J. Eukaryot. Microbiol., 2008 The Author(s) DOI: 10.1111/j ...xrm.phys.northwestern.edu/research/pdf_papers/2008/twining_jem_200… · The biogeochemical cycles of many elements in

Exploring Ocean Biogeochemistry by Single-Cell Microprobe Analysis ofProtist Elemental Composition1

BENJAMIN S. TWINING,a STEPHEN B. BAINES,b STEFAN VOGTc and MARTIN D. de JONGEc

aDepartment of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina, andbDepartment of Ecology and Evolution, Stony Brook University, Stony Brook, New York, and

cX-ray Science Division, Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois

ABSTRACT. The biogeochemical cycles of many elements in the ocean are linked by their simultaneous incorporation into protists. Inorder to understand these elemental interactions and their implications for global biogeochemical cycles, accurate measures of cellularelement stoichiometries are needed. Bulk analysis of size-fractionated particulate material obscures the unique biogeochemical roles ofdifferent functional groups such as diatoms, calcifying protists, and diazotrophs. Elemental analysis of individual protist cells can beperformed using electron, proton, and synchrotron X-ray microprobes. Here we review the capabilities and limitations of each approachand the application of these advanced techniques to cells collected from natural communities. Particular attention is paid to recent studiesof plankton biogeochemistry in low-iron waters of the Southern Ocean. Single-cell analyses have revealed significant inter-taxa differ-ences in phosphorus, iron, and nickel quotas. Differences in the response of autotrophs and heterotrophs to iron fertilization were alsoobserved. Two-dimensional sub-cellular mapping indicates the importance of iron to photosynthetic machinery and of zinc to nuclearorganelles. Observed changes in diatom silicification and cytoplasm content following iron fertilization modify our understanding of therelationship between iron availability and silicification. These examples demonstrate the advantages of studying ocean biogeochemistry atthe level of individual cells.

Key Words. Iron fertilization, phytoplankton, plankton, Southern Ocean, synchrotron X-ray fluorescence.

THE BIOGEOCHEMISTRY OF PLANKTONIC PROTISTS

THE field of biogeochemistry concerns, in part, the impact ofbiological processes on the chemical composition of the en-

vironment. Many critical transformations of biologically activeelements in natural ecosystems are mediated by protists.Autotrophic protists are often responsible for entry of elementsinto food webs. As consumers, mixotrophic and heterotrophicprotists play a key role in returning elements to the abiotic world.Our knowledge of the mechanisms driving these transformationsis poised to rapidly expand in the near future as a molecular levelunderstanding of cellular physiology, biochemical composition,and gene expression develops. Such an understanding will beneeded if we are to anticipate the consequences of anthropogenicalterations to global geochemical cycles. Many of the changesalready underway will in turn impact protist biology and ecologyin the future in ways that may depend on their elementalrequirements.

The importance of protists to global biogeochemistry is perhapsmost obvious in the sea. Covering 70% of the Earth’s surface, theoceans play numerous critical roles in global biogeochemistry andclimate. The oceans buffer temperature fluctuations, provide animportant food supply for maritime countries, and mediate chang-es in the global C cycle. The oceans have absorbed approximately120 petagrams (1 Pg 5 1015 g) of anthropogenic carbon dioxide inthe past two centuries, about half of the total amount released byhuman activities during this time (Sabine et al. 2004). Carbon insurface waters is ‘‘pumped’’ to deep waters and the sea floor viathe sinking of planktonic organisms. Through this ‘‘biologicalpump’’, phytoplankton play a major role in the global C cycle(Longhurst 1991; Sarmiento and Gruber 2006). Calcifying protists

such as coccolithophores and foraminifera further impact theocean C cycle through the formation and export of external shellscomposed of CaCO3 (Langer, this issue). Ocean sediments insome regions are described as calcareous ‘‘oozes’’ because of theabundance of calcareous shells of protists. The growth and me-tabolism of marine phytoplankton may also impact global climatethrough the production of dimethylsulphide (DMS). Certainphytoplankton species produce DMS and its precursor dime-thylsulphoniopropionate (DMSP) for osmonic regulation and an-tioxidant protection in the cell (Sunda et al. 2002). PhytoplanktonDMS can diffuse into the overlying atmosphere and may serve asa significant source of non-sea salt sulfate aerosols and cloudcondensation nuclei (Charlson et al. 1987). This process may im-pact global climate through the enhancement of cloud albedo(Bates, Charlson, and Gammon 1987).

Marine protists also control the biogeochemical cycling ofmany elements in the ocean besides C. The nutrient elements N,P, Si, Fe, Mn, Ni, Cu, and Zn are accumulated by phytoplankton insurface waters and exported to depth as sinking biogenic particles.This process results in the surface-depleted ‘‘nutrient profile’’characteristic of many bioactive elements (Donat and Bruland1995). Protistan metabolic activities can also alter the chemicalform of many elements. Eukaryotic phytoplankton reduce nitrateto ammonia upon uptake, convert silicic acid to opaline shells, andmodify the chemical speciation and reactivity of dissolved metals(e.g. Coale and Bruland 1988; Rue and Bruland 1995). Grazingprocesses can further change the physico-chemical speciation ofmetals and non-metals (Hutchins and Bruland 1995; Sato, Takeda,and Furuya 2007).

Numerous approaches have been taken to study the biogeo-chemical interactions between ocean protists and their marine en-vironment. Incubations with isotopically labeled compounds haverevealed differences in the relative availability of different nutri-ent substrates to ambient phytoplankton populations (Dugdale andGoering 1967; Hutchins et al. 1999; McCarthy 1972), enzymeactivities have been measured as markers of nutrient utilizationand limitation (Glibert et al. 2004; Webb, Moffett, and Waterbury2001), and gene expression has been used to determine nutrientcycling by specific phenotypes in field assemblages (Church et al.2005). Comparisons of nutrient stoichiometries have also beencritical to our understanding of ocean biogeochemistry. Alfred

Corresponding Author: B. S. Twining, Department of Chemistry andBiochemistry, University of South Carolina, Columbia, South Caroli-na—Telephone number: 11 803 777 7765; FAX number: 11 803 7779521; e-mail: [email protected]

1Invited presentation delivered during the joint annual meeting of thePhycological Society of America and the International Society of Pro-tistologists, Providence, Rhode Island, August 5–9, 2007.

