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Optical imaging of individual scattering/absorbing nanoparticles has recently emerged as an important research topic in intracellular and biological assay imaging (see Optical scatter imaging. This is prompted by interests in detecting nanoparticles in biological assays, for example, immunolabel nanoparticles with a size of 40 nm to 80 nm (Schultz et al 2000). However, demands placed on the mobility and non-invasiveness of nanoparticles for use within cells (for example, Chithrani et al 2006) necessitate further reduction of nanoparticles' sizes. This reduction requires, in turn, more sensitive methods of detecting and characterising such small particles.
In detecting a particle by optical means, its scattering efficiency as well as the absorption efficiency are important factors. The scattering efficiency decreases with the relative particle size, x, as ~x4 (the Rayleigh law of scattering for x << 1, Eq. 3 in Mie theory: Small-particle limit equations). When expressed for the scattered intensity as a function of the absolute particle size D, this law becomes ~D 6. The absorption efficiency decreases much slower (as ~x) in this particle size range. This raises a fundamental question of what is the size of the smallest scattering/absorbing nanoparticle detectable by optical means? Here, we briefly discuss the progress on lowering that particle size.
In a typical diffraction-limited microscopy system, a nanoparticle is imaged as a diffraction-limited spot whose brightness varies according to the scattering efficiency of the nanoparticle according to the Rayleigh law of ~x6 (for example, Colpin et al 2006) but whose size is independent of the particle-size. Fig. 1 illustrates a dramatic difference in the heights of the irradiance peaks across images of particles with different sizes, whereas widths of the irradiance peaks (i.e. the particle image sizes) are roughly equal.
The detectability of a nanoparticle in a biological assay background depends not only on the brightness of the particle image but also on the contrast it yields against that background. In the 80's, an intriguing technique of video-enhanced contrast optical microscopy emerged (Allen 1985). By using this technique, individual fine sub-cellular structures, such as microtubules and 10-nm vesicles were successfully imaged. It was a truly remarkable achievement considering noisy video cameras and limited computing power of that time.
Other techniques, developed to minimize the effect of light-scattering by the nanoparticle environment, also helped to push down the size limit of a particle which can be detected optically. For example, photothermal imaging (for example, Zharov and Lapotko 2005) allowed detecting 2.5 nm light-absorbing gold nanoparticles embedded in a polymer film (Boyer et al 2002) and 10-nm light-absorbing gold nanoparticles in a biological cell environment (Cognet et al 2003). According to the authors' interpretation, this technique exploits a much slower decrease of the absorption efficiency with particle size (~x, see Eq. 4 in Mie theory: Small-particle limit equations) than that of the scattering efficiency (~x4). Hence, illumination of a particle by a heating beam causes sizeable heating of the immediate environment of the particle although the scattering of light by the particle may be difficult to detect. A resulting thermally-induced change in the refractive index of that environment can be sensed by a probe beam at a longer wavelength to minimize light scattering by the particle environment.
A combination of confocal microscopy and interference microscopy enabled Lindfors et al (2004) to detect 5-nm gold nanospheres and 10-nm dielectric nanoparticles. Such progress suggests that detection of non-labelled viruses may be feasible. Another approach to minimize the background light scattering and hence maximize the particle detectability has been demonstrated by Ignatovich and Novotny (2006), who enhanced a technique of using the interference of the back-reflected illuminating wave and the scattered wave (Batchelder and Taubenblatt 1989). Ignatovich and Novotny used a split detector to differentiate between a steady interference of the illuminating and background-scattered light and a time-dependent interference signal resulting from a passage of a particle moving in a microfluidic channel across the field-of-view of the detector. This effectively elliminated the background contribution and permitted to detect ~10-nm particles within several milliseconds.
It seems that careful design of a microscopy imaging system should enable detection of individual macromolecules, including proteins, and therefore, has an invaluable niche in biomedical imaging and nanoparticle characterization. Note that detection of a small light-scattering particle often depends on an ability to discriminate between the particle and other scattering objects, for example, intracellular constituents in the living cell. Therefore, detection of light-scattering particles with a size of several nanometers may be possible only in a highly homogeneous non-scattering environment.
| CITATION: Zvyagin A. V., Plakhotnik T. 2006. Optical scatter imaging: Detection limits (www.tpdsci.com/Tpc/OSIDetLim.php). In: Top. Part. Disp. Sci. (www.tpdsci.com). |
HISTORY: Published: 24-Nov-2006 Modified: 15-Jun-2007 Peer-reviewed: 14-Mar-2007 |
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