Metal Nanoparticles Offer New Imaging Method
As biochemical investigations focus on ever-smaller structures, methods for detecting elements as small as single molecules must be developed. The ideal label would be small enough to have no impact on molecular activity, yet would have a constant interaction cross section large enough to make the molecule easily detectable. Researchers in France and the Netherlands have recently demonstrated the imaging of nanometer-size metal particles, an approach that promises high contrast, small size and a constant detection cross section for single-molecule studies.
Metallic nanoparticles could replace fluorescent labels as biomolecular markers. In a proof-of-principle test, the signatures of the nanoparticles are clearly visible. Courtesy of Brahim Lounis.
Fluorescent dyes are commonly used in such work. And although they can be chemically grafted to molecules of interest, they are subject to photobleaching, which renders them undetectable. Semiconductor nanocrystals show potential as molecular labels but can suffer from "blinking" effects that vary their visibility with time. Rather than depending on the scattering cross section, which rapidly decreases with particle size, the proposed technique would detect the local increases in sample temperature resulting from the relatively high absorption cross section of the metal nanoparticles.
To demonstrate the concept, researchers at Centre National de la Recherche Scientifique in Talence, France, and Leiden University in the Netherlands imaged 20-, 10- and 5-nm-diameter particles of gold in a polyvinyl alcohol film. They focused the acousto-optic modulated output from a Coherent Inc. argon-ion laser operating at 514 nm onto the sample and monitored the temperature of the surrounding film.
The volume heated depends on the modulation frequency of the incident beam, and the researchers detected localized heating using a polarization interference method. They split the 632-nm output from a JDS Uniphase Corp. HeNe laser into two orthogonally polarized beams, focused them onto two adjacent spots on the sample and then recombined the reflected beams, realigning their polarization. The variation in phase difference between the two red beams was revealed as a signal modulation at the frequency of the heating beam.
The researchers maximized the intensity of the return signal by overlapping the green spot with one of the red spots and by adjusting the modulation frequency to match the heating volume and spot size. By scanning the sample through the fixed beams, they created images of the nanoparticles with a signal-to-noise ratio of approximately 10.
The initial success led the researchers to examine the impact of background scattering on the technique. The addition of 300-nm-diameter latex spheres significantly increased scattering but did not affect the imaging of the nanoparticles.
With the current setup, the increase in local temperature can be as large as 15 K, which could cause distress to biological samples. Brahim Lounis, a professor at the center and a member of the project team, said that the researchers hope to improve the signal-to-noise ratio and to reduce label heating. He believes that the approach, coupled with an appropriate detection technique for this photothermal effect to detect particles as small as 2 nm, will offer a superior alternative to fluorescence imaging.
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