in flux and area determination (No. 1 above). (3) When lines far apart in kinetic energy are compared, validity of the method requires that Xl/X2 in the material analyzed be the same as that in the materials from which standard intensity data were derived. Calculations based on the work of Penn indicate that the ratio of X 1 / X 2 for two lines can be variable by as much as 10% for E1/E2 = 2, or 22% for E1/E2 = 6. For most line pairs the effect is minimal. (4)When lines of widely different kinetic energies are used, adsorbed contamination layers can present problems. In the Na 2s/Na 1s line pair (Mg x rays) a 10% adsorbed hydrocarbon layer (C 1s signal equaling 10% of the infinitely thick value) should give an enhancement in I(Na 2s)/l(Na 1s) (Mg x rays) of 31%. Most line pairs are much closer in kinetic energy, so that variation in contamination normally contributes a few percent to the error. (5) The photoelectron yield, y, is greatly diminished in compounds of transition metals because of prevalent multielectron processes. To the extent that these effects are variable among different chemical forms of such an element, quantitative applications in the general sense will be difficult with these elements. There seems to be no evidence here for strongly varying y among nearest neighbors in 2 or for serious variability in elements other than the transition elements. (6) The possible variable screening of photoelectron emission by nearest neighbor atoms is not evident in this study, but further work might establish whether it is a real effect and worthy of concern. (7) Use of internal standards comprising a second phase has no validity for quantitative analysis, because of variability in absolute values of electron mean free paths in different
materials, and probably more importantly, variability in friability and plasticity among different materials.
ACKNOWLEDGMENT Many of the spectra were obtained by R. H. Raymond of this laboratory, and some by David Zatko of the University of Alabama while he was in residence at this laboratory. The cage-type ligand compounds were kindly furnished by Lon J. Wilson of Rice University.
LITERATURE CITED (1) C. D. Wagner, Anal. Chem., 44, 1050 (1972). (2) C. K. JLrgensen and H. Berthou, Chem. Soc. Faraday Dlsc.,54, 269 (1973). (3) H. Berthou and C. K. JLrgensen, Anal. Chem., 47, 482 (1975). (4) R. S. Swingle 11, Anal. Chem., 47, 21 (1975). (5) K. T. Ng and D. M. Hercules, J . Nectron Spectrosc. Relat. fhenom., 7, 257 (1975). (6) V. I. Nefedov, N. P. Sergushin, J. M. Band, and M. B. Trzhaskovskaya, J . Electron Spectrosc. Relat. fhenom., 2, 383 (1973). (7) W. J. Carter, G. K. Schwekzer, and T. A. Carlson, J . Electron Spectrosc. Relat. Phenom., 5, 827 (1974). (8) C. J. Powell, Surf. Sci.,44, 29 (1974). (9) P. C. Kemeny, A. D. Mclachlan, F. L. Elattye, R. T. Poole, R. C. G. Leckey, J. Liesegang, and J. G. Jenkin, Rev. Sci. Instrum., 44, 1197 (1973). (10) D. R. Penn, J . Nectron Spectrosc. Relat. fhenom., S, 29 (1976). (11) D. M. Wyatt, J. C. Carver, and D. M. Hercules, Anal. Chem., 47 1297 (1975). (12) J. C. Helmer and N. H. Weichert, Appl. fhys. Lett., 13, 266 (1968). (13) J. H. Scofleld, J . Electron Spectrosc.Relat. fhenom., 8, 129 (1976). (14) F. W. Lytle, Appl. fhys. Lett., 24, 45 (1974). (15) J-T J. Huang, J. W. Rabalais, and F. 0.Ellison, J . Electron Spectrosc. Relat. fhenom., 6, 85 (1975). (16) R. F. Rellman, A. Msezane, and S. T. Manson, J . Nectron Spectrosc. Relat. fhenom., 8 , 389 (1976). (17) C. S. Fadley, Chem. fhys. Lett., 25, 225 (1974). (18) M. 0. Krause, J. phys. (faris), Colloque C4, Suppl. 70,32 C4-87 (1971).
RECEIVED for review December 9,1976. Accepted May 4,1977.
