Anal. Chem. 1992, 64, 2972-2976
2972
Detection of Copper in Isolated Plant Cells by Resonance Ionization Mass Spectrometry N. 5. Nogar* and R. C. Estler**+ Chemical and Laser Sciences Division, MS J565, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
J. Conia*and P. J. Jackson Life Sciences Division, MS M880, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
Detection of copper In m a t e d plant celb at the mlllhrdar leveland below k demonstratedby resonancelonlzatkn mass spectrometry (RIMS). Datura bmoxk MIII. cell swpe” cultureswere malntalned In the presence of 1 mM CuSO. and converted Into protoplastsby enzymatk dlgestlon of the cell wall. Allquots contalnlng 10-100 protoplasts were subsequently examlned by RIMS to detect the mlnute amounts of copper isotopes that were accumulated by the colk. These results suggest opportunltles to measure trace metal concentratlons at the rlngle cell level. Slngle cell mass spectra should not only p r o w Information about the phyrldogkal state of isolatedgrowlng celk but ako refled the Intracellular content of small cell populatlonr when exposed to chemkak or toxlc compounds.
INTRODUCTION The detection of trace metals in single cells is a difficult and important analytical task. Apart from the inherent challenge of performing the measurement,there are a number of compelling reasons for determining this property on a cell by cell basis. Such an analysis would allow LIS to answer questions not normally accessible to methods which integrate the signal over an ensembleof cells. Thus, it may be possible to establish distributions of elemental concentrations within a cell population and of correlations between measured characteristics of a cell population and this distribution. The mass spectrum of a single cell contains information on both its intracellular cation contenta and on the bioorganic matrix. The physiological state of a cell may express itself by the magnitude and variation of metal ion concentrations; this is particularly true for metals present in macroscopic concentrations, such as Na, K, and Ca. A measurement of trace metal concentrations may provide information on transport mechanisms and uptake in the cell or the host organism. Nutritional and metabolic processes may be reflected in the concentration of these species. In addition, the presence of certain toxic metals in cells may reflect exposure of the cell or host organism, either intentionally, as in chemotherapy, or unintentionally, as in exposure to toxic waste. Significant progress has been made recently in the interrogation of single cells. In a significant recent report, electrochemical measurementswere performed on single cells using microelectrode techno log^.^ Electrochemicalmethods have also been used in the interrogation of ion channel activity in single cells.2 In addition, several reports have appeared t Permanent address: Department of Chemistry, Fort Lewis College, Durango, CO 81301. $Permanent address: Cell Robotics, 2715 Broadbent Pkwy, NE, Albuquerque, NM 87107. (1) Bailey, F.; Malinski, T.;Kiechle, F. Anal. Chem. 1991,63, 395-8. (2) Neher, E.; Sakmann, B. Nature 1976,260,799.
describing the use of maw spectrometry in the analysis of cella. Laser microprobe mam spectrometry (LAMMA) has been used to measure the ratio of sodium to potassium in Escherichia coli cells exposed to varying levels of a nitrofuran derivative3 and in mycobacterial cells (Myobacterium tuberculosis H 37 Ra).4-6 Uptake of uranium into the alga Dunaliella has been explored by LAMMA.’ Microprobe methods have also been used for qualitative investigation of vegetative bacterial cells.