Olson, RJ, Sosik HM, Chekalyuk AM
Woods Hole Oceanographic Institution
This research has been supported by grants from the Department of Energy (DE-FG02-93ER61693) and NSF (OPP-9530718 and OCE-9819206).
Background: Active fluorescence techniques are becoming commonly used to monitor the state of the photosynthetic apparatus in natural populations of phytoplankton, but at present these are bulk water measurements which average all the fluorescent material in each sample. Here we describe two instruments which combine individual-cell "pump-during-probe" (PDP) measurements of chlorophyll (Chl) fluorescence induction, on the time scale of 30 to 100 ms, with flow cytometric or visual characterization of each cell.
Methods: In the PDP flow cytometer, we measure the time course of Chl fluorescence yield during a 150 ms excitation flash provided by an argon ion laser; each particle is subsequently classified as in a conventional flow cytometer. In the PDP microfluorometer, individual cells in a sample chamber are visually identified, and fluorescence excitation is provided by a blue light-emitting diode which can be configured to provide a saturating flash and also a subsequent series of short flashlets. This sequence allows both saturation and relaxation kinetics to be monitored.
Results: Phytoplankton from natural samples and on-deck iron-enrichment incubation experiments in the Southern Ocean were examined with each PDP instrument, providing estimates of the potential quantum yield of photochemistry and the functional absorption cross section for photosystem 2, for either individuals (for cells larger than a few micrometers) or populations (for smaller cells).
Conclusions: Results from initial field applications indicate
that single-cell PDP measurements can be a powerful tool for investigating
the nutritional state of phytoplankton cells and the regulation of phytoplankton
growth in the sea.
(Click on images for better resolution)
Fig. 1. Schema of the PDP microscope. In early work, the LED was controlled by an analog circuit based on a 555 timer chip, and signals were captured with a digital oscilloscope and subsequently transferred to a personal computer (PC). Longpass, shortpass, bandpass and neutral density filters are indicated as LP, SP, BP and ND, respectively.
Fig. 2. Optical layout of the PDP flow cytometer. L = lens; M = mirror; D = dichroic; S = splitter; F = filter.
Fig. 3. Timing diagram for the PDP flow cytometer. (A) represents schematic signals from the three laser beams. Panels B-G denote the timing of the control and signal processing functions.
(A) The IR, PDP, and classification beams are aimed and focused while observing signals with an oscilloscope. Scattered light from a particle passing through the IR beam triggers the system (time = 0). For clarity, the PDP signal is shown exaggerated in size relative to the other two signals. The shape of the unmodulated PDP beam profile (i.e., a signal from a bead with the modulator kept open) is indicated by a dotted line; the modulated signal would cause a vertical rise in the bead signal at 550 ms. The signal shape generated by the passage of a healthy cell through the modulated PDP beam is indicated by the solid line.
(B) The system is prevented from responding to new trigger signals during the period required for measuring and storing the data from the triggering particle (33 ms).
(C) The IR laser is turned off after triggering, to reduce background during the subsequent measurements.
(D) PDP signal acquisition (200 ms) begins as the cell approaches the measuring region.
(E) After 50 ms of PDP background data has been acquired, the PDP modulator opens to start the fluorescence induction measurement.
(F) The classification beam shutter (which has a minimum 800 ms delay) opens after the PDP measurement is completed.
(G) The classification pulse stretchers are reset after the opening of the shutter, but before the cell passes through the strong beam. The IR pulse stretcher is reset after the IR laser has been turned back on for the next sample.
Fig. 4. PDP microfluorometric measurement of a pair of Phaeocystis cells collected in the Ross Sea, south of New Zealand.
(A) The cell as it appeared under measurement magnification; a subset of this view was assayed using the aperture as shown in Fig. 4B.
(B) The same field at 10-fold lower magnification reveals the entire Corethron cell as well as part of a Phaeocystis colony.
(C) Time course of fluorescence intensity for the cell, reference (excitation flash) and blank (empty field of view), averaged over 50 flashes.
(D) Time course of cell fluorescence yield, averaged over 50 flashes. Least squares fit of the data to a biophysical model yielded an estimate of Fv/Fm of 0.47.
(E) Time course of cell fluorescence yield for the first flash in the measurement series. Though the single-flash data are noisier, the estimate of 0.50 for relative variable fluorescence was close to that for the averaged data above.
Fig. 6. Pump-during-probe microfluorometry results for Fragilariopsis spp. cells from the initial sample and from control and iron enriched bottles on day 5 of an iron enrichment bottle incubation experiment south of the Polar Front in the northern Ross Sea. While iron-treated cells exhibited the highest average Fv/Fm , the distribution of values was wide in each case; some cells in the initial sample were quite "healthy" even though the average Fv/Fm was low. Likewise, even in the iron-enriched sample, a few cells were in poor condition.
Fig. 7. Pump-during-probe microfluorometry of Fragilariopsis spp. (A) from the iron-enriched bottle of Fig. 6, showing the post-saturation decay in fluorescence yield. (B) Reference signal from flash sequence. (C) Cell fluorescence. (D) Fluorescence yield.
Note in (D) the change in scaling of the time axis between the saturation and decay phases. In (B) and (C) the decay phase signals are shown with the same time resolution as for the saturation phase data, but the 100 ms intervals between flashlets are omitted.
Fig. 8. PDP flow cytometric analysis of the phytoplankton in a water sample from 30 m depth south of the Polar Front in the northern Ross Sea. Each dot in panels A and B represents a particle with the indicated light scattering and fluorescence values. Color coding indicates categories of cells selected through multiparameter analysis. The fluorescence data for each group selected was averaged (C) and normalized to that of the beads to give fluorescence yield (D). The yields in (D) have been scaled to Fm to facilitate comparisons of curves between populations. The number of particles in each group were: beads = 1852, pennates = 359, cryptophytes = 332 , "small phytoplankton" = 6493, and "large phytoplankton" = 438.
Individual cell fluorescence induction curves from the largest cells
measured (such as the one indicated by the asterisk in (A) had reasonable
signal-to-noise ratios (E); Fv/Fm for this cell was
estimated at 0.44 (F).
Fig. 9. Comparison of bulk (FRR, squares) and single-cell (PDP flow cytometer, circles) measurements of relative variable fluorescence yield (Fv/Fm) during an iron enrichment bottle incubation experiment south of the Polar Front in the northern Ross Sea. Iron-enriched (filled symbols) and control (open symbols) bottles were sacrificed at each time point and measurements made within 1 h; results for replicate bottles for the 2- and 5-day time points are indicated. For this "bulk" comparison, all the cells in the flow cytometer signature were considered as a single population; both approaches indicate an increase in the Fv/Fm of the total phytoplankton population after iron enrichment.
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