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For some people, the annual battle against allergies means keeping constant vigil over the daily pollen reports. But current methods to classify the myriad species of pollen are laborious and time-consuming and can make daily pollen reports imprecise. In a new AC paper, Franziska Schulte, Janina Kneipp, and colleagues at Humboldt University of Berlin and the BAM Federal Institute for Materials Research and Testing (Germany) describe their efforts to accelerate the pollen identification process by using Raman spectroscopy (2009, DOI 10.1021/ac801791a). Current pollen detection systems are based on morphological information, explains Kneipp, who leads the project. Pollen samples are trapped from the air, pretreated (in some cases), and then analyzed microscopically. The method works well, she adds, but it can be slow. Raman spectroscopy, by comparison, does not require sample pretreatment and is very fastOa spectrum can be obtained in seconds or less. “Our research is directed toward the development of a fast automated on-line detection of pollen species based on Raman spectrometric fingerprints,” Kneipp says. “The Raman spectra are treated as patterns that can be recognized by identification algorithms, which are more objective and less time-consuming than visual inspection.” The researchers collected pollen from 15 different tree species during the flowering season, snap-freezing and freezedrying the samples to preserve the morphological features as well as the potentially labile biological molecules of interest. They then irradiated the samples with 633 nm laser light to deplete signals from the spectrally dominant carotenoids. “Excitation of preresonant Raman scattering cannot be avoided and leads to intense carotenoid signals that superimpose and obscure the normal Raman bands of all the other pollen constituents,” Kneipp explains. “Photo4
ANALYTICAL CHEMISTRY /
JANUARY 1, 2009
JANINA KNEIPP
Classifying pollen with Raman
Researchers are using Raman spectroscopy to identify and characterize individual pollen grains. Inset: A photomicrograph of a rye pollen grain.
destruction is one way to get rid of the carotenoids.” The researchers acquired Raman spectra of individual pollen grainsO91 samples in totalOand compared the spectral signatures arising from cellular components such as proteins, nucleic acids, and carbohydrate polymers. They then performed cluster analysis on the spectra to generate a dendrogram of the various species. The samples were divided into three large clusters that were further differentiated into smaller groups of closely related species. This division was in good agreement with the taxonomic differences of the trees from which the pollen grains were collected. In one case, the resolution between the spectra was sufficient to distinguish related species growing in different geographic regions. The researchers found that two Eurasian maple species produced very similar Raman spectra, and yet these fingerprints were quite distinct from that of a North American maple. Whether these differences represent environmental or evolutionary effects is unknown. “We can only guess on the speciation process,” Kneipp says. “It would indicate that the European spe-
cies may have closer phylogenetic connections, but this is highly speculative and would need to be confirmed by other methods.” Key to the pollen identification effort was the use of freshly harvested pollen samples. Several different attempts to use Raman spectroscopy to analyze commercially available pollen samples had noted significant background fluorescence that complicated signal analysis and biomolecule identification. “The chemistry of pollen changes with time, preparation, and storage,” Kneipp explains. “We have done experiments on aging pollen samples and have observed a rise in fluorescence over a period of just weeks.” The new technique opens the door to deeper analysis of pollen species that may be too difficult to differentiate visually, Kneipp adds. Critical to that potential will be expansion of the researchers’ spectral database on different pollen species and a better understanding of pollen biochemistry. That understanding will be greatly facilitated by the development of chemical maps that will allow the researchers to correlate the presence of different biochemical constituents and their locations on different morphological structures within the pollen grains. In the current work, the researchers sectioned rye pollen grains and rasterscanned the sections by measuring Raman spectra across a predefined grid. By focusing on individual Raman shifts, the researchers could see which biomolecules colocalized. For example, they could correlate the presence of aliphatic carbon chains with internal pollen structures likely to be membranes and with the outer pollen capsule, which comprises an ill-characterized mixture of biopolymers such as phenolic compounds and long-chain fatty acids. Kneipp says that the chemical mapping effort continues and will be presented more fully in a subsequent publication. —Randall C Willis
10.1021/AC8022668 2009 AMERICAN CHEMICAL SOCIETY
Published on Web 11/19/2008