* More recently, resonance ionization maw spectrometry (RIMS) has been suggested as a promising tool for the interrogation of cells,s11 and even subcellular components. RIMS utilizes a multistep, multiphoton process to effect ionization of a selected element or compound in a highly selective manner. Typically, a pulsed (or, in some cases, continuous-wave12)laser is tuned to resonance with a (single or multiple photon) transition in the analyte of interest. This resulta in selective excitation of that analyte; subsequent absorption of one or more photons results in ionization of the target atom or molecule. The narrow bandwidth of available dye lasers, together with the sharp resonances typical of gasphase atoms can result in extraordinary selectivity for the ionization process. Selectivities in excess of 106 are routinely reported, and may be as high as 102O in some cases.13 This is of critical importance in interrogating complex biological system, where the background due to isobaric metale or organic fragments may be substantial. In addition, the resonance ionization process can be extremely efficient; it is possible to convert -100% of the atoms within the focal volume of the laser into i0n8.l~ Overall conversion efficiency ( a t o m into ions) is typically S10-6,14J6 due to spatial and temporal overlapof the sample plume and laser beam. When combined with mass spectral sorting, and high efficiency electron multipliers for detection, RIMS offers the opportunity for unparalleled sensitivity. (3) Lindner, B.; Seydel, U. Int. J. Mass Spectrom.Zon Phys. 1983,48, 265-8. (4) Seydel, U.; Lindner, B. Biomed. Enuiron. Mass Spectrom. 1987, 16,457-9. (5) Seydel, U.; Lindner, B. Znt. J. Quuntum Chem. 1981,20,605-12. (6) Seydel, U.; Lindner, B.Freseniw’Z.Anal. Chem. 1981,308,263-7. (7) Sprey, B.; Bochem, H.P. Fresenius’ Z. Anal. Chem. 1981, 308, 239-45. (8) Bohm, R. Fresenius’ Z. Anal. Chem. 1981,308,253-7. (9) Hrubowchak, D. M.; Ervin,M. H.; Wood, M. C.; Winograd, N. Anal. Chem. 1991,63,1947-63. (10) Arlinghaua, H. F.;Thonnard, N.; Spaar, M.T.;Sachleben,R. A.; Larimer, F.W.; Foote, R. S.;Woychik, R. P.; Brown, G. M.; Sloop, F. V.; Jacobson, K. B. Anal. Chem. 1991,63, 402-7. (11) Botter, R.; Dimicoli, I.; Mona,M.;Piuzzi,F.Adu. Mass Spectrom. 1989, l l A , 294-313. (12) Miller, C. M.; Nogar, N. S. Anal. Chem. 1983,55, 1606-8. (13) Huret, G. S.;Payne, M.G.; Kramer, 5. D.;Young,J. P.Reu. Mod. Phys. 1979,51, 767-819. (14) Miller, C. M.; Nogar, N. S. Anal. Chem. 1983,55,481-8. (15) Downey, S.W.; Nogar, N. S.;Miller, C. M.Znt. J. Mass Spectrom. Zon Processos 1984, 61, 337-45. @ 1992 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
We report here on the detection of copper atoms in cells of Datura innoxha (jimsonweed). This tsst system was chosen for several reasons. The cells are rather large, typically exhibiting diameters -30 pm. In addition, numerous strains have been shown to be metal tolerantPJ1 cells have been cultured in solutions containing heavy metale a t concentrations approaching millimolar18.19 as well as in extreme pH solutions.20 In addition, methods are well-developed for culturing these cella in a variety of media.16J192 Lastly, copper was chosen for a test case because of past experience in RIMS detection and analysis of copperBsu and because copper is an important trace nutrient.2k2I
EXPERIMENTAL SECTION RIMS Instrumentation. The measurementsdescribed below utilized a tunable dye laser for excitation, and a 0.4-m time-offlight (TOF) mass spectrometer for ion sorting and detection. This instrument has been described in greater detail previously;lS@ only pertinent aspects are presented here. Pulses, at 20 Hz and 12-nsduration, from an excimer laser-pumped dye laser (Lambda Physik Model 101/2002) were focused to a spot =0.7 mm in diameter in the source region of the mass spectrometer, immediately adjacent to (in some cases, grazing) the sample fiiament, and retroreflected with a dielectricmirror. Ionization of ground-state