Anyone who uses fluorescence detection of single molecules to investigate protein structures and dynamics within cells eventually faces the problem of isolating individual molecules from areas with high concentrations of proteins, such as muscle fibers. Researchers at the Mayo Clinic in Rochester, Minn., led by Thomas P. Burghardt, have developed a variation of total internal reflection microscopy that enables them to form images from sample volumes as small as 3 al. The scientists report in the March 24 issue of Biophysical Journal’s BioFast that they coated standard glass coverslips with a 30- to 40-nm layer of aluminum and placed fluorescently tagged rabbit muscle fibers in a watertight chamber atop the slide. They acquired images of the fibers that were on the metal film with through-the-objective total internal reflection excitation using an Olympus 1.45-NA objective and an inverted microscope from Carl Zeiss Inc. of Oberkochen, Germany. For comparison, epifluorescence images of the fibers were acquired in the absence of the metal film using a Zeiss confocal laser scanning microscope and a 63x, 1.20-NA objective. Fluorescence detection was by either an avalanche photodiode from PerkinElmer OptoElectronics of Fremont, Calif., or a 16-bit CCD camera. For the technique, excitation light from a 514-nm laser passes through the glass-metal and metal-water boundaries, totally internally reflects across the layers and emerges as refracted rays in the water medium. The metal film permits negligible light transmission for all incidence angles, but light entering at about 66° induces enhanced fluorescence transmission via the resonant excitation of the aluminum’s surface plasmons at the metal-glass boundary. The surface plasmons create an evanescent field that not only excites fluorophores in the sample volume within a short distance of the boundary, but also decays exponentially as it goes deeper into the sample. The fluorophore emissions that are transmitted above the critical angle for total internal reflection are collected by the objective. All other emissions are reflected by the aluminum film and, thus, do not enter the objective. This reduces the background fluorescence, providing a larger signal-to-noise ratio and reducing the detection volume.