Molecular diffusion in biological membranes is a determining factor in cell signaling and cell function. In the past few decades, three main fluorescence spectroscopy techniques have emerged that are capable of measuring molecular diffusion in artificial and biological membranes at very different concentration ranges and spatial resolutions. The widely used methods of fluorescence recovery after photobleaching (FRAP) and single-particle tracking (SPT) can determine absolute diffusion coefficients at high (>100 microm(-2)) and very low surface concentrations (single-molecule level), respectively. Fluorescence correlation spectroscopy (FCS), on the other hand, is well-suited for the intermediate concentration range of about 0.1-100 microm(-2). However, FCS in general requires calibration with a standard dye of known diffusion coefficient, and yields only relative measurements with respect to the calibration. A variant of FCS, z-scan FCS, is calibration-free for membrane measurements, but requires several experiments at different well-controlled focusing positions. A recently established FCS method, electron-multiplying charge-coupled-device-based total internal reflection FCS (TIR-FCS), referred to here as imaging TIR-FCS (ITIR-FCS), is also independent of calibration standards, but to our knowledge no direct comparison between these different methods has been made. Herein, we seek to establish a comparison between FRAP, SPT, FCS, and ITIR-FCS by measuring the lateral diffusion coefficients in two model systems, namely, supported lipid bilayers and giant unilamellar vesicles.

A fluorescence correlation spectroscopy (FCS) setup is built with an electron multiplying charge-coupled device camera. Although the instrument has a limited time resolution of 4 ms, compared to 0.1-0.2 mus for common instruments using avalanche photodiodes, it allows multiplexing of FCS measurements, has a software-adjustable pinhole after data collection, performs flow speed as well as flow direction measurements in microchannels and could be used to do spectral FCS. Measurements are performed on fluorescent dyes and polystyrene beads in high-viscosity media and on epidermal growth factor receptors in Chinese hamster ovary cells. Using real measurements on single spots, multiplexing of focal spots and detection elements are simulated and the results are discussed.

Background: Antimicrobial peptides are found in all kingdoms of life. During the evolution of multicellular organisms, antimicrobial peptides were established as key elements of innate immunity. Most antimicrobial peptides are thought to work by disrupting the integrity of cell membranes, causing pathogen death. As antimicrobial peptides target the membrane structure, pathogens can only acquire resistance by a fundamental change in membrane composition. Hence, the evolution of pathogen resistance has been a slow process. Therefore antimicrobial peptides are valuable alternatives to classical antibiotics against which multiple drug-resistant bacteria have emerged. For potential therapeutic applications as antibiotics a thorough knowledge of their mechanism of action is essential. Despite the increasingly comprehensive understanding of the biochemical properties of these peptides, the actual mechanism by which antimicrobial peptides lyse microbes is controversial.