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A simple grating-based instrument for spectral microscopy
CHRISTOPHER LAFRATTA
monochromator is used as a filter in an Spectral measurements can add useful inoptical design, an aberration often ocformation about the chemical composition curs because light diffracts off the gratof a sample such as a biological cell or a ing at a non-incident angle. This creates pharmaceutical tablet. And now, spectral images can be obtained from a wide field of view with an instrument that incorporates a diffraction-based monochromator in a simple optical design. The setup is called a chromatically resolved optical microscope, or CROMoscope, and is described in a new AC paper (DOI 10.1021/ac9011655) by David R. Walt, Michael R. Webb, and Christopher N. LaFratta at Tufts University. The CROMoscope is less expensive and less complex than many spectral imaging instruments currently in use. “Although there are a number of commercial spectral imaging systems available, the components of this system are inexpensive, so it should make spectral imaging more accessible,” says Walt. Combining a monochromator with a microscope Walt and his colleagues needed this allows sequential acquisition of monochromatic type of instrument for several projects. images. For example, a main focus of the researchers is collecting spectral informaa horizontally stretched or compressed tion from microarrays by taking meaimage. To overcome this aberration, the surements at many different spatial sites researchers used an out-of-plane Littrow and wavelengths, says Walt. In another configuration. project, a collaboration with George The microscope’s optics retroreflect light Whitesides’s group at Harvard Univerin the diffraction axis. In the nondiffracting sity, observing signals at different waveaxis the grating is tilted, separating the incilengths with a wide field of view is dent and reflected light. The optical design needed to detect light emissions from ensures that light remains collimated when it various metal salts. (See the AC news exits the monochromator, which is necessary story “Infochemistry: the new word on for imaging. The microscope uses interthe block”.) Webb, the first author of the CROMo- changeable infinity-corrected objectives, and a CCD camera acquires the images. scope paper, constructed the microscope The instrument’s spectral resolution is with items from around the lab. “It was a proportional to the iris aperture, and the classic confluence of the need to solve a researchers measured a spectral resolution problem coupled to having the right person down to 2.4 nanometers (nm), with a 1-milthinking about it,” says Walt. limeter (mm)-diameter aperture. The spatial The CROMoscope is basically a miresolution is highly dependent on the nucroscope with a monochromator in the merical aperture (NA) of the objective-iris infinity space (where filters are usually combination. The microscope resolved placed). The monochromator acts as a 2.2-micrometer bars on a standard blackfilter, allowing sequential acquisition of and-white target (USAF 1951) with a 0.5 monochromatic images, and has the adNA 50⫻ objective and 3- and 5-mm irises. vantage of being less expensive than the The researchers found that CROMoacousto-optic tunable filter or liquidcrystal tunable filter alternatives. When a scope measurements from colored inks on 10.1021/AC9016665 2009 AMERICAN CHEMICAL SOCIETY
Published on Web 08/04/2009
glass and chloroplasts from an Elodea leaf matched well with those from conventional instrumentation and with published spectra, respectively. Stephen A. Boppart of the Beckman Institute for Advanced Science and Technology at the University of Illinois Urbana⫺Champaign sees the advantages of the CROMoscope. “This technique offers high spectral resolution, broad spectral range, and is sensitive to changes in sample absorption over very short distances,” he says. Boppart adds that the CROMoscope “is a cost-effective solution for acquiring hyperspectral image data from brightfield microscopes. Further refinement and optimization of the technique will further improve its usefulness for more quantitative applications.” The researchers acknowledge that the technique has some room for improvement. Acquiring the spectral images of the colored inks and chloroplast took ⬃10 minutes; this could be shortened to a few seconds by automating the grating rotation and image acquisition, they say. Other improvements might include correcting the somewhat problematic image distortions due to mechanical scanning misalignment, the nonuniform wavelength-dependent grating efficiency over the broad spectral range, and the chromatic aberrations in the optics used to image across such a broad spectral range, says Boppart. Ammasi Periasamy of the W. M. Keck Center for Cellular Imaging at the University of Virginia says that the technique is a simple way to obtain a spectral image of a microscopic sample. He points out that the optical setup is less expensive than a commercial unit and is easy to couple to any microscope. Walt says that the instrument is adaptable to a variety of applications. For example, with different form factors, it could be used for both macro- and microscopic imaging. —Nancy D. Lamontagne
SEPTEMBER 1, 2009 / ANALYTICAL CHEMISTRY
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