Saturating-flash fluorescence techniques are used to monitor the state of the photosynthetic apparatus in phytoplankton under natural conditions. At present these are bulk water measurements, which produce estimates of average properties of all the fluorescent particles present in a sample. Here we describe an improved approach for single-cell measurements of phytoplankton. We have combined individual- cell 'pump-during-probe' measurements of chlorophyll fluorescence induction on the time scale of 30 to 100 microseconds with flow cytometric characterization of each cell, to obtain population-specific photosynthetic characteristics. The results provide information about the potential quantum yield of photochemistry, the fraction of functional reaction centers, and the functional absorption cross section for photosystem 2

Chekalyuk, Alexander M.

Olson, Robert J.

Sosik, H. M.

English

Columbia University Academic Commons

PUMP-DURING-PROBE FLUOROMETRY OF PHYTOPLANKTON: GROUP-SPECIFICPHOTOSYNTHETIC CHARACTERISTICS FROM INDIVIDUAL CELL ANALYSISChekalyuk, AM., Olson, R.J., Sosik, H.M.Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USAABSTRACTSaturating-flash fluorescence techniques are used to monitor the state of the photosynthetic apparatus in phytoplanktonunder natural conditions. At present these are bulk water measurements, which produce estimates of average properties ofall the fluorescent particles present in a sample. Here we describe an improved approach for single-cell measurements ofphytoplankton.We have combined individual-cell "pump-during-probe" (PDP) measurements of chlorophyll (Chl) fluorescence inductionon the time scale of 30 to 100 microseconds [1, 2] with flow cytometric (FC) characterization of each cell, to obtainpopulation-specific photosynthetic characteristics. The results provide information about the potential quantum yield ofphotochemistry (I ), the fraction of functional reaction centers (f), and the functional absorption cross section forphotosystem 2 (aps2).Key words: phytoplankton, photosynthesis, fluorescence, technique, pump-during-probe, inductionI. CHL FLUORESCENCE INDUCTION AND PHOTOSYNTHETIC CHARACTERISTICSThe biophysical background of the PDP approach is discussed in [1 , 2) To a first approximation, fluorescence inductionat the time scale of 30-100 ts under supraoptimal light intensity I can be described ascIf (t) = (em' ( m ) at )1 (1)where cI0 and tm are initial and maximum values of ChI fluorescence yield. and a is the rate constant of closing of PS2reaction centers (RCs, a = PS2 I). The magnitudes of tm , (t)o and a can be retrieved using nonlinear regression of ameasured PDP induction curve to equation (1), and the photosynthetic characteristics can be estimated asct = (m o )/ 1:m f =(?max 1 )/( (tl)p I ), 3PS2 a I I. (2)The maximum P52 photochemistry efficiency for phytoplankton, pma IS known to be 0.65 [3J.2. THE EXPERiMENTAL TECHNIQUEWe measure the time course of chlorophyll fluorescence yield during a 100 s excitation flash provided by a weak 488 nmargon ion laser whose beam passes through an electro-optical modulator, while a strong laser beam for measuringconventional FC parameters is blocked by an electromechanical shutter (Figs. 1, 2). A cell detector based on an infrareddiode laser triggers the PDP and subsequent FC measurements. Since both analog and photon counting detectiontechniques are used, cells ranging in size from <0.7 tm (prochlorophytes) to >30 tm can be assayed.Fig 1. Schema of the flow cell with the IR, PDP andFC FC laser beams.