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J. Eukaryot. Microbiol., 55(3), 2008 pp. 151–162r 2008 The Author(s)Journal compilation r 2008 by the International Society of ProtistologistsDOI: 10.1111/j.1550-7408.2008.00320.x

Redfield famously noted similarities in the stoichiometries of Nand P in bulk surface plankton and dissolved nutrients at depth(Redfield 1934; Redfield 1958), and this approach has since beenapplied to a number of other bioactive elements, including tracemetals (e.g. Loscher 1999; Sunda 1997).

THE NEED FOR A SINGLE-CELL APPROACH

The biogeochemical cycles of bioactive elements are linked bytheir simultaneous incorporation into protists. Therefore, in orderto understand these elemental interactions, accurate measures ofcellular element stoichiometries are needed. These are typicallyobtained for field communities using bulk size-fractionation (Col-lier and Edmond 1984; Copin-Montegut and Copin-Montegut1983; Martin and Knauer 1973). However, this macroscopic ap-proach obscures significant internal processes and limits our abil-ity to link ecology and biogeochemistry. Protists play uniqueecological roles in pelagic plankton communities. A simplifiedmodel of such a system is shown in Fig. 1. Photoautotrophs formthe base of the food web and provide organic substrates to he-terotrophic bacteria, protists, and mesozooplankton, as well asmixotrophic protists (Caron 2000). Photoautotrophs occur in eachof the traditional plankton size classes (0.2–2, 2–20, 20–200 mm).Heterotrophic nanoflagellates, dinoflagellates, and ciliates con-sume autotrophs and bacteria, moving carbon through the foodweb and also contributing dissolved organic compounds for useby bacteria and dissolved inorganic and organic nutrients for useby autotrophs. These trophic relationships are organized by size inFig. 1, although it is unlikely that predation and grazing occuronly between these clear size delineations. Each size class con-tains both autotrophs and heterotrophs (and likely mixotrophs),and bulk elemental analyses performed on all cells collected on0.2-, 2-, and 20-mm membranes would obscure differences be-tween ecological functional groups in the same size range. Be-cause of the presence of abiotic particulate material and thesorption of dissolved and colloidal metals to filters, bulk chemi-

cal analyses often cannot be used to estimate the average cellularelemental stoichiometries for the entire protistan community.Even in open-ocean high-nutrient low chlorophyll (HNLC) areas,biogenic iron may only account for a small fraction of total par-ticulate iron (Strzepek et al. 2005). Further complicating matters,delicate cells may burst upon contact with the filter membraneunder vacuum.

Diverse pelagic protists from the same size class can performunique biogeochemical roles. Eukaryotic autotrophs such as co-ccolithophores and diatoms produce mineral shells composed ofcalcite and silicate, respectively, that provide relatively dense‘‘ballast’’ to the cells. It is thought that these taxa are more like-ly to sink from surface waters and contribute to carbon export thannon-ballasted autotrophs (Armstrong et al. 2002; Klaas and Ar-cher 2002). Coccolithophores and diatoms will also impact carbonand silicon geochemical cycles differently than non-mineralizedtaxa by removing silicon and additional carbon from surface wa-ters and reducing alkalinity. Given their different biogeochemicalroles, it is desirable to separate ballasted and non-ballasted cellsfor elemental analysis. Diazotrophic (N-fixing) organisms such asTrichodesmium and Crocosphaera are also present in the samesize classes (and in the case of the endosymbiont Richellia, in thesame organism) as non-diazotrophs, but they clearly play a uniqueand very important role in nitrogen biogeochemistry. Given thehigh iron requirements for nitrogenase (Kustka et al. 2003),diazotrophs are also likely to impact iron biogeochemistry differ-ently than other cells types (Berman-Frank et al. 2001; Falkowski1997). Specific taxa can also impact sulfur geochemistry, becauseprymnesiophytes such as Emilinia huxleyii and Phaeocystis sp.produce far more intracellular DMS/DMSP than other algal spe-cies (Matrai and Keller 1994; Stefels and Vanboekel 1993). Toadvance our understanding of biogeochemistry, we must collectelemental information at the level of ecological and biogeochem-ical functional groups. That is, since taxonomy influences bio-geochemistry, biogeochemical measurements should reflecttaxonomic differences.

Fig. 1. Description of common particulate material in pelagic waters, including a hypothetical food web demonstrating the diversity of ecologicaland biogeochemical functional groups contained within common size classes. Picoplankton (o2mm) are typically composed of heterotrophic and au-totrophic prokaryotes, including Synechococcus, Prochlorococcus, and some pico-eukaryotes. Nanoplankton (2–20mm) generally comprise the bulk ofthe protist biomass. This size class includes heterotrophs, mixotrophs, and ‘‘naked’’ and ‘‘ballasted’’ autotrophs. Microplankton (420mm) can includelarge diatoms, dinoflagellates, foraminifera, radiolarians, and zooplankton nauplii.

152 J. EUKARYOT. MICROBIOL., 55, NO. 3, MAY–JUNE 2008

Recent modeling efforts have begun to recognize these variedbiogeochemical roles and have incorporated multiple planktonfunctional groups. Moore, Doney, and Lindsay (2004) and Mooreet al. (2002) include three phytoplankton groups—small phyto-plankton, diatoms, and diazotrophs—and assign unique parame-ters to each, including different nutrient uptake kinetics, grazingmortality, and minimum cell quotas. Salihoglu and Hofmann(2007) further separate the picoautotrophs into low light-adaptedProchlorococcus, high light-adapted Prochlorococcus, and Syne-chococcus, in addition to autotrophic eukaryotes and diatoms.Others have chosen to separate the behavior of herbivorous mi-crozooplankton grazers and bactivorous heterotrophic nanoflagel-lates (Lancelot et al. 2000). In order to accurately parameterizethese models and ground truth the output, it is necessary to makeelemental measurements of each relevant group. Given the broad-ly overlapping sizes of the autotrophic and heterotrophic func-tional groups, group quotas cannot be resolved with standard bulkchemical analysis techniques.