Surface Studies of Mammalian Cells Grown in Culture by X-ray Photoelectron Spectroscopy Merle M. Millard" and James C. Bartholomew Western Regional Research Center, U.S. Department of Agriculture, Berkeley, California 947 10 and Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720
The surface elemental composltlon of Balb 3T3 A31 HYF mouse flbroblast cells and transformed MSV/MLV Balb 3T3 A31 HYF cells was obtained using x-ray photoelectron spectroscopy (XPS). The surface composltlon of the cells was sensitlve to the nature of the speclmen preparation; however, the oxygen to nitrogen (O/N) atom ratlo varied between 3 and 1 for washed cells and fixed cells. The oxygen to nitrogen atom ratlo was generally lower for transformed cells. Cell coverage on glass substrate was estimated to be 95% by comparlng the slllcon 2p slgnal from clean glass and glass substrate overlaid with cells. The change In concentratlon of phosphorus and sodium to a depth of approximately 40 nm Into the surface of the cells was obtained comblnlng XPS surface analysls data and oxygen plasma etching. The change in phosphorus concentratlon was llnear wlth tlme and falrly similar for the cells before and after tranformatlon.
Among the changes that occur in a cell after malignant transformation are alterations in the cell surface. This conclusion has been drawn primarily from three types of observations. First, new antigens appear on the surface of 1290
ANALYTICAL CHEMISTRY, VOL. 49, NO. 9, AUGUST 1977
malignant cells that are missing from their normal counterpart (1-10). Second, various plant lectins agglutinate malignant cells at much lower concentrations than required to agglutinate normal cells (11-19). And third, the carbohydrate content of the cell surface is altered in malignant cells (2(t28). Despite the large amount of information regarding changes in the macromolecular structure of the cell surface induced by malignant transformation, little is known about the elemental composition of the surface of transformed and nontransformed cells. The major reason this information is lacking has been that techniques for analysis of surface elemental composition have not been applied to such biological samples. X-ray photoelectron spectroscopy (XPS) is a relatively new technique capable of analyzing the surface of solid materials (29). The discrete energies of core electrons photo-ejected from the elements on the surface can yield information on both elemental composition and chemical bonding. Only the region less than 10 nm into the surface is analyzed; therefore, the technique is blind to substances situated more than a few nanometers below the surface. In a relatively brief time, a considerable number of experiments have applied XPS to the structural and analytical characterization of metals, refractory oxides, and polymer surfaces (30). The unique surface analysis
Table I. Description of Cell Specimen Preparations Specimen 1. Normal cells 2. Normal cells
3. Normal cells
4.Normal cells 5. Transformed cells
6. Normal cells
7 . Normal cells 8. Transformed cells 9. Normal cells
Preparation growth medium decanted away washed with isotonic saline solution washed with distilled water not fixed not fixed fixed with glutaraldehyde ethanol critically dried with Freon 113
10. Normal cells 11. Transformed cells 12. Transformed cells capabilities of XPS have, however, been neglected as a method to study the surface of biological materials. The binding of magnesium ions to cell walls of gram positive bacteria, as well as the chemical nature of osmium tetraoxide fixation and staining of membranes (31),has been studied using XPS (32). Human blood cells (33), various bacteria cells (34), and bacterial spores (35) have been surface-analyzed using XPS. The combination of a surface analysis technique with a method of removing the outer surface of a specimen allows measurement of the change in concentration of various elements as a function of depth into the specimen. Oxygen plasma has been used to gently etch outer layers of organic material from the surface of biological specimens without altering the surface morphology of inorganic components (36). XPS and oxygen plasma have been combined to surface analyze and depth profile bacterial cells (34). Electron microscopy was used to ascertain the uniformity of plasma oxidation of bacterial cells, and changes in dimensions of polystyrene spheres were used to calibrate the oxidation process (35). Meisenheimer et al. (33)used argon ion etching and XPS to depth profile red blood cells. We have explored the use of XPS and oxygen plasma etching to study the surface of Balb 3T3 A31 HYF cells before and after viral transformation. Tissue culture cells have the desirable property of forming a monolayer of cells on the substrate, and their surface morphology is relatively well known from scanning electron microscope studies (8,37,38,). It would seem natural to apply XPS to the study of mammalian cell membranes to learn more about what components are on the surface of these cells and how the surface may change with different growth states.
