Linear discriminant analysis (LDA) of single-cell fluorescence excitation spectra (λ_em = 680 nm) for five species of marine phytoplankton was used to determine whether intra-species variation among single cells precluded discrimination among species. Single-cell spectra were recorded in an optical trap with a custom-built spectral fluorometer. For nitrogen (N)-replete cells, separation of all five species ( Emiliania huxleyi, a coccolithophore, Thalassiosira pseudonana, a diatom, Dunaliella tertiolecta, a chlorophyte, Amphidinium carterae, a dinoflagellate, and Rhodomonas salina, a cryptophyte) was possible using only a portion of the excitation spectra (570-610 nm). This wavelength region gave perfect classification of species with a minimum Fisher ratio of 62. For four species ( E. huxleyi, T. pseudonana, D. tertiolecta, and A. carterae), variations in fluorescence excitation spectra as cells were starved of N did not impact the classification process adversely within the chosen spectral window. R. salina cells grown with and without N showed significant differences in their fluorescence excitation spectra but could still be classified if a different spectral window (490-570 nm) was used. Overall, we conclude that intra-species variation among single-cell fluorescence excitation spectra does not preclude discrimination among species.

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