There is substantial evidence from laboratory culture work thatelement quotas are dynamic and can vary significantly in protistsas a function of taxonomy, trophic function, and nutrient sub-strate. Much of the work has focused on the micronutrient iron.Brand (1991) found the iron requirements of eukaryotes to besignificantly lower than those of prokaryotes. Minimum iron quo-tas (often normalized to cellular carbon or phosphorus) requiredfor growth have been shown to be 2- to 4-fold lower in oceanicdiatoms compared to coastal diatoms (Maldonado and Price 1996;Sunda, Swift, and Huntsman 1991). It also appears that mixotro-phic and heterotrophic phagotrophs require 2- to 3-fold more ironthan autotrophic protists (Chase and Price 1997; Maranger, Bird,and Price 1998; Sunda et al. 1991). The cell quotas of other bio-active metals also vary between taxa. Diatoms contain less iron,cobalt, copper, and molybdenum (normalized to P) than dinofla-gellates grown in the same media (Ho et al. 2003), and seleniumconcentrations in phytoplankton can vary by more than four or-ders of magnitude among species at ambient levels of selenite(Baines and Fisher 2001). Furthermore, phytoplankton metal quo-tas are sensitive to the ambient nutrient environment. The addi-tional iron required for nitrate reductase raises iron quotas ofdiatoms grown on nitrate compared to those grown on ammonium(Maldonado and Price 1996). Trichodesmium iron requirementsincrease 5-fold during nitrogen fixation as a result of nitrogenasesynthesis (Kustka et al. 2003). Other trace nutrients such as man-ganese, copper, and nickel may also vary in cells in response toiron status and nutrient utilization patterns (Peers and Price 2004;Peers, Quesnel, and Price 2005; Price and Morel 1991). Further-more, phytoplankton have the ability to accumulate and storeconsiderable amounts of trace metals in response to environmen-tal concentrations (Sunda and Huntsman 1998a).

Further complicating the extrapolation of culture studies to thefield is the uncertainty surrounding bioavailability of Fe and othertrace elements in the field. In the sea, the dissolved fraction of Feand many other trace elements exists predominantly as complexeswith dissolved organic ligands (Coale and Bruland 1988; Ellwoodand Van den Berg 2000; Rue and Bruland 1995; Saito, Rocap, andMoffett 2005). In the free-ion model, the ligand-bound metal isassumed to be unavailable to cells (Sunda and Huntsman 1998a).This assumption allows modelers to calculate Fe quotas usingsimple Michaelis–Menten uptake kinetics, measured Fe concen-trations, and presumed concentrations of Fe-binding ligands (e.g,Salihoglu and Hofmann 2007). However, at least some Fe boundto organic ligands in nature may be directly accessible to protists(Maldonado and Price 1999; Maldonado et al. 2005). Alternative-ly, phagotrophic protists may sidestep the dissolved pool by di-rectly ingesting colloidal Fe or bacteria (Chase and Price 1997;Maranger et al. 1998). Iron is not the only element for which bio-

availability of dissolved fractions is open to question. Both nitro-gen and selenium are accumulated not only as inorganic ion, butalso in the dissolved organic form (Baines et al. 2001; Bronk et al.2007).

Given the plasticity of protist metal quotas and the uncertaintyregarding bioavailability of dissolved trace elements, it is prob-lematic to assume that field quotas match those measured in lab-oratory cultures. Perhaps more importantly, valuable informationregarding phytoplankton physiology, biogeochemical cycling,and ambient nutrient bioavailability can be gained from measure-ments of in situ protist cell quotas. The elemental content of in-dividual protists cells can be assayed through one of severalmicroscope-based technologies (Table 1). Light microscopy canbe used in conjunction with fluorescent dyes to detect variousmetal ions within cells (Kikuchi, Komatsu, and Nagano 2004;Thomas et al. 1999; Yang et al. 2005). Such dyes are commer-cially available, utilize relatively common light and confocal mi-croscopes, and have become widely used. However, only labileforms of the metals that are available to bind with the probes aredetected, so absolute quantification of metal quotas is difficult.Fluorescent probes are also prone to interferences from competingmetal ions, further complicating quantitation. More promising foranalytical work are microscopy techniques which base detectionon the characteristic radiation absorption and fluorescence prop-erties of individual elements.

A REVIEW OF APPLICABLE MICROPROBE APPROACHES

Fluorescence X-rays are emitted when atomic electrons transi-tion from outer shells to lower energy inner shell vacancies cre-ated by incident ionizing radiation. The fluorescence energiesassociated with these transitions correspond to the difference be-tween inner and outer shell electron binding energies, and are thusunique to each element, making it possible to deduce the cellularelemental composition from the X-ray fluorescence spectrum. Forexample, for iron the predominant X-ray fluorescence emissionhas an energy of 6.404 keV, corresponding to the transition of anL-shell electron into a K-shell vacancy. The number of detectedX-ray photons for each element generally scales directly as afunction of atomic abundance, thus elemental content can bequantified in a straightforward manner. The yield of fluorescencephotons is lowest for the lighter elements since the excess energyof the relaxed electron for such elements is more likely to be car-ried away through ejection of a secondary emitted electron (Augerelectron) rather than via a fluorescent X-ray photon.

