evanescent field penetration depth compared to conventional SPR. Furthermore, our setup is based on a high numerical aperture microscope objective allowing us to discriminate individual cell activity within the evanescent field at a high spatial resolution. We evaluated the performance of our imaging system by detecting individual cell-cell responses within a cell monolayer exposed to endotoxins. Our findings indicate that the sensor signal during toxin exposure originates from the appearance of intracellular gaps. Further, we followed the activation of a G-protein coupled receptor (GPCR) and show that during a GPCR mediated contraction, the sensor signal represents a cell mass redistribution towards the cell center.