Tweaks Improve Fluorescence Imaging

A simple modification to a fluorescence microscope greatly improves single-molecule imaging. Adam E. Cohen, Sabrina R. Leslie, and Alexander P. Fields of Harvard University achieve these improvements using a technique called convex-lens-induced confinement (CLIC) (Anal. Chem., DOI: 10.1021/ac101041s). In the CLIC device, the curved side of a plano-convex lens rests on a planar cover slip, creating a wedge-shaped nanoscale gap to trap molecules for single-molecule fluorescence imaging. The small dimensions of the gap improve rejection of background fluorescence and increase the achievable observation time. For example, background rejection with CLIC was 20-fold higher than with total internal reflection fluorescence imaging, another sensitive method. And because freely diffusing molecules are confined to the focal plane, the imaging time is 10,000-fold longer with CLIC than with confocal fluorescence correlation spectroscopy, yet another fluorescence method. In addition, the CLIC device excludes molecules larger than the gap depth and thus provides a rough estimate of the size of macromolecules. The accuracy of the size measurement depends on how well the curvature of the lens is known.