Characteristic X-rays can be generated by one of several formsof higher energy ionizing radiation, and each technique has ad-vantages and disadvantages (Table 1). Electron microprobes arecommercially available and fairly common, and electron beamscan be easily focused, although the high flux requirements to de-tect trace elements in biological samples limit the achievable spa-tial resolution in analytical mode to 20 nm typically. Electrons arerapidly absorbed by light elements (Zo10) in biological samples,typically limiting the depth of penetration to several micrometersat best. More importantly, multiple inelastic scattering will causethe electron beam to broaden rapidly, thus increasing the excitedvolume in the sample significantly and lowering achievable spa-tial resolution (Ingram et al. 1999). To circumvent this problemthin sections (o100 nm) are typically used, and larger protist tar-gets may need to be embedded and sectioned prior to analysis.Lower ionization cross-sections for high mass (Z420) transitionmetals and significant Bremsstrahlung background combine tolimit the sensitivity of electron microprobe X-ray microanalysis(XRMA) for the heavier bioactive metals (e.g. Mn, Fe, Cu, Zn).

Cellular elemental quantification and sub-cellular mapping canalso be formed by measuring energy loss of electrons as they pass

153TWINING ET AL.—SINGLE-CELL ELEMENT ANALYSIS OF PROTISTS

through thin biological samples. Electron energy loss spectrosco-py (EELS) measures the energy loss of electrons at a singlefocused spot while the energy of the incident electron beam issystematically shifted across the absorption edge for the elementof interest (Table 1). A closely related technique, energy-filteredtransmission electron microscopy (EFTEM), involves the collec-tion of a series of images of the whole specimen at specificenergies spanning the absorption edge. The ‘‘stack’’ of hyper-spectral images can then be processed to produce element maps ofextremely high spatial resolution (Leapman 2004). These tech-niques are also capable of remarkable elemental sensitivity; forexample EELS has been used to detect single atoms of Ca and Fein biomolecules mounted directly onto TEM grids (Leapman2003). These techniques require samples o100 nm in thicknessto avoid multiple scattering interactions between the electrons andthe sample matrix, so their applicability to natural protist samplesis limited.

Protons may also be used to generate characteristic X-ray flu-orescence in protist cells, a technique termed proton-inducedX-ray emission (PIXE). Although less common than analytical elec-tron microprobes, nuclear (proton) microprobes are more readilyavailable and less expensive than synchrotron facilities. A a resultof their higher momentum, protons generate less Bremsstrahlungbackground than electrons and have higher sensitivity for transi-tion metals (Garman 1999). Lower absorption coefficients alsoeliminate the need for sample sectioning for most protist samples.Protons can be focused to only � 1 mm at the required fluxes, andlarger beam sizes are commonly used to analyze marine samples(Iwata et al. 2005; Pallon et al. 1999). Proton-induced X-rayemission is therefore generally inappropriate for studies of sub-cellular element distributions. Radiation damage can also be sig-nificant. Proton-induced X-ray emission analyses can be supple-mented with direct carbon and nitrogen measurements via protonbackscattering, and sample thickness can be quantified by mea-surement of proton absorption by the sample (scanning transmis-sion ion microscopy) (Pallon et al. 1999).

Synchrotron-based X-ray fluorescence (SXRF) microprobes arerapidly becoming a powerful tool for single-cell element analysis.With low Bremsstrahlung background and high ionization cross-sections for heavier elements, SXRF has the highest sensitivity fortransition metals (Sparks 1980). Improvements in Fresnel zone-plate optics are allowing spatial resolution to approach that ofXRMA (Wang, Yun, and Jacobsen 2003; Yun et al. 1999). Thedevelopment of high brightness third-generation synchrotronX-ray sources has provided the needed sensitivity to measuretransition metal stoichiometries in cells collected from the mostpristine regions of the ocean. Radiation damage is significantlyreduced in SXRF. Synchrotron-based X-ray fluorescence analysesdo not require high vacuum conditions and can be performed onfrozen-hydrated samples to reduce radiation damage and preservecell structure optimally. However, it is often most practicalto prepare dried protist samples in order to immobilize mobilespecies and preserve elemental composition until analysis. Syn-chrotron-based X-ray fluorescence measurements can also be sup-plemented with microscale chemical speciation measurementsthat provide redox and coordination environment information(e.g. Bacquart et al. 2007).

A representative SXRF spectrum for a marine protist is shown inFig. 2. The spectrum shown is the sum of 156 individual spectracollected during a raster scan of a small pennate diatom collectedfrom the equatorial Pacific Ocean. The Ka fluorescence peaks for Si,P, S, Cl, Ar, K, Ca, Mn, Fe, Co, Ni, Cu, and Zn are clearly visible, aswell as Kb peaks (for Ca and Cu especially). Using custom designedsoftware (Vogt 2003), each peak in the spectrum is iteratively fit toan exponentially modified Gaussian curve to find the peak areas. Atthe same time background is estimated from the data using a peakstripping algorithm (SNIP; Ryan et al. 1988). Fitting of a complexseries of peaks to data can be time consuming and prone to artifactsdue to the number of free parameters. To make the fitting proceduretractable and efficient, ratios of the Ka and Kb peak areas for an el-ement are tightly constrained according to the ratios observed in thethin-film X-ray fluorescence standards from NIST. Also, relative

Table 1. Comparison of some available techniques for trace element mapping in protists.

