Molecular fingerprints acquired on a small scale
Microscopy technique measures nanobeads on viruses
At Max Planck Institut für Biochemie in Martinsried, Germany,
a team has succeeded in taking some very small fingerprints, using scattering-type
near-field scanning optical microscopy (s-NSOM) to measure the infrared spectra
of individual nanobeads and viruses. The particles were as small as 18 nm.
The near-field technique complements other methods
of detecting nanoparticles, such as light microscopy, by providing a means to identify
the makeup of particles without the need to label them first. Researcher Markus
Brehm noted that this could be helpful when characterizing engineered nanodevices
or when researching biological specimens. “The technique will be useful whenever
chemical identification of material is needed at nanoscale resolution,” he
In using s-NSOM, he exploited the fact
that a sharp, conductive probe tip confines nearby electromagnetic fields and enhances
them. These near-field augmentations make it possible for the tip to sense local
properties of particles and materials.
The optical properties of a sample
near the probe are determinable by measuring the light scattering from the tip.
In addition, the sharpness of the tip — not the wavelength of the scattered
light — determines the lateral resolution. Thus, resolutions of less than
20 nm are possible using visible or infrared illumination. In their experiments,
the researchers used tips with a diameter of roughly 30 nm, as measured by electron
The investigators report in the July
issue of Nano Letters that they used a line-tunable carbon monoxide laser
from Invivo GmbH of Adelzhausen, Germany, that produced a beam of ∋6 μm
wavelength. They also used cantilevered, platinum-covered silicon tips from MikroMasch
Inc. of Wilsonville, Ore., which they mounted in a custom-built scanning force microscope.
They operated the microscope in a tapping mode, forcing the tips up and down about
25 nm at 33 kHz. When capturing infrared spectra, they moved the sample beneath
To create a scattering near-field effect,
they focused the laser onto the moving tips. Some of the light reflected back, and
they superimposed this onto a reference beam in a Michelson interferometer. They
captured the resulting signal using a mercury-cadmium-telluride infrared detector
from Kolmar Technologies Inc. of Newburyport, Mass. By regularly shifting the reference
beam’s phase, they separated amplitude and phase from each other in the interferometric
signal. After demodulating this signal, the researchers had the s-NSOM output.
In a series of studies, they first
looked at spherical beads of polymethyl methacrylate (PMMA) that varied in diameter
from 30 to 70 nm. They also examined tobacco mosaic viruses, which were cylindrical
and had a diameter of 18 nm. They chose these subjects for the demonstration in
part because they had distinct vibrational resonances in the spectral region covered
by the laser that was being used. They put the particles into suspension, dispersed
that across a silicon or gold-coated silicon substrate, and then dried it. They
mapped the substrate and the particles using their s-NSOM setup.
Brehm reported that the researchers
did find something unexpected when they looked at their results. “We were
quite surprised by the strength of the spectral signature,” he said.
He added that this outcome was predicted
when they used a simple dipole model on the various electromagnetic interactions.
Whether in theory or practice, the enhancement from near-field effects was pronounced,
upping the signal scores of times. In conventional infrared transmission spectrometers,
a sample thickness of ~1 μm would be needed to generate a signature of
Near-field scanning optical microscopy
was used to visualize tobacco mosaic virus, with an additional component that measured
the light scattered from polymethyl methacrylate (PMMA) particles placed on the
virus. Numbers indicate the height of the PMMA particles in nanometers. Courtesy
of the American Chemical Society.
As might be predicted given the strength
of the signal, the researchers were successful in acquiring telltale infrared signatures.
For example, they found a phase contrast peak with the substrate near 1730 cm—1
for a 68-nm PMMA bead, which agrees with known absorbance spectra. They also found
a phase contrast peak near 1660 cm—1 for virus particles, again in agreement with
known absorbance spectra. By comparing the s-NSOM results with the size of the particles
as determined by other methods, the researchers estimated that the spatial resolution
of their instrument was ~30 nm. They also calculated that the amount of sample
needed for a measurement was about 10—20 l.
Although the demonstration was promising,
some improvements must be made before the technique can be widely applied. The clear
difference between the substrate, which has no spectral features in the frequency
region of the probe beam, and the virus particles varied according to the infrared
frequency. Thus, to build up a complete spectrum of a single virus, the investigators
had to repeat the scan using a different frequency. Each pass took 10 minutes, along
with some laser configuration time. Consequently, extracting the spectrum of a single
particle took a day — too long for many applications. It also is difficult
to maintain the setup for that long.
Brehm reported that the researchers
were working on enhancements to the technique that would eliminate this problem.
“The key is to move from our sequential way of taking spectra — taking
images repeatedly at different wavelengths — to a parallel method that takes
a complete spectrum at each pixel during scanning,” he said.
He noted that progress has been made
by combining infrared frequency-comb spectroscopy with near-field microscopy. The
group also is working with theoreticians to improve the modeling of the scattering
from the tip-particle geometry substrate, something that will be necessary to more
completely interpret the measurements.
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