Multimodal imaging of fluid transport in living epithelial sheets

Abstract

Detailed knowledge on the biophysical mechanisms and local intercompartmental dynamics by which fluid and solute transport is achieved and regulated at the cellular and subcellular level is lacking. In this paper, cultures of immortalised human retinal pigment epithelium (RPE) cells have been established on coverslips coated with 50nm gold and poly-Z-lysine. These cultures divide and grow to confluence over the course of approximately 4 weeks, displaying tessalated cuboidal morphology, apical-basal polarity, and formation of tight junctions progressively over this time. Apposition to the substrate is very close, the cells being visible under evanescent field methods such as total internal reflection microscopy (TIRM), surface plasmon resonance (SPR) imaging, or TIR fluorescence, when fluorophores are introduced intracellularly. In confluent RPE cultures the formation of tight junctions isolates the apical and basolateral fluid compartment. Since the basolateral fluid space is thus restricted to that lying beneath the cells or in the intercellular space, the volume becomes very restricted and the basal cell membrane resides within the evanescent field. Evanescent field imaging techniques thus become sensitive to changes in this compartmental volume, and will be used to monitor physiological alterations occurring in response pharmacological manipulation. Use of gold-coated substrates allows SPR imaging, which gives contrast to refractive index within the evanescent field. Electrical connection to the gold also allows the measurement of transepithelial voltage and resistance from the preparation. To complete the picture, the apical membrane of the epithelial layer will be addressed by scanning ion conductance microscopy (SICM). This technique measures high-resolution 3D profiles of tissue architecture in live cells. By exploiting the gold substrate electrode this method will be further used to map the resistivity and voltage fields associated with the intercellular pathway to dissect transepithelial ion transport at the subcellular level.