Studies of the interactions between cells and surrounding environment including cell culture surfaces and their responses to distinct chemical and physical cues are essential to understanding the regulation of cell growth, migration, and differentiation. In this work, we demonstrate the capability of a label-free optical imaging technique-surface plasmon resonance (SPR)-to quantitatively investigate the relative thickness of complex biomolecular structures using a nanoimprinted plasmonic crystal and laboratory microscope. Polyelectrolyte films of different thicknesses deposited by layer-by-layer assembly served as the model system to calibrate the reflection contrast response originating from SPRs. The calibrated SPR system allows quantitative analysis of the thicknesses of the interface formed between the cell culture substrate and cellular membrane regions of fixed Aplysia californica pedal ganglion neurons. Bandpass filters were used to isolate spectral regions of reflected light with distinctive image contrast changes. Combining of the data from images acquired using different bandpass filters leads to increase image contrast and sensitivity to topological differences in interface thicknesses. This SPR-based imaging technique is restricted in measurable thickness range (∼100-200 nm) due to the limited plasmonic sensing volume, but we complement this technique with an interferometric analysis method. Described here simple reflection imaging techniques show promise as quantitative methods for analyzing surface thicknesses at nanometer scale over large areas in real-time and in physicochemical diverse environments.