EXPERIMENTAL Cells. All cells were carried in 100-mm polystyrene dishes (Falcon, Oxnard, Calif.) and incubated at 37 OC in a 5% COP incubator. The medium used to grow the cells was Eagle's minimal medium (39) (GIBCO, Grand Island, N.Y.) containing 10% fetal calf serum. Balb 3T3 A31 mouse fibroblasts were obtained from Helene Smith of the Naval Biomedical Research Laboratory (Oakland, Calif.) and cloned prior to use to give Balb 3T3 A31 HYF, the clone used in all these experiments. All viral transformants used in these experiments were derived from Balb 3T3 A31 HYF. The cells were judged free of mycoplasma by incorporation of 3H-thymidine(20.1 Ci/mM; New England Nuclear, Boston, Mass.) into the nucleus of cells and not the cytoplasm (40). The Balb 3T3 A31 HYF cells will subsequently be referred to as normal. Viral Infection. The Moloney strain of murine sarcoma virus (MSV/MLV) used in these experiments was obtained from Adeline Hackett of the Naval Biomedical Laboratory (Oakland, Calif.) and carried in high passage mouse embryo cells. The focus forming units were measured on Balb 3T3 A31 HYF as described by Calvin et al. (41). The continuous line of MSV/MLV Balb 3T3 A31 HYF was obtained by infecting 2 X 10' Balb 3T3 A31
Substrate polystyrene polystyrene polystyrene polystyrene polystyrene glass mounted on gold holder glass on metal mount glass on metal mount glass glass glass glass
Angle between specimen plane and detector 90
90 90 90 90 90 90
90 90
15 90 15
HYF cells 24 h after seeding with approximately 6 X lo5 focus forming units of MSV/MLV. After the cells reached saturation density they were transferred, for a total of 4 passages, by seeding cells at approximately their saturation density. At passage number 4, the cells were suspended in medium containing 20% serum and 10% dimethyl sulfoxide (DMSO) and frozen at 203 K in a liquid nitrogen freezer. The cells used in these experiments were one passage beyond the frozen stocks. The MSV/MLV Balb 3T3 A31 HYF cells will subsequently be referred to as transformed. Specimen Preparation. Specimen Substrates. A signal is obtained in the spectrometer from a surface with a circular area 6.35 mm in diameter. Cells were grown as confluent monolayers on polystyrene tissue culture dishes, and 6.35-mm disks cut from the bottom of the dish. To minimize contamination due to handling, cells were later grown on precut 6.35-mm polystyrene disks. A similar procedure was used with glass substrates. Initially, cells were grown on glass microscope cover slips, and 6.35-mm disks cut from the coverslips containing the cells. Later, cells were grown on precut 6.35-mm diameter disks. Glass substrates were cleaned and sterilized for cell growth by treatment with an oxygen plasma. Unfixed Cells. Cells were grown on polystyrene culture dishes. While in the dish, the culture medium was decanted away, and the cells were given several washes with isotonic saline or distilled water. These liquids were decanted away, and 6.35-mm disks cut from the bottom of the dishes. The disks were introduced into the spectrometer moist and evacuated to Torr in 3-5 min. Fixed and Critical-Point Dried Cells. The cells were fixed at pH 7.35 in 2% glutaraldehyde in growth medium without serum and dehydrated by washing in 0.1 M sodium cacodylate buffer, pH 7.4 followed in order by water, 20, 50, 70, 95, and 100% ethanol. To prepare these cells for critical point drying, they were washed in 25,50, and 75% Freon 113 in ethanol. Table I contains a description of the specimens studied in this paper. These specimens will be referred to by number in subsequent tables and discussion. X-ray Photoelectron Spectra. Core binding energies were measured with a standard DuPont 650 electron spectrometer with a magnesium anode. The x-ray source was operated at standard maximum voltage and current. Grazing-angle spectra were obtained using a machined sample mount designed so that the electron take-off angle, between the electron analyzer entrance and the plane of the sample surface was 15' (42-44). The 15' mount exposes a bullet shaped area to radiation, and cells were grown on bullet shaped pre-cut glass surfaces for the grazing angle spectra. Electron lines from the surface of cells were obtained using a routine scan rate of 1 eV/cm. Data were accumulated over the energy region between 550 and 50 eV using a multichannel analyzer. Data were collected using half of the memory address group consisting of 512 channels. The scan rate was fixed at 0.1 s per channel, and data were accumulated for 16 scans. Samples were maintained at ambient temperature in the spectrometer. Line intensities were determined by manually estimating a baseline and measuring the peak height at half-width. Elemental ANALYTICAL CHEMISTRY, VOL. 49, NO. 9, AUGUST 1977