Typ.spacialresolution(nm)

Typ.samplethickness(mm)

Resolutionlimitation

Sample preparation Advantages (1)/disadvantages (� )

Light microscope 200 30 Wavelength Requires use of fluorescent dyesCells generally need to behydrated

1 Changes in living cells can be monitored1/� Can only detect ‘‘labile’’ ions insolution and not total element content�Absolution quantification is difficult

Analytical electronmicroprobe (XRMA,EPXMA)

20 0.1 Samplethickness

Thick samples need to besectionedSamples typicallyneed to be dried, or imagedfrozen-hydrated

1 Simultaneously detect 410 elements�Analyses are slow� Significant radiation damage

EELS/EFTEM 2 0.005–0.05 Radiationdamage

Requires ultrathin sections �Only some elements are readily accessible(e.g. P, Fe)�Co-localization can be difficult (EFTEM)or slow (EELS)

Proton microprobe 1,000 50 Radiationdamage;Flux

Samples typically need tobe dried

1 Simultaneously detect420 elements1 high sensitivity�Analyses are slow� Significant radiation damage

Synchrotron X-raymicroprobe

30–200 10 Optics(currently)

Samples typically need tobe dried, or imaged frozen-hydrated

1 Very high sensitivity, low background,selective excitation of analytes1 Simultaneously detect410 elements1m-XANES for chemical state mapping�Analyses are slow

XRMA, X-ray microanalysis; EPXMA, electron probe X-ray microanalysis; EELS, electron energy-loss spectroscopy; EFTEM, energy-filtered transmissionelectron microscopy; PIXE, proton-induced X-ray emission; SXRF, synchrotron X-ray fluorescence; m-XANES, micro X-ray absorption near-edge structure.


positions of the peaks are fixed in accordance with the known X-rayemission lines for each element. Quantitation is achieved by com-paring the areas under the curves with corresponding peaks from theNIST standard.

Synchrotron-based X-ray fluorescence also provides two-dimensional maps of element distribution in target cells. Givena typical focused beam spot size of 200–300 nm for 10 keV hardX-ray microprobes, informative maps of sub-cellular elementlocalization can be generated for larger protists. An example isshown in Fig. 3. This silicoflagellate was collected from theSouthern Ocean during the Southern Ocean Iron Experiment (SO-FeX) project. The edge of a neighboring pennate diatom can beseen to the right of the cell. The chloroplasts are clustered in themiddle of the cell. Silicon maps onto the silicate test, while S isbroadly indicative of the internal cytoplasm components. In thiscell Fe and Zn are most highly localized in the chloroplasts, al-though Zn distributions are often found to correlate with P in othercells. In addition to providing information about the biologicalfunctions of the elements in target cells, these maps are extremelyuseful for identifying extra-cellular abiotic particles that can eas-ily confound bulk analyses (Twining et al. 2003b).

SINGLE-CELL ANALYSIS OF PLANKTON BY ELECTRON

MICROPROBE

Electron microprobe X-ray fluorescence analysis, often termedXRMA, energy-dispersive X-ray microanalysis or electron probeX-ray microanalysis has been used to study the elemental com-position of eukaryotic algae and cyanobacteria collected fromfreshwater systems. Sigee, Levado, and Dodwell (1999) analyzedthe dinoflagellate Ceratium hirundinella sampled from severaldepths in a stratified lake and consistently detected Mg, Si, P, Cl,K, and Ca in the cells. Sodium, Al and Fe were occasionally de-tected, as well, but transition metals generally occur below thedetection limits of XRMA. Minor decreases in some elementswere observed below the epilimnion, but elemental ratios wereconsistent throughout the water column. Concentrations of indi-vidual elements varied significantly between cells from the samepopulation (location and depth), but concentrations were normallydistributed. Other researchers have also noted significant intra-population variability in elemental composition (Fagerbakke,Heldal, and Norland 1991; Villareal and Lipschultz 1995).

A draw-back of XRMA is the limited depth of penetration ofelectrons into target cells. As mentioned above, impinging elec-trons are strongly absorbed by biological materials. Beam pene-tration will vary as a function of the accelerating voltage,specimen density, and the critical excitation energies of elementsin the specimen. Typically, XRMA measurements are limited tothe upper 1 mm of the target cell (Krivtsov, Bellinger, and Sigee2000; Reed 1975). Therefore, while XRMA is useful for studyingthe surface composition of eukayotic cells (Krivtsov et al. 2000;Sigee and Levado 2000), interpretation of XRMA data as whole-cell concentrations can be misleading. Diatoms are more prob-lematic than other cell types as a result of their dense silicate test.Tien (2004), for example, analyzed planktonic and epilithic dia-toms from a polluted river with XRMA, and presented the result-ing concentrations as ‘‘intracellular chemical levels’’. However,given the limited beam penetration into the 10–15mm diam.diatoms, the characteristic X-rays were likely not representativeof the intracellular matrix. A comparison of the reported elementconcentrations for planktonic diatoms collected from the same siteshowed Hg and Pb concentrations (4.4 and 2.7 mg/g, respectively)to actually be higher than P concentrations (1.7 mg/g) in the samecells. Iron concentrations in epilithic diatoms were comparable toP concentrations. These findings depart significantly from our un-derstanding of metal stoichiometries in organisms (Outten andTwining 2007), and such intracellular concentrations are biolog-ically implausible. It is likely that the measured metals are insteadsorbed to the outer surfaces of the cells, and the resulting stoic-hiometries may not be representative of the rest of the cell.

Because of these limitations, marine scientists have used elec-tron microprobes primarily to analyze prokaryotes (Fagerbakke,Heldal, and Norland 1996; Gundersen et al. 2002; Heldal, Nor-land, and Tumyr 1985; Vrede et al. 2002). Heldal et al. (2003)measured C, N, P, and S in single cells from several strainsof cultured Prochlorococcus and Synechococcus. The range ofC:N:P stoichiometries spanned the Redfield ratio, but inter-population variability was observed. Carbon:phosphorous ratiosranged from 156 � 6 to 215 � 9 and 73 � 8 to 350 � 30 forProchlorococcus and Synechococcus, respectively. Sodium, Mg,Cl, K, and Ca were also detected in the Prochlorococcus cells, butthe bioactive transition metals were not detectable via XRMA.Reliable XRMA measurements of trace metals such as Mn and Feappear limited to metal-sequestering bacteria which contain1,000- to 10,000-fold more metal in extracellular appendagesthan typical microorganisms (Heldal et al. 1996).

