Nanometer-Resolved Mapping of Cell-Substrate Distances of Contracting Cardiomyocytes Using Surface Plasmon Resonance Microscopy.

Abstract

It has been shown that quantitative measurements of the cell-substrate distance of steady cells are possible with scanning surface plasmon resonance microscopy setups in combination with an angle resolved analysis. However, the accuracy of the determined cell-substrate distances as well as the capabilities for the investigation of cell dynamics remained limited due to the assumption of a homogeneous refractive index of the cytosol. Strong spatial or temporal deviations between the local refractive index and the average value can result in errors in the calculated cell-substrate distance of around 100 nm, while the average accuracy was determined to 37 nm. Here, we present a combination of acquisition and analysis techniques that enables the measurement of the cell-substrate distance of contractile cells as well as the study of intracellular processes through changes in the refractive index at the diffraction limit. By decoupling the measurement of the cell-substrate distance and the refractive index of the cytoplasm, we could increase the accuracy of the distance measurement on average by a factor of 25 reaching 1.5 nm under ideal conditions. We show a temporal and spatial mapping of changes in the refractive index and the cell-substrate distance which strongly correlate with the action potentials and reconstruct the three-dimensional profile of the basal cell membrane and its dynamics, while we reached an actual measurement accuracy of 2.3 nm.