Adding metal film to total internal reflection microscopy enhances images
Lynn M. Savage
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.
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