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RESEARCH PROFILES Inverted confocal Raman microscope is uplifting news Waste outlet
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The news was good for Joel Harris’s research team at the University of Utah. During last year’s annual Federation of Analytical Chemistry and Spectroscopy Societies conference in Detroit, Mich., postdoctoral fellow Michael Houlne received rave reviews after showing preliminary research on a new Raman microscopy configuration involving optical trapping and levitation of small structures. “We got a real charge last fall,” recalls Harris. “[Houlne] presented these results there and got some very positive feedback from the Raman spectroscopy community on it.” In this issue of Analytical Chemistry (4311–4319), Harris and colleagues introduce an inverted confocal Raman microscope for analyzing chemical reactions on single, optically trapped, 5-µm silica particles. The researchers used optical trapping and levitation to move the particle away from the coverslip and into solution to avoid fluorescence interference from the coverslip. Allowing the light to come up from below, which “inverts” the microscope, has its advantages, says Harris. The diffusion of reagents into the particle isn’t hindered by a surface, and reaction conditions mimic those of particles dispersed in solution, reports Harris’s group. The optical trapping and levitation also maintain optical alignment. The particle is centered laterally along the optical axis and stays within the focal plane of the objective, where optical forces and light collection are maximized. Combined with optical trapping and levitation, the sensitivity and selectivity of an inverted confocal Raman microscope offer more than just low background noise, explains Houlne. “I think one of the remarkable things about the experiment is that we were able to get an unenhanced Raman signal with an integration time anywhere from 15 to 30 seconds, depending on what the sample is.” Harris and Houlne add that only a few picograms of sample are required for a Raman spectrum.
Flow cell
Sample inlet
Band pass filter
Transfer optics
Quartz window
Dichroic beam splitter
Silicone rubber gasket #11/2 cover slip
Kr+ laser 647.1 nm
4 beam expander
High pass filter Eyepiece
Notch filter Transfer optics
Image splitter
Imaging camera
Monochromator CCD
Block diagram of the confocal Raman microscope.
In 2000, Harris’s group began its quest to find convenient ways of studying surface chemistry on an individual particle basis. “I became intrigued with the fact that more chemistry is being delivered as dispersions of solid materials in liquids,” says Harris. “In other words, things like coatings, inks, adhesives, [and] even synthetic products are more and more being processed and delivered as solid particles—fine particles—suspended . . . often in aqueous solution.” But when those materials are dispersed in water-based solvents, they are no longer single-phase products, an analytical chemistry challenge Harris’s group wanted to tackle. The researchers used a Kr+ laser operating at 647.1 nm as the excitation source and adjusted the laser focal spot ~20–30 µm above the coverslip to view the sample. In preliminary experiments, the optically trapped silica particles were used to monitor reactions in a solid-phase peptide synthesis scheme. Harris’s group set up a two-step addition-and-removal process, developed by C. J. Welch and co-workers at Regis Technologies, Inc., (Morton Grove, Illinois) for preparing
peptide stationary phases on silica for chiral separations. The first reaction tested was the addition of 9-fluorenylmethoxycarbonyl (fmoc)-protected phenylalanine to a propylamine-primed silica surface. The second reaction scheme involved the removal of the fmoc-protecting group. “That same beam that’s hanging onto the particle is also examining the chemistry of that particle,” says Harris. Optical trapping and levitation, which have been applied to Raman spectroscopy as far back as the 1980s, use forces derived from photon momentum transfer at a tight focus to localize or control the position of a particle, Harris explains. Harris says that turning confocal Raman microscopy upside down, so to speak, was inspired by the work of Nancy Thompson at the University of North Carolina–Chapel Hill. Prompted by Thompson’s research, his group built an inverted microscope for internal-reflection fluorescence spectroscopy and for looking at fluorescent molecules visiting surfaces. The configuration worked well, and Harris’s group was hooked. The rest is history. a —Cheryl M. Harris
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