Real-Time Observation of Molecular Transport across Biological Membranes with Non-Linear Optical Spectroscopy and Fluorescence Microscopy

Abstract

Molecular transport in biological systems occurs predominantly at the cellular membrane. Real-time observation of transport therefore demands a surface sensitive technique, capable of discriminating against an otherwise dominating bulk background signal. Towards this end, we have employed time-resolved second-harmonic scattering (SHS) to characterize the adsorption, as well as the transport efficiency and kinetics, of cationic dyes through living bacterial cells. SHS stems from a second-order non-linear optical response. In the presence of strong electric fields, coherent signal is produced whenever SHS-active molecules adsorb to surfaces or interfaces. For systems containing dual-surfaces, in close proximity, adsorption of SHS-active molecules to both sides results in coherent signal cancellation. Transport through membranes, as monitored by SHS, is characterized by a signal rise and decay directly proportional to the rates of adsorption and transport, respectively. The figure below depicts the dramatically different transport dynamics of malachite-green and crystal-violet in E.coli(mc4100). Both dyes rapidly transport through the outer membrane whereas only MG, well-known to stain even the outer coating of endospores, is observed to penetrate the cytoplasmic membrane. SHS results will be discussed and compared to complementary bright-field microscopy experiments.View Large Image | View Hi-Res Image | Download PowerPoint Slide