— —PDPI IR—0The optical setup (Fig. 2) includes an Ar ion laser (488 nm, 350mW), splitters Si and S2 (20% transmission), an electro-mechanicalshutter (100 p.s opening time) which is normally closed to preventdistortions in PDP induction, an electro-optic modulator (1 p.s openingtime) which produces a 1 00-p.s PDP pulse, and an infrared (785 nm, 50mW) laser. Spherical lenses (LI, L2, f= 80 and 100 mm) andcylindrical lenses (L3-Ll0, f= 50mm) are used to shape the beams.00840 SPIE Vol. 2963 • 0277-786X/97/$1OOODownloaded from SPIE Digital Library on 23 Nov 2010 to 129.236.10.169. Terms of Use:  http://spiedl.org/termsLII1+;Fig. 2 Schema of the optical part of the PDP flow cytometer.The achromatic lens Li 1 (N 38 mm) provides focusing ofthe IR, PDP and conventional FC beams as presented in Fig. I.Lenses L7 and L8 increase the vertical divergence ofthe PDP beam, resulting in vertical widening ofthe PDP laser spot inthe flow cell and an almost rectangular PDP "pulse" with opening ofthe modulator. Translations of L3, L5 and L8 allowindependent adjustment of the horizontal size of the PDP and FC laser spots. Dichroic filter DI serves to direct the IR beamto the flow cell. Lens Li2 (from a Becton-Dickinson FACScan) and spherical lens Ll3 (f =30 mm) are used for collection ofside and forward optical signals from the the flow cell. Blue and IR forward scattered (FS) signals are split by dichroic filterD2 and detected by a photodiode (FSD) and an avalanche photodiode (IRD), respectively. Neutral density filters Fl and F2are used for adjustment of the PDP beam and FS signals to appropriate levels. Dichroic filters D3, D4, D5, and a mirror M5provide splitting of the side optical signals and, after filtering with F4, F5, F6 and F7 filters, direction to PMT detectors forPDP (PDPD), side scattering (SSD), red fluorescence (RFD), and orange fluorescence (OFD).Fig. 3 Simplified schema of the PDP FC electronics.841M5—'D4D3Ar laser'I1 \ MlM2D2 F2L13 cZJ'fl1\1SL4 L3 L2 LIF 1 0 lShutterFU< /Ll2 LI!1 (\ .VDI/SiJ I1__JBI FlowCell\L8 L7 L6 L5 Fl( ri nil t\fl/S2Ll0 L9NM4f1-E.j_IR laserDownloaded from SPIE Digital Library on 23 Nov 2010 to 129.236.10.169. Terms of Use:  http://spiedl.org/termsA simplified schema of PDP FC electronics is presented in Fig. 3 . It consists of the IR triggering channel, a conventionalFC measuring part, and the PDP measuring channel. Since FC channels for measuring forward scattering, sidescattaring, orange and red fluorescence are almost identical, we show just one "FC" channel in Fig. 3 . 4.5-decadelogarithmic amplifiers (Log Amp) are used to maximize the dynamic range in both IR and FC channels. Stretcher modulescatch and hold the peak amplitudes of cell-produced pulses in IR and FC channels. An 8-channel 100-KHz ADC computerboard (Slow DAB) measures these amplitudes on triggering from a timing pulse generator. This generator also providesresets of the stretchers after each measuring cycle, self-blocking to avoid triggering by new cells during event processing,blockingthe IR laser to reduce background when measuring the PDP and FC response, pulsed opening of the PDPmodulator (PDP Mod) and the FC beam shutter (FC shutter), and triggering of data acquisition in the PDP channel. Thischannel includes a PMT (PDP Det), a 60-Mhz analog ADC (Fast DAB) and time-resolved photon counting (PhC DAB, 2-jis resolution) boards installed (along with the Slow DAB) in a 100-MHz Pentium personal computer (PC). Fig. 4 shows atiming diagram of the system.IRA1FS PDP FC0 500 600 900 Time,sJ Generator triggering by IR peak detector I0 3000 Time, sActivation of the shutter0 2000 Time, tsSelf-blocking of generator triggering 120I20000IR laser OFF25 2000 Time, .tsPDP Modulator ON500 600 Time, jisResets of stretchers FC fi850JR20000Acquisition trig. JPDP450[]FC1000 Time, tsFig. 