neutral Cu atoms was effected by pulses from a single dye laser tuned to a two-photon resonance at A = 464nm, with pulse energies in the range 3 mJ I E p h I6 mJ. The use of a single-color, two-photon transition greatly simplifies the experimental apparatus and may lead to a greater utility of the RIMS technique in biochemical applications (as opposed to multicolor, multilaser ionization schemes). The extraction electric field was approximately 110 V/cm, followed by a drift tube at a potential of -500 V. Time lag focusing, synchronized to the laser trigger, was used to discriminateagainst thermal ions and thus improve the signal to noise ratio. A pair of deflection plates between the extractor and the flight tube could be voltage adjusted to maximize the transmission of ions to the detector. Detection electronics consisted of a channel electron multiplier (Gaillileo),a preamplifier (Comlinear Corp.) and discriminator, a digital storage oscilloscope (Tektronix 2430), a gated counter (Stanford Research SystemsSR400),and a controlling computer system (MacintoshPlus/IOTech 488 bus controller). The mass signal is routed to both the gated counter and storageoscilloscope. Individual mass spectra corresponding to single laser shots are transferred to the computersystem from the storage scope where they are summed during the filament heating cycle of the experiment. Simultaneously, the ion counts are accumulated for sequential, preset time intervals (usually 10 e), providing a signalhime histogram of the same experiment. Total signals for a given run are obtained by summing the normalized signal histograms from the gated counter over the entire heating cycle. Maintenance of the Cell Suspension Culture. The D. innoxia Mill. cell line was grown in the dark at 30 O C with (16)Jackson, P. J.; Naranjo, C.; McClure, P. R.; Roth, E. J. Cell. Mol. Biol. Plant Stress 1985,146-60. (17)Delhaize, E.;Jackson, P. J.; Lujan,L. D.; Robinson, N. J. Plant Physiol. 1989,89,700-6. (18)Jackson, P. J.; Roth, E. J.; McClure, P. R.; Naranjo, C. M. Plant Physiol. 1984,75,914-8. (19)Jackson, P. J.; Torres, A. P.; Delhaize, E.; Pack, E.; Bolender, S. L.J.Environ. Qual. 1990,19,644-8. (20)Brachet, J.; Coseon, L. J . Exp. Bot. 1986,37,650-6. (21)Wylegalla,C.;Meyer,R.;Wagner, K. G.Planta 1985,166,44641. (22)Conia, J.; Alexnnder, R. G.; Wilder, M. E.; Richards, K. R.; Rice, M. E.; Jackson, P. J. Plant Physiol. 1990,94,1568-74. (23)Apel, E. C.; Aaderson, J. E.; Estler, R. C.; Nogar, N. S.;Miller, C. M. Appl. Opt. 1987,26,1045-50. (24)Engleman, R.J.; Keller, R. A.;Miller, C. M.; Nogar, N. S.;Paisner, J. A. Nucl. Imtrum. Methods Phys. Res., Sect. B. 1987,B26, 448-53. (25)ZincandCopper;Praesad,A.S.,Oberlas,D.,Eds.;TraceElementa in Human Health and Disease; Academic, New York, 1976; Vol. 1. (26)Copper in Animals and Man; Howell, M. E., Ed.; CRC Press: Boca Raton, FL, 1987. (27)Roles of Copper in Lipid Metabolism; Lei, K. Y .,Ed.; CRC Press, Boca Raton, FL,i9%. (28)Nogar, N. S.;Downey, S. W.; Miller, C. M. Anal. Chem. 1985,57, 1144-7.
2973
agitation.% As described elsewhere, the cell suspension was maintained as exponentially growing culture by regular transfer at 2-day intervals into fresh culture medium.18 This culture medium was a modified Gamborg's mediumBsupplementedwith 1mM CuSOd (finalconcentration),where they were cultured for 24 h. Protoplast Preparation. Protoplast isolation was obtained as previously described.30 Protoplasts were isolated from D. inrwxia cell culturesby mixing an equal volume of cell suspension with a solution containing 600 mM KC1, 70 mM CaClg, 0.5% (w/v) Cellulase (Worthington Biochemical Co., Freehold, NJ), 0.05% (w/v) Pectolyase (Sigma Chemical Co.), 0.08% (w/v) dithiotreitol, and 0.6% (w/v) poly(ethy1ene glycol) 8OOO (Sigma Chemical Co.). The pH of the solution