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J Biosci Bioeng. 2009 Jan;107(1):90-4. doi: 10.1016/j.jbiosc.2008.10.001.

Classification of pollen species using autofluorescence image analysis.

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  • 1Electronics and Optics Research Laboratories, Kowa Company, Ltd., 3-3-1 Chofugaoka, Chofu, Tokyo 182-0021, Japan.


A new method to classify pollen species was developed by monitoring autofluorescence images of pollen grains. The pollens of nine species were selected, and their autofluorescence images were captured by a microscope equipped with a digital camera. The pollen size and the ratio of the blue to red pollen autofluorescence spectra (the B/R ratio) were calculated by image processing. The B/R ratios and pollen size varied among the species. Furthermore, the scatter-plot of pollen size versus the B/R ratio showed that pollen could be classified to the species level using both parameters. The pollen size and B/R ratio were confirmed by means of particle flow image analysis and the fluorescence spectra, respectively. These results suggest that a flow system capable of measuring both scattered light and the autofluorescence of particles could classify and count pollen grains in real time.

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