Quantitative Reflection Imaging for Morphology and Dynamics of Live Aplysia Californica Pedal Ganglion Neurons Cultured on Nanostructured Plasmonic Crystals

摘要

We describe a reflection imaging system which consists of a plasmonic crystal, a common laboratory microscope, and bandpass filters for use in the quantitative imaging and in-situ monitoring of live cells and their substrate interactions. Surface plasmon resonance (SPR) provides a highly sensitive method to monitor changes in physicochemical properties occurring at metal-dielectric interfaces. Polyelectrolyte thin films deposited using the layer-by-layer (LBL) self-assembly method provide a reference system to calibrate the reflection contrast changes that occur when the polyelectrolyte film thickness changes, as well as provide insight into the optical responses that originate from the multiple plasmonic features supported by this imaging system. Finite-difference time-domain (FDTD) simulations of the optical responses measured experimentally from the polyelectrolyte reference system are used to provide a calibration of the optical system for subsequent use in quantitative studies investigating live cell dynamics in cultures supported on a plasmonic crystal substrate. Live Aplysia californica pedal ganglion neurons cultured in artificial sea water were used as a model system through which to explore the utility of this plasmonic imaging technique. Here, the morphology of cellular peripheral structures < ~80 nm in thickness were quantitatively analyzed and the dynamics of their trypsin-induced surface detachment visualized. These results illustrate capacities of this system for use in investigations of the dynamics of ultrathin cellular structures within complex bioanalytical environments.