was adjusted to 5.5 using a solution of 1M HC1. The enzyme solution was sterilized by passage through a 0.2-pm Nylon membrane (Corning,NY)prior to use. Cells were incubated in the presence of the enzymes with agitation for 40 min at 30 "C. After digestion of the cell wall, the protoplast suspension was filtered through a 65-pm Nylon-mesh fiiter. The protoplasts were rinsed twice with culture medium by successivecentrifugations (lOOg,2 min). This culture medium was similar to the one used for cell suspension culture but was without added copper. The protoplasts were then suspended in this medium at an initial density of about 30 OOO/mL. For the detection of copper concentration within small populatione, aliquots of protoplastswere further diluted in the culturemedium lacking copper. Mass Spectral Analysis. Protoplasts in the culture medium were diluted and applied to the sample fiiaments. Here, 20-pL aliquots were air-dried onto Re-ribbon fiiaments (0.5cm X 0.075 cm X 0.0025 cm) for subsequent mass spectral analysis. Depending on the level of dilution and the enzymaticactivity,these samples contained single cells, a number of dispersed cells (typically3-10),orcellclusters. Agivensampleplatformisplaced within the ionization region and resistivelyheated to provide the source of neutral Cu analyte atoms. Filaments were used only for a single experimental determination to avoid cross-contamination. To minimize shot-to-shot signal variations the excimer laser/ dye laser was operated at an intensity level sufficient to effect saturation of this two-photon transition.u For the 0.7-mm spot diameter used in these experiments,this corresponds to a pulse energy 1 5mJ, at a laser bandwidth S0.3 cm-I. For the purposes of spectroscopically tuning the dye laser to the appropriate transition, a continuoussource of the naturally occurringisotope neutrals was available by resistively heating a f i i e n t spiked with Cu from a standard sample; this source was removed prior to protoplast analysis. Following opticaltuning, each protoplast sample was loaded into the spectrometer for analysis. Operationally, the sample was f i i t heated for 3 min at a temperature estimated to be 600 OC and for 5 min 650 O C (below the temperature required for detection of thermal evaporation of Cu neutrals), in order to drive off high-volatility impurities and ensure orderly evaporation of the analyte. After this preheatingcycle the temperature was stepped to 760 O C to induce atomizationand begin data collection. Data collectioncontinued until the sample was depleted.
RESULTS AND DISCUSSION Ionization of ground-state (3d104s1,2S1p) neutral Cu was effected by a two-photon transition (A = 464 nm) to an upper electronic state (3d105S1,2S1p, 43137 cm-l) followed by the absorption of a third photon of the same frequency. Figure l a shows the detected ion signal as a function of wavelength for the 2 + 1ionization of a copper sample (-1 pg) obtained from an ICP standard. The inset in this figure also shows a schematic of the relevant energy level diagram. The large feature a t 463.65 nm corresponds to the two-photon resonant transition described above, while the small feature a t 463.55 ~~
(29)Gamborg, 0.L.; Miller, R. A,; Ojemos, K. Exp. Cell. Res. 1968, 50.151-8. '(30)Conia, J.; Neeman, A.; Anderson, P.; Jackson, P. J. Manuscript in preparation.
2974
ANALYTICAL CHEMISTRY, VOL. 64,
NO. 23, DECEMBER 1, 1992
r---‘“1 60
\
,
,, 1.0
-
0.8
-
0.6
-
0.4
-
0.2
-
0.0
-
.021
11 0
463.50
463.55
483.60
463.65
463.70
Wavelengthlnm. MasslDa 5,O
6,:
515
I 11 5
7,O
I
I
12 0
12 5
7,5
1 130
:8
-
,
I 135
Flight T i m d u s e c
+
Flgue 1. (a, Top) optical spectrum for the 2 1 bnkatkn of copper, with a s 1 m p M energy level diagram as the Inset. (b, Bottom) time of-fllght mass spectrum of resonantly photohized copper.