PLANKTON ELEMENTAL ANALYSIS WITH PIXE

Within the marine sciences, PIXE has primarily been used tostudy the elemental composition of inorganic particulate materialcollected on aerosol filters (e.g. Reis et al. 2006), but severalgroups have applied PIXE for element analysis of algae. Zhanget al. (1997) investigated the binding of Ag, Ba, Cd, Cu, Hg, andPb to the cell wall of Chlorella vulgaris under controlled labora-tory conditions. The metals were easily detected in the samples,which were composed not of individual cells but of a layer offreeze-dried cells collected on a membrane filter. A similar ap-proach was used by Iwata and colleagues to measure metal bio-accumulation by actively growing phytoplankton cultures (Iwata2001; Iwata et al. 2005). These researchers used a 2-mm protonbeam to assay mats of cells collected on filters. More recently,Gisselson, Graneli, and Pallon (2001) mapped C, N, P, S, Cl, K,and Ca concentrations in individual cells of Dinophysis norvegicacollected from the Baltic Sea. Carbon and N were quantified byproton backscattering, and the heavier elements were assayed withPIXE. Absolute cellular quotas (mol/cell) of C, N, and P werelower when measured with the nuclear microprobe than with

Fig. 2. Synchrotron-based X-ray fluorescence spectrum for a pennatediatom collected from the surface waters of the equatorial Pacific Ocean.Fluorescence data are plotted in gray, and model functions are shown asblack lines of varying style: fitted model (solid), Ka peaks (dashed), Kb

peaks (dotted) and background (dash-dot) features unique to the detector.The elements corresponding to the Ka peaks—which are used for quan-tification—are listed at the top of the graph.


traditional bulk techniques, but C:P and N:P ratios were similar.More intra-population variability was observed for N quotas (6.4-fold variation) than C or P quotas (2.2- and 3.4-fold, respectively),which the authors suggested might indicate mixotrophic orheterotrophic ingestion of prey by some of the D. norvegica cellsto obtain N.

SINGLE-CELL SXRF ANALYSIS OF MARINE PROTISTS

Synchrotron X-ray fluorescence was first used to analyze plank-tonic protists in culture and collected from the coastal Atlantic

(Twining et al. 2003a). Following method development (Twininget al. 2003b), the first large-scale field sampling program cameduring SOFeX (Coale et al. 2004). Single autotrophic andheterotrophic protist cells were collected within and outside thefertilized patch and analyzed with SXRF. Here we will summarizeand expand upon the results of this study, which demonstrates thepower of the single-cell approach to studying ocean biogeochem-istry.

The importance of Fe to the functioning of HNLC regions wasnot apparent until the adoption of stringent trace metal ‘‘clean’’techniques to oceanographic sampling and analysis protocols

Fig. 3. Light, epifluorescence, and false-color SXRF element maps of a silicoflagellate cell collected from the Southern Ocean. Warmer colors(yellow and red) indicate higher concentrations of elements. The color scales are different in each element map, so a red pixel in the Si map does notnecessarily indicate the same concentration as a red pixel in the Fe map. Scale bar 5 20 mm.


(Martin and Fitzwater 1988; Martin, Gordon, and Fitzwater 1990;Martin, Gordon, and Fitzwater 1991). The resulting observationsof low dissolved Fe led Martin (1990) to hypothesize that varia-tions in delivery of airborne Fe-rich dust to the Southern Oceanover time may have caused past glacial periods by generatinglarge blooms of phytoplankton. This hypothesis has stimulated aseries of large-scale Fe addition experiments, modeling exercises,and even proposals to geoengineer atmospheric CO2 by adding Feto stimulate phytoplankton growth and reduce atmospheric CO2

(Kintisch 2007). Cellular Fe:C ratios in the plankton are of par-ticular interest because they affect the relationship between C se-questration and Fe addition, and therefore the economic viabilityof attempts to bioengineer atmospheric carbon through Fe fertil-ization. Quotas of other nutrient elements such as P, Mn, Ni, andZn are also important for their potential to limit cellular growth.Cellular quotas of these elements were measured with SXRF onsingle diatom, autotrophic flagellate and heterotrophic flagellatecells.

Iron quotas were detectable even under HNLC low Fe condi-tions, and community wide averages of Fe:C agreed well withradioisotopic measurements of C and Fe uptake (Twining et al.2004b). However several interesting differences between celltypes emerged. First, diatoms collected from unfertilized watershad lower cellular Fe:C (with carbon estimated from biovolume)than did flagellates (Fig. 4). While cell volume-normalized ele-mental contents might be expected to be lowest for diatoms withlarge vacuoles, the differences in Fe:C ratios may reflect moreinteresting ecological or biological distinctions. For example, di-atoms are constrained to obtain Fe from the dissolved fraction andmay thus be more susceptible to limitation than flagellates, whichin the open ocean are widely believed to feed upon other cellswhich have already done the work of concentrating Fe from theenvironment (Maranger et al. 1998). Indeed, the Fe:C ratios ofheterotrophic flagellates more closely resembled the uptake ratioof Fe and C into the bacterial size fraction as determined byradioisotopes. Alternatively, the higher Fe content in heterotrophsmay reflect a greater requirement. Respiratory enzymes that makeup the electron transport system have a higher Fe content thandoes the photosynthetic apparatus that makes up much of auto-trophic cell mass (Chase and Price 1997; Raven 1988).

Cell types differed in a number of other important respects aswell. For example, estimated P:C and P:S ratios were lower fordiatoms than for flagellated cells (Fig. 4), a finding in oppositionto culture studies indicating that diatoms as a group have high Pcontents (Ho et al. 2003). Diatoms also exhibited much higher Niand Zn quotas. Elemental fluorescence maps indicate that most,but not all, of the Ni in diatoms was associated with the frustule,and in particular with the densely silicified girdle region (Twininget al. 2003b). The only known Ni enzyme in eukaryotes is urease,which can be used to assimilate reduced urea nitrogen from theenvironment. Although nitrate was present at non-limiting con-centrations (Coale et al. 2004), urease expression in culturedphytoplankton is not generally suppressed in the presence of ni-trate (Lomas 2004; Peers, Milligan, and Harrison 2000). Further-more, diatoms in the Southern Ocean may favor the use of reducedN when cellular energy is limited by light and Fe availability(Price, Ahner, and Morel 1994). In contrast with Ni, Zn seemed tobe associated mostly with the nucleus (Twining, Baines, and Fish-er 2004a), perhaps reflecting the importance of Zn-containing fin-ger proteins in the overall Zn budget of the cell. This observationis interesting because most discussion of Zn in phytoplankton re-volves around carbonic anhydrase, a well described enzymeknown to contain Zn, and does not consider the role of Zn fingerproteins (Morel et al. 1994; Saito, Goepfert, and Ritt 2008).