4 Timing diagram of the PDP electronics.842Downloaded from SPIE Digital Library on 23 Nov 2010 to 129.236.10.169. Terms of Use:  http://spiedl.org/termsA specially developed data acquisition software runs under Windows and allows real-time control of Fast DAB , SlowDAB, PhC DAB, and storage of both the PDP induction curve and FC data for each cell in the PC memory and on thehard drive. Due to substantial amount of information to be acquired (about 1 .6 Kbyte per cell) ,we use a CD recordingsystem for permanent data storage after finishing the measurement. Typically we accumulate 1 ,000 to 25,000events fromeach sample . An analysis of conventional FC data allows specification of cells or cell groups in the sample for retrievingindividual or group-specific photosynthetic characteristics. In analog mode, either individual cell curves (for cells > 5tm) or curves averaged over a group of cells can be reconstructed; in photon counting mode data from hundreds tothousands of cells are accumulated for each PDP induction curve.We measure the fluorescence time course of red-fluorescing latex microspheres, added to the sample, to monitor the shape ofthe excitation "pulse", i.e. the light intensity profile in the PDP area within the core ofthe flow cell. The PDP inductioncurves are then normalized to this profile to compensate for deviations from the "ideal" rectangular shape. The intensity ofthe PDP laser beam was adjusted with neutral-density filters (Fl in Fig. 2) to obtain cell fluorescence rise times ofapproximately 30-100 its, and the rate ofsample introduction was adjusted to ensure that only one cell was in the PDPbeam at any given time.3. LABORATORY TESTS OF THE PDP APPROACHThe results ofpreliminary laboratory tests ofthe PDP approach were presented in (Olson et a!. 1995, 1996). In particular, forsix species ofphytoplankton grown under different conditions of light intensity and nutrient depletion, estimates of thequantum yield ofphotochemistry in PS2 by PDP measurements made on individual cells were well correlated with estimatesderived from DCMU-enhancement measurements ofbulk samples (Fig. 5). Part ofthe measurements was made with amicroscope-based PDP system (Olson Ct al. 1 995, 1 996). No obvious differences were observed between the two PDPinstruments or among the six species tested. These results indicate that the microscope- and flow cytometer-based PDPtechniques, applied to individual cells, provide reliable information about the photosynthetic characteristics ofphytoplankton.We have recently tested the use ofa compact solid-state green0 7 laser (100 mW, 532 nm) in the PDP FC system. Although we.observed an apparent decrease in the efficiency of Chlfluorescence excitation (due to the lower absorption cross-0.6 section as compared to the blue region ofthe spectrum), thegreen laser appeared to be suitable for both FC and PDP0.5 measurement ofrelatively big (>1 m) cells. In Fig. 6 wea. present results of a sample containing a mixture of Dunaliellalertiolecta (5-7 im cells, DT) and Nannochioris sp. (2-3 m0.4 cells, N). l-im red beads (BD) were used for monitoring thePDP profile in the flow cell. This profile was used to0.3 normalize PDP intensity profiles for the two species (panel Bin Fig. 6). Processing of the PDP induction curves (see Fig. 6-C) was done based on equations (2). Due to unfavorable0.2 nutrient conditions both species indicated moderatephotosynthetic performance:0.1-- I • I0.1 0.2 0.3 0.4 0.5 0.6 0.7 DI N(DCMU) cI 0.51 0.38p f 0.56 0.32Figure 5. Correlation between PDP -based estimatesof 1 and estimates derived from bulk measurements In fact, just one third of PS2 reaction centers were active inof DCMU enhancement of fluorescence. the case of Nannochioris sp., and about 50% for Dunaliellatertiolecta.843UUU• I • I •y0.97x-0.01r2=0.83Downloaded from SPIE Digital Library on 23 Nov 2010 to 129.236.10.169. Terms of Use:  http://spiedl.org/termsFig. 