nm is currently unidentified. In this case, the signel processing electronicswere set to accept signale from both W u and GCu. This sample was used to tune the laser to the appropriate wavelength and to optimize transmissiw of the ion optics. The ionization signal exhibits a spectral width (fwhm) of -0.4 cm-l, slightly greater than that of the exciting dye laser (=0.3 cm-9, suggesting that either or both of the twophoton absorption or the ionization step was saturated. Previous experiments23and calculationsu suggest that the two-photon step is saturated and the ionization step is 190% saturated. Figure l b is a mass spectrum obtained for a single sample, showing incomplete resolution of the two copper isotopes. This is due to two constraints: first, the sample platform intruded into the extraction region, disrupting the normal electric fields; second, the ion optics were optimized for transmission efficiency rather than for resolution. It is useful to calculate the signal size anticipated for these analyses. The protoplasts are on the order of 30 pm in diameter. These cells are aseumed to equilibrate with the culture medium during growth. It is likely that at least the cells on the outer edge of the cell clusters are in equilibrium; those in the interior of cell clusters may assimilate less copper than those on the outside. Cells achieving the equilibrium concentration should contain =(loW3M)(1.4 X 16’’ L)(6 X loBmol-’) = 8 X 1Og copper atoms, or about 1pg. We assume the probability of evaporation of -0.5, and the combined ionization-transmission-detection efficiency, for a t o m in the ionizationvolume,of =0.1. For the geometry discussed above, the spatial overlap of the laser beam with the sample plume, assuming an isotropic spatial distribution, is -0.2, while the
temporaloverlap (with continuousevaporationand irradiation at 20 Hz) is =2 X le7.This yields an overall detection efficiency of 2 X lo+, or about 20 copper atoms detected per protoplast on the sample platform. Samples containing the plant protoplasts were dried on the filaments in room air at ambient or slightly elevated (100 OC < 2‘9 temperature in order to fix them on the filament. Because of the limited capabilities in this study for cell handling, sorting, and counting, it was not poeeible to reliably place a single protoplast on the sample platform. Extreme dilutions and careful manual sample selection typically allowed a minimum of 10 f 10 cells to be placed on the platform. Figure 2a,b show samples before heating. In the former case, no protoplasts (cells)were present on the sample platform; in the latter case, several individual cella were present, as well as at least one two-cell cluster. Note that the protoplasts appear larger than 30 pm, due to the fact that they are flattened on the sample platform, as effectively twodimensionalentities. Figure 2c showsa sample platform after analysis. The surface appears “carameliZBd*,presumably due to the relatively high concentration of sucrose in the culture medium. It is obvious from the bubbled appearance of the surface where protoplasts had been prior to evaporation. The temporal evolution of the copper signal is displayed in Figure 3, for a sample loading of =10 protoplasts and for a sample of culture medium with no added copper. The signal from the copper can be seen to rise and fall in a period of about 2-3 min, while the blank exhibits a minimal signal over asimilar period. The rising edge of the waveform corresponds to the heating rate for the f i i e n t and equilibration of the copper to this temperature. The anticipated ratas of evaporation for elemental copper applied to the filament can be calculated using standard techniquedl and the assumption of equilibrium evaporation from a source of elementalcopper. The decay of the signal is due to exhaustion of the sample and ia roughly consistent with the calculated decay ratas for samples heated to this temperature. The transient signal from the blank is likely due to radiative evaporation of copper deposited on nearby parta of the time-of-flight ionization region by past samples and removed by flash heating during the warm-up cycle of the sample filament. It is tempting to estimate the totaiCu content in the protoplasts by comparing these signaVtime histograms, but such analysis may be dangerous at this stage of investigation since the rate of Cu evaporation from this complex matrix ae well as the mechanism and efficiency of Cu atomization may be quitedifferent. Further study is required prior to making such comparisons. After decay of the copper signal from the cell matrix, a new signal appeared at masses 66/67, lasting for many minutes. This signalwas photogenerated but consistedof both resonant and nonresonant contributions. A brief s w e y of the optical spectra suggested the presence of bandheads, indicating that the spectra may be due to a small molecule. The ionization of this molecular species must be due to accidental coincidences of the chosen excitation wavelength with rovibronic transitions in the molecular species. At the present, we are unable to unambiguously identify the species. In Figure 4 we display the mass spectra due to a loading of =lo0 protoplasts, 10protoplasts, and a blank. Protoplasts were counted with an optical microscope for the smaller sample,estimated by optical microscopyfor the larger sample and verified absent for the blank. The total signal for the 100 cell sample generated several thousand counts, in rough agreement with the signallevel calculated above. The sample containing 10cells produced 530detected ions, while the blank produced 30counts. If we assume that the copper was evenly (31)Dushman, S . ScientificFoudation.9 of Vacuum Technique;John Wiley & Sons, Inc.: New York, 1962.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
2975
Flgure 2. Micrographs of (a, left) the sample platform with only growth medium applied, (b, middle) several protoplast in the growth medlum, and (c, right) a sample platform after heating. The bubbled appearance of the surface in some areas of (c) indicates where protoplasts had been prior to evaporation. The width of the platforms displayed in these images is 250 pm. 120
I
100
-
90 -
a
80 -
0
70 -
U
aJ 0 c
60
-
50
-
40
-
30
-
20
-
ii m
0-
2
0 0
I
I
I
1
n
110-
l'OI
0.9
0,8 1-100 protoplasts of Datura innoxia
0.6
-
-0.1
I-0
1
2
3
4
i
11.0 5
Elapsed Time in Minutes
Flgure 3. Temporal evolution of the Cu signal for a filament loading of =10 protoplasts and for a control sample containing an amount of culture medium equal to that of added copper. The fast rise of the signal for the protoplastsample occurs at the temperature jump of the filament following a preheat cycle.