Measured elemental ratios differed from expected values inseveral important respects. Cellular Fe:C quotas ranged between6 and 14 mmol mol� 1, 2–4 times higher than the 3–5 mmol� 1

Fe mol� 1 C estimated by Moore et al. (2004) for Southern Oceanplankton. This discrepancy may reflect inter-specific differencesin the Fe quotas of Southern Ocean phytoplankton compared tothe cultured species used to parameterize most models. It also re-mains unclear how to extend culture work performed in EDTA-buffered media to natural systems where the bioavailability ofdissolved Fe is largely unknown. Furthermore, higher Fe quotasmight result from physiological adjustments to low-light condi-tions in the Southern Ocean (Sunda and Huntsman 1997). Asmight be predicted for a region with excess dissolved phosphate,cellular P was also unusually high in Southern Ocean cells, being42-fold more abundant in flagellates than predicted by Redfield.Diatom Zn:P ratios were elevated relative to bulk plankton fromthe Pacific Ocean but similar to bulk plankton from SouthernOcean (Twining et al. 2004a). This suggests there may be uniqueecological or biogeochemical aspects of Southern Ocean taxa thatare reflected in the elemental composition. Measured diatom Znquotas were more than an order of magnitude higher than thosereported for cultured diatoms by Ho et al. (2003). This may indi-cate greater bioavailability of Zn bound to natural organic ligandsthan typically assumed. Diatom Zn quotas in culture may alsohave been lowered by relatively high Mn concentrations in themedia, as Mn can inhibit Zn uptake in marine phytoplankton(Sunda and Huntsman 1998b). These comparisons highlight theuncertainties introduced when culture data is applied to naturalsystems and the benefit of field measurements.

The cellular concentrations of several elements changed mark-edly in response to Fe addition. Iron cellular concentrationschanged most, increasing in all cell types by a factor of 3–6 overthe course of 6 days and two additions. While the Fe content ofdiatoms and autotrophic flagellates increased after both Fe addi-tions, the Fe:C ratio in heterotrophic flagellates exhibited a de-layed response which probably reflected a particulate food supplythat was continuously changing in Fe content (Fig. 5). Such metalcycling within plankton communities is extremely difficult tostudy without single-cell analytical tools, because the dominantautotrophs and heterotrophs have similar sizes. Interestingly, Pand S content of diatoms increased markedly after Fe addition,perhaps reflecting an increase in cytoplasm within the frustule. It

Fig. 4. Comparison of P, Mn, Fe, Ni, and Zn quotas (normalized to C)in diatoms and flagellated cells collected from the Southern Ocean. Foreach element the bar represents the difference between the logged meanquotas of diatoms and flagellated cells (all quotas were log-transformed tostabilize variance). Quotients greater than 0 indicate higher quotas in di-atoms, while negative quotients indicate higher quotas in flagellated cells.Statistical significance is denoted with asterisks (�Po0.05, ��Po0.01).Modified from Twining et al. (2004a, 2004b).


may be that reduction of the cytoplasmic mass (or cell volume) isa general response to Fe limitation. In addition to these changes,cellular concentrations of the bioactive metals Mn, Ni, and Zn alsoincreased 1.5- to 4-fold after Fe addition. In general, these resultssuggest that relaxation of Fe limitation caused a physiologicalcascade in protist cells leading to a high growth, high metabolicrate condition.

Some of the most interesting findings regarded cellular silica indiatoms. Diatoms in the Southern Ocean were heavily silicifiedprior to Fe fertilization. The average diatoms had Si:C ratios of0.5 mol mol� 1, which is nearly 4 times the typical value for di-atoms in culture (Brzezinski 1985). These measurements implythat SiO2 constitutes � 20% of wet mass and � 50% of the drymass of the diatoms in the Southern Ocean. The high degree ofsilicification may explain why the Southern Ocean plays such animportant role in the global C cycle since dense silica ballast en-hances sinking rates of aggregates and fecal material produced inthese regions. It is widely believed that high silicification is typ-ical of Fe-limited regions because silica incorporation is inexpen-sive in energy terms and tends to continue even as cellular growthrates slow in response to limitation by other nutrients (Francket al. 2000). However, Fe addition in the Southern Ocean pro-duced only a transient 40% decline in Si per cell, after which cel-lular Si contents rebounded to match those of cells collectedbefore fertilization and from a control patch after the experiment.There was a 2-fold decline in Si:P and Si:S ratios, but this reflect-ed the increase in cellular P and S rather than a decrease in Si.Clearly, the maintenance of high cellular silicification in theSouthern Ocean requires an explanation other than Fe limitation.

The SXRF data collected during the SOFeX project demon-strate the power of single-cell element measurements for studyingthe role of protists in the biogeochemistry of natural systems. Thisproject has been recently followed up by SXRF studies in theEastern Equatorial Pacific, which is also believed to be Fe-limited,and in the Sargasso Sea, which is nutrient poor but generally con-sidered Fe-replete. These analyses are ongoing and as yet unpub-lished, but they generally confirm many trends noted for diatomsin the Southern Ocean, most notably higher than expected cellular

Fe, lack of an effect of Fe availability on cellular silicification, anincrease in cellular S and P content when Fe limitation is relieved,and high cellular Ni and Zn. Large differences among cell typeswere also noted, although not all patterns conformed to those ob-served in the Southern Ocean. In addition, recent improvements inanalytical sensitivity of the SXRF microprobe have allowed traceelement contents to be measured in prokaryotes for the first time,and these have proven to be distinct from those of eukaryotic cells.

