Construction, figures of merit, and testing of a single-cell fluorescence excitation spectroscopy system

Abstract

Characterization of phytoplankton community composition is critical to understanding the ecology and biogeochemistry of the oceans. One approach to taxonomic characterization takes advantage of differing pigmentation between algal taxa and thus differences in fluorescence excitation spectra. Analyses of bulk water samples, however, may be confounded by interference from chromophoric dissolved organic matter or suspended particulate matter. Here, we describe an instrument that uses a laser trap based on a Nikon TE2000-U microscope to position individual phytoplankton cells for confocal fluorescence excitation spectroscopy, thus avoiding interference from the surrounding medium. Quantitative measurements of optical power give data in the form of photons emitted per photon of exposure for an individual phytoplankton cell. Residence times for individual phytoplankton in the instrument can be as long as several minutes with no substantial change in their fluorescence excitation spectra. The laser trap was found to generate two-photon fluorescence from the organisms so a modification was made to release the trap momentarily during data acquisition. Typical signal levels for an individual cell are in the range of 10(6) photons/s of fluorescence using a monochromated 75 W Xe arc lamp excitation source with a 2% transmission neutral density filter.

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

“…Due to the large cell-to-cell fluorescence variability described by Hill et al., 33 combined with the temporal variability observed of phytoplankton fluorescence reported by Swanstrom et al., 28 the best discrimination of cells via fluorescence comes from an intensity-independent measure such as an intensity ratio with the measurements made close together in time to maximize their correlation.…”

“…The simplest, least expensive, and most readily available form of a near-binary optical filter is of the single bandpass or band-blocking type, and we have chosen bandpass filters to create discriminant ratios for signal-to-noise (S/N) reasons and their broader availability. Due to the large cell-to-cell fluorescence variability described by Hill et al, 33 combined with the temporal variability observed of phytoplankton fluorescence reported by Swanstrom et al, 28 the best discrimination of cells via fluorescence comes from an intensity-independent measure such as an intensity ratio with the measurements made close together in time to maximize their correlation.…”

Development ofIn SituSensors for Chlorophyll Concentration Measurement

“…Because of their size and power requirements, broadband lamps are seldom used for in situ measurement at present. However, some special applications such as single-cell fluorescence excitation spectroscopy systems [54] and multivariate optical computing instruments [55] require super high power Xe arc lamps (75 W).…”

“…In order to avoid interference from CDOM or suspended particulate matter, an instrument based on a Nikon TE2000-U microscope to position individual phytoplankton cells for confocal fluorescence excitation spectroscopy was designed by Hill et al [54,95].…”

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

“…Natural fluctuations in environmental variables such as light and nutrients will influence the magnitude and shape of the fluorescence excitation spectra, but surprisingly little is known about the cell-to-cell variability of fluorescence excitation spectra for an individual species. In a recent report, 23 we described development of an instrument capable of capturing a single phytoplankton cell in an optical trap and measuring its fluorescence excitation spectra. Here, we report results from the use of that instrument to investigate the within-species variability of single-cell fluorescence excitation spectra for five marine phytoplankton species: Emiliania huxleyi (a coccolithophore), Thalassiosira pseudonana (a diatom), Dunaliella tertiolecta (a chlorophyte), Rhodomonas salina (a cryptophyte), and Amphidinium carterae (a dinoflagellate) (Table I).…”