High spatial resolution observation of single-molecule dynamics in living cell membranes

Abstract

Self-organized lipid bilayers together with proteins are the essential building blocks of biological membranes. Membranes are associated with all living systems as they make up cell boundaries and provide basic barriers to cellular organelles. It is of interest to study the dynamics of individual molecules in cell membranes as the mechanism of how biological membranes function at the single molecule remains to be elucidated. In this letter we describe a study in which we incubate rat basophilic leukemia cells with a fluorescently labeled cell membrane component on a surface containing zero-mode waveguides (ZMWs). We used the ZMW to confine fluorescent excitation to an approximately 100-nm region of the membrane to monitor lipid diffusion along the cellular membrane. We showed that confinement with a ZMW largely reduced fluorescent contributions from the cytosolic pool that is present when using a more standard technique such as laser-induced confocal microscopy. We show that optical confinement with ZMWs is a facile way to probe dynamic processes on the membrane surface.

FIGURE 1

Schematic diagram of the ZMW along with an RBL cell labeled with DiI-C18. The cell has an approximate dimension of 15 μm.

FIGURE 2

Photon-burst scan of fluorescence from DiI-C18 molecules diffusing in the membrane of RBL cells within a zero-mode waveguide.

FIGURE 3

(a) FCS curves for DiI-C18 diffusion in RBL cells placed on a microscope coverslip. (b) FCS curves for diffusion within a zero-mode waveguide.