FUTURE METHODOLOGICAL DEVELOPMENTS

Synchrotron X-ray microprobes offer arguably the best combi-nation of analytical sensitivity, analytical breadth and spatial res-olution while imparting the least radiation damage to protistsamples. There are several limitations to their usefulness for en-vironmental and other microbiologists, however. First, SXRF typ-ically cannot measure the major components of organic matter (C,N, and O), which complicates interpretation of cellular concen-trations and two-dimensional element maps. Second, localizationof elements within the cell is currently only possible in two di-mensions, which makes it difficult to determine how much of anelement is associated with, for example, the cell surface or cellularorganelles embedded in protoplasm. Third, routine spatial resolu-tions are currently too low to resolve cell structures smaller thanthe nucleus or chloroplast.

Estimation of cellular mass simultaneous with SXRF analysesis possible in one of two ways. Just like visible light passingthrough water or air, X-rays passing through biological samplesare scattered in proportion to the X-ray density of the substance.These scattered X-rays are registered by the detector and the areaunder the Compton scattering peak can be used as a proxy for cellmass at a particular pixel. Without a vacuum chamber, the methodis fairly insensitive, because background scattering peaks due toHe atmosphere are large, resulting in a high ratio of noise to signaland high error. Also, the XRF detector is typically positioned tominimize detection of scattered radiation (and thus improve sen-sitivity), though use has been made of a second detector at a po-sition optimized for detection of increased scatter (Golosio et al.2003). A much more sensitive method that can be used for thinbiological samples without a vacuum is X-ray phase contrast mi-croscopy (Hornberger, Feser, and Jacobsen 2007). X-rays are sub-ject to phase shift as they pass through biological samples to adegree dependent on the thickness and composition of the sample.Using a specially designed and fabricated segmented detector po-sitioned downstream of the sample source, differences in phasecan be recorded to produce a differential phase contrast image(Feser et al. 2006). Given assumptions about the C, O, and Ncomposition of the sample, and direct measurements of other el-ements available from the fluorescence spectra, the phase shift canbe reconstructed (de Jonge et al. submitted; Hornberger et al.2007) and used to estimate cellular mass at each pixel. Becausethe method uses the same X-ray beam to construct a map of flu-orescence and phase contrast (Hornberger et al. 2006), the twomaps coincide perfectly (Fig. 6). This method has been calibratedon latex beads, and a comparison with standard chemical deter-minations of elemental composition of algal cells is underway(Hornberger et al. 2007).

X-ray fluorescence computed tomography (XFCT) offers thepossibility of visualizing both elemental distributions and phasecontrast maps in three dimensions (Boisseau and Grodzins 1987;Golosio et al. 2003; La Riviere and Vargas 2006; La Riviere et al.2006). In XFCT the target is repeatedly imaged in two dimensionsunder different angular positions. Once the desired angular rangeis covered, the three-dimensional elemental or mass distributionthat best reconciles all of the images is determined. This processcan be complicated for larger cells due to the absorption of lower

Fig. 5. Carbon-normalized Fe quotas for diatoms (white circles), au-totrophic flagellated cells (white triangles), heterotrophic flagellated cells(gray triangles) and the mean of all three cell types (black circles) fol-lowing fertilization of Southern Ocean waters. Cellular Fe was measureddirectly with SXRF. Cellular C was calculated from cellular S using a C:Sstoichiometry of 100. Points are geometric means ( � SE). Modified fromTwining et al. (2004b).


energy fluorescent X-ray photons by cellular organic matter (Go-losio et al. 2003). However, recent developments in statistical fit-ting techniques that also estimate this self absorption usingpenalized-maximum likelihood have been able to correctly recon-struct the three dimensional test structures for which self absorp-tion is significant, making quantitative tomography possible (LaRiviere et al. 2007). This approach effectively increases the size oftargets that can be imaged. Anticipated improvements in terms ofX-ray source brightness should improve the speed of such tech-niques in the future.

The focused spot sizes (i.e. spatial resolution) of hard X-raymicroprobes continue to improve with advances in X-ray optics(Hignette et al. 2005; Kang et al. 2006). Current instrumentationat third-generation synchrotron facilities is capable ofo200 nmspatial resolution with 10 keV X-rays, with ongoing efforts toreduce this to 30 nm. Beamlines utilizing intermediate energyX-rays (2–3 keV), such as 2-ID-B at the Advanced Photon Source,have already achievedo100 nm resolution (Fig. 6D) (McNultyet al. 2003). This level of focus will allow elemental measure-ments in viruses and the smallest prokaryotes, which can beimportant food resources for protists. It will also enable moreprecise sub-cellular element mapping in eukaryotes, expandingour ability to study the biological roles of metals in protists.

CONCLUSIONS

No microprobe technique is perfectly suited to all potentialenvironmental applications. Electron microprobes are widely

available and are useful when studying major elements in smallprokaryotes. Proton microprobes can detect a wide range of ele-ments but at limited spatial resolution. Synchrotron-based X-rayfluorescence combines unmatched sensitivity for transition metalsand spatial resolution that approaches XRMA, at the expense oflight element quantification and instrument availability. Each ofthese microprobe techniques can be used to help open the ‘‘blackbox’’ of plankton elemental composition with single-cell mea-surements. Only by making geochemical (elemental) measure-ments of individual functional groups will we advance ourunderstanding of the relationships between protist biology, ecol-ogy, and biogeochemistry.

ACKNOWLEDGMENTS

We would like to thank Ian McNulty for collaboration ondifferential phase contrast experiments at beamline 2-ID-B. Thiswork was supported by grants from NSF to BST (OCE 0527062)and SBB (OCE 0527059). Use of the Advanced Photon Sourcewas supported by the U.S. Department of Energy, Office ofScience, Office of Basic Energy Sciences, under Contract No.DE-AC02-06CH11357.

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Received: 02/01/08; accepted: 02/02/08


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