6 PDP FC measurement of a mixture of Dunaliellatertiolecta (DT) and Nannochioris sp. (N), and I -.tm redbeads (BD). The solid-state green laser (100 mW, 532nm) was used. Panel A: FC red fluorescence vs. FC sidescattering (5,000 events); B: PDP intensity profiles fordifferent sample componetnts, reconstructed based onindividual PDP curves; panel C: PDP induction curvesfor two species, obtained by normalizing correspondingPDP intensity profiles to BD PDP profile.844A]2 / // /32.5a 25=0.50-20FC Side Scattering (log scale)Fig. 7 Analysis of group-specific PDP induction curvesfor a natural sample of sea water (279 Oceanus Cruise,June 1996, Gulf Stream, Chi maximum at 80 m).Panel A: prochlorophytes (Pr) and small eukaryoticphytoplankton (EC) are well defined along with 2-tmNile Red beads. Panel B: PDP intensity profiles fordifferent sample components. Panel C: PDP inductioncurves for two phytoplankton groups (Pr and EC) and acurve averaged over these two groups (TS).HFC Side Scattering (log scale)a=0==a===a0==5004003002001000.100-20 0=ei 501000. 50------—4.— _L._20 40 60 80Time, microseconds100 120-50 4 .4.._ 4 .. 4-20 0 20 40 60 80Time, microseconds100 1202.5 -- I.20 0 20 40 60 80 100 120Time, microseconds0 20 40 60 80 100Time, microseconds120Downloaded from SPIE Digital Library on 23 Nov 2010 to 129.236.10.169. Terms of Use:  http://spiedl.org/terms4. FIELD APPLICATIONThe operation ofthe PDP flow cytometer was tested at sea during R/V Oceanus cruise 279 (June 1996). An example ofgroup-specific PDP induction curves for natural populations is presented in Fig. 7. Note the change in relative intensity ofred fluorescence per particle between beads and phytoplankton cells in the FC (upper panel) and PDP (middle panel) modes.This can be explained by strong saturation of Chl phytoplankton fluorescence in the powerful FC laser beam. Normalizationofthe group-specific PDP induction profiles to the PDP profile for beads (middle panel) allows us to obtain thephytoplankton group-specific PDP induction curves (lower panel) and photosynthetic characteristics presented in the table:Prochlorophytes Eukaryotic cells Total samplePS2 efficiency, cI 0.52 0.31 0.42Fraction ofactive RCs, f 0.59 0.27 0.43Abs. cross-section, a.u. 0.16 0.11 0.14The photosynthetic functional state ofthe smallest phytoplankton cells (Pr) appeared to be better than that ofthe largereukaryotic group (59% vs. 27% offunctional RCs) in the same sample, and the TS column indicates intermediate values, aswould be obtained with techniques based on bulk measurements. This example illustrates how group-specific analysis ofphotosynthetic characteristics can improve evaluation ofthe "health" ofthe phytoplankton community.5. CONCLUSIONBy combining pump-during-probe measurements of individual phytoplankton cells with conventional flow cytometricmeasurements of light scattering and fluorescence, we can obtain information about the photosynthetic characteristics ofdifferent groups of cells in natural populations. This capability will be valuable in investigations of the regulation ofphytoplankton growth and productivity; for example, it should help to elucidate the responses of phytoplankton of differentsize classes to nutrient limitation.6. REFERENCES1. Olson, R.J., A.M. Chekalyuk, and H.M. Sosik. "Phytoplankton photosynthetic characteristics from fluorescence inductionassays of individual cells." Limnol. Oceanogr. 41: 1253-1263 (1996).2. Olson, R.J., A.M. Chekalyuk, H.M. Sosik, and M.Yu. Gorbunov. "Pump-during-probe technique for measuringphotosynthetic characteristics of individual algal cells using flow cytometry and microfluorometry". In "Photosynthesis:from light to biosphere", P. Mathis (ed.), Kluwer Acad. Publ., Dordrecht/Boston/London, Proc. of the X mt. PhotosynthesisCongress, Montpellier, France, 20-25 Aug. 1995, 5: 743-748 (1995)3. Kolber, Z.S., and P.G. Falkowski. "Use of active fluorescence to estimate phytoplankton photosynthesis in situ." Lininol.Oceanogr. 35: 72-79 (1993)845Downloaded from SPIE Digital Library on 23 Nov 2010 to 129.236.10.169. Terms of Use:  http://spiedl.org/terms

Pump-During-Probe Fluorometry of Phytoplankton: Group-Specific Photosynthetic Characteristics from Individual Cell Analysis

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Pump-During-Probe Fluorometry of Phytoplankton: Group-Specific Photosynthetic Characteristics from Individual Cell Analysis

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