Linear Discriminant Analysis of Single-Cell Fluorescence Excitation Spectra of Five Phytoplankton Species

Abstract

Linear discriminant analysis (LDA) of single-cell fluorescence excitation spectra (λem=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.

Fluorescence Excitation Spectroscopy for Phytoplankton Species Classification Using an All-Pairs Method: Characterization of a System with Unexpectedly Low Rank

“…Cells were grown in batch culture according to the protocol described in Bruckman et al. 30 Bulk fluorescence excitation spectra with excitation over the range 400–630 nm and emission detected at 680 nm for each species were obtained using a Hitachi F4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) modified for a 180° backscatter configuration (see Supplemental Material Fig. S1 for a diagram) that more closely approximated conditions in the FIP.…”

Asymmetric Versus Symmetric Filter Wheels and Associated Processing Algorithms: Results from Asynchronous Fluorescence Imaging Photometer Measurements of Phytoplankton

“…The T. pseudonana strain (CCMP 1335) used in this demonstration was acquired commercially from the National Center for Marine Algae and Microbiota (NCMA, East Boothbay, Maine) and was cultured using the procedure described by Bruckman et al. 4 Sets of 2000 images of each species were collected using each of the two filter wheel conditions along with 100 background images and 100 flat field images collected according to the procedure described by Pearl et al. 3 Image sets collected with the new asymmetric filter wheel design were processed using a new method described below, while images collected with the symmetric filter wheel design were processed using both the automated algorithm, described by Pearl et al., 3 and a modified version of the algorithm described below.…”

“…In past reports, our lab has described the use of a filter wheel on the excitation arm of an asynchronous fluorescence imaging photometer (FIP) for classification of phytoplankton cells. [1][2][3][4] The asynchronous character of the measurements resulted from the fact that phytoplankton entered the field of view of the instrument at random times, so there was no exact correlation between the appearance of a phytoplankton cell and the position of the filter wheel. Instead, the position of the filter wheel had to be inferred from measurements of the phytoplankton fluorescence recorded on a single charge-coupled device (CCD) camera frame as the cell passed through the field of view of the camera.…”

Single-Cell and Bulk Fluorescence Excitation Signatures of Seven Phytoplankton Species During Nitrogen Depletion and Resupply

“…12,13 Most of our work has been conducted with phytoplankton cultures grown under non-varying incident irradiance, fixed light-dark cycles, and under nutrient-replete conditions. 8,14 Extension of our results from cultures to natural populations requires an understanding of how environmental fluctuations might affect relative pigment concentrations and hence fluorescence ratio signatures for cells. Cellular pigment ratios are known to change in response to light and nutrient status in at least some phytoplankton taxa.…”

“…6,7 We have previously reported even greater levels of heterogeneity in single-cell fluorescence, possibly due to varying fluorescence quantum efficiency. 8 Cellular pigment concentrations also vary with incident irradiance and/or nutrient supply 9,10 and this could likewise affect fluorescence intensities.…”