distributed among the cells, this implies a signal to noise ratio for a single cell of 53/(30)1/2= 10. This indicates that we have the ability to detect transition metals in single cells at the millimolar and below level. Numerous experiments were conducted in which samples containing 3-20 cells were interrogated, with detectable signals observed for all samples. Signal levelswere in rough agreementwith those quoted above, though a cell count and associated numerical analysis were conducted in only two cases. Significant improvements in sensitivity may be possible through the use of a multichannel plate detector in place of the electron multiplier and by the use of a higher repetition rate laser. During these experiments, it was assumed that the cells in culture came into chemical equilibrium with the surrounding growth medium. That is, the copper concentration within the cell was the same as that in the medium. It is not clear that this is necessarily the case: cells may concentrate, or in some casesdilute, trace metals, relative to the growth medium. Future experiments may test this by exposing the cells for
11.5
,
,
,
,
i
12.0
,
,
,
,
l
12.5
,
,
,
,
l
13.0
,
,
,
,
~
13.5
Time-ot-Flight (us)
Flgure 4. Timeof-flight mass spectra for a sample containing onty the c u k e medium(lower trace), a sample Containing10 protopakts(middle trace), and a sample containing =lo0 protoplasts (upper trace).
shorter periods of time in an attempt to force intercellular variation in metal content. Under these circumstances, one might expect, based on simple diffusion arguments, that cells in the interior of clusters will take up less copper than those on the exterior. A correlationwould then be expected between the distribution of measured per-cell signals and the cluster size distribution.
CONCLUSIONS Copper was detected by RIMS from an ensemble of 10 cells cultured in a medium containing M copper. Calculations indicate that the copper in a single cell could easily be detected at this concentration level. Single cell interrogation was not possible in this case because of difficulties in sampling. Manual selection of the samples proved to be satisfactory for the handling of aliquota containing from 10 to 100 protoplasts. Using diluted protoplast suspensions, cell handling with a micropipet was effective in applying tens of cells onto the filament. However, this method was extremely time consuming and inadequate for the selection of one unique cell. In order to make such
2978
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, lQg2
a task achievable, one should consider other nondestructive methods to handle and manipulate a single cell. It should be possible to choose from a complex cell suspension one particular cell and, by observation under a microscope, place this cell on the platform for further mass spectrometric measurement. Recently, the optical tweezers has emerged as a new tool for cell Use of this device appears to be a powerful approach in the selection and micromanipulation of fragileprotoplastswithout inflicting damage to them. Detection of intracellular amounts of metal could be particularlyuseful for the analysis of the perturbation in plant (32) Block, S. M. In Optical Tweezers: A New Tool for Biophysics, Foskett, J. K., Grinstein, S.;John Wiley & Sons, Inc.: New York, 1990, pp 375-402. (33) Conia, J.; Jackson, P.; Lower, W. Unpublished work.
cella of planta exposed to toxic chemicals in the laboratory and to environmentalcontaminants in the field. For example, it has been suggested elsewhere when wing pollen grains as the bioassay that characterization of microspore populations should be of considerable interest for environmental testa.=
ACKNOWLEDGMENT The authors thank the excellent technical assistance of Johnny Anderson, as well as the mistance of B. Perez-Lopez in instrument calibration and data acquisition.
RECEIVED for review June 11, 1992. Accepted August 31, 1992. M S t W NO. CU,7440-50-8.
