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Confocal Raman Microscope Analyzes Reactions

Photonics Spectra
Nov 2002
Gary Boas

A team at the University of Utah in Salt Lake City has developed an inverted confocal Raman microscopy technique that may enable researchers to study chemical reactions on individual, optically trapped particles. The setup promises to improve solid-phase chemical delivery systems such as colloidal inks, adhesives, coatings and time-release medications that are prepared as suspended particles in aqueous solutions.

Solid-phase synthesis is increasingly popular in the pharmaceutical and biotechnology industries as a means to produce molecular libraries for high-throughput screening. It is preferred over traditional, solution-phase methods because it employs relatively small amounts of reagents and because it makes it relatively simple to isolate the products from the reaction mixture.

Nevertheless, solid-phase synthesis offers little information about the surface chemistry of the substrates, the effects of the substrates on reaction kinetics and the variation in reactivity among individual particles. Therefore, it is desirable to be able to characterize the composition and reactivity of the materials spectroscopically.

Researchers at the University of Utah have demonstrated an inverted confocal Raman microscope configuration that simultaneously traps and analyzes chemical reactions on particles. Courtesy of Joel M. Harris.

In a demonstration of the technique, the research team employed a Nikon microscope in inverted configuration, using a Coherent Kr-ion laser and a Nikon 100X, 1.4-numerical aperture, oil-immersion objective to trap 5-µm silica particles. The 647-nm trapping light also excited Raman scattering from the target, which the same objective collected and imaged onto the entrance slit of a Chromex monochromator. An Andor CCD camera detected the dispersed spectrum.

The researchers found that confocal Raman microscopy combined with optical trapping offers a sensitive means to monitor the kinetics of solid-phase synthesis reactions, an improvement over the wet techniques currently used, said researcher Joel M. Harris. Those wet techniques, such as high-performance liquid, thin-layer and gas chromatography, require additional steps to cleave the product from the solid support for ex situ analysis. Moreover, he said, confocal Raman microscopy enables online, real-time analysis and requires only a few picograms of material.

Potential applications include drug delivery via biodegradable polymers, particle-labeling efficiency experiments, and polymer swelling and degradation studies. But two technical challenges remain to be addressed before the technique may be employed for single-particle analysis, Harris said. The researchers are investigating means to isolate a test particle and to add and remove reagents, and they hope to better characterize the illumination and collection efficiency for absolute quantitation.

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