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line widths were approximately 2.0 eV. Atom ratios were determined by dividing the estimated line intensity by sensitivity factors taken from Wagner’s data (45). Surface Etching for Depth Profile Analysis. The surface of cells was oxidized and etched by exposure to oxygen plasma generated at ambient temperature using a “Plasmod” plasma system manufactured by Tegal Corp. (Richmond, Calif.). This system consists of a cylindrical glass chamber approximately 3 inches in diameter and 6 inches in length. A gas may be introduced into the chamber, and the chamber is evacuated by means of a mechanical vacuum pump. Up to 100 W of radiofrequency power at 13.56 MHz can be coupled into the gas flow system through external electrodes surrounding the cylindrical chamber. The gas pressure in the system during routine operation conditions was typically around 1Torr. In order to assume that sample oxidation occurred under the mildest possible conditions, samples were oxidized at the lowest power setting obtainable for the instrument. This power level was probably below 5 W. RESULTS AND DISCUSSION Technical Considerations. Specimen Preparation. The specimen was held a t a pressure of approximately Torr in the photoelectron spectrometer. Excluding the possibility of maintaining specimens a t low temperature in the instrument, the specimen must be evacuated and dehydrated. The disruptive effects of dehydration and evacuation on biological samples are well documented in the electron microscopy literature (37,38,46-48). After some preliminary studies on cells introduced directly into the spectrometer and evacuated, we adopted standard procedures used to fix and critical point dry specimens prior to introduction into the spectrometer. Specimen Substrates. Different substrates were used to overcome the difficulties resulting from interferences of elemental line intensity contribution from the substrate and the specimen covering the substrate. The spectrometer had a variable carbon contamination which always seemed to contribute a signal at 285 eV. Because of the uncertainty involved in assigning line intensities from the carbon line at 285 to the specimen, this line was not used to characterize the specimen surface. Glass and polystyrene were used as substrates. Polystyrene has the disadvantage of contributing a substrate carbon line, while glass contributes an interfering line due to oxygen. The silicon line from glass is unique to the substrate and was used to estimate the degree of surface coverage by cells. The difference in the silicon intensity from clean glass disks and disks covered with cells was taken as a measure of surface coverage. The contribution of the oxygen signal from the glass substrate was estimated by measuring the oxygen to silicon line intensity ratio for clean glass and assuming that the silicon intensity from the substrate containing cells had associated with it a similar oxygen intensity. Polystyrene has a negligible oxygen signal, and the oxygen intensity characteristic of cells could be obtained using polystyrene as the substrate. The oxygen signal from cells on polystyrene and from cells on glass with the appropriate substrate correction were in reasonable agreement. Electron Escape Depths. A unique and important feature of XPS is the fact that only the outer surface of the sample is analyzed. Although there is some uncertainty in the escape depth of electrons photoejected from the surface, there is general agreement that electrons escape from a region less than 10 nm from the surface (49),and an escape depth of around 5 nm is usally quoted for electrons ejected from organic substrates such as organic polymers (50). It is possible to decrease the effective sampling depth into the surface of the sample by decreasing the angle between the plane of the sample and the observation direction of the energy analyzer component of the spectrometer (42-44, 51). Normal measurements are made with the sample 90” to a line normal to the entrance region of the energy analyzer. The effective 1282
ANALYTICAL CHEMISTRY, VOL. 49, NO. 9, AUGUST 1977
0
J
550
\jka 50 eV Figure 1. Wide scan spectra from nmmal cells washed in various ways. Effect of cell surface cleanup on surface composltbn. Sample numbers refer to Table I(a) sample 1, (b) sample 2, (c) sample 3, (d) sample 6
sampling depth can be decreased by a factor of four by making measurements on a wedge shaped sample mount so that the angle between the plane of the sample and the direction of observation is 15’. Data collected with the sample mounted at an angle of less than 90” are usually referred to as grazing angle measurements. When spectra were measured with the sample flat, low intensity electron lines due to silicon were detected if the cells were grown on glass coverslips as a substrate. This signal could arise from areas not covered by cells or the thin outer portions of the cells. A distinction between these two alternatives could not be made. Cells are thickest a t the center, and this regular array could lead to shadowing effects; however, little systematic work has been published dealing with these questions (42, 52). Radiation Damage. Another possible difficulty in studying biological specimens, such as cells, is the alteration of the sample due to decomposition as a result of exposure to x-rays. In these studies, the intensities of lines arising from the surface did not change appreciably during the time the sample was exposed to radiation, and radiation decomposition did not appear to be a serious problem. Preliminary Results on Vacuum Dehydrated, Unfixed Normal Cells. In order to ascertain the sensitivity of electron line signals to the external composition of the cell surface, wide scan spectra were obtained over the region 550 eV to 50 eV for normal cells after several cleanup procedures. Wide scan spectra are presented in Figure 1 for cell samples No. 1-3, unwashed, washed with isotonic saline solution, and washed with distilled water. The position, intensity, and the structural interpretation for the photoelectron lines obtained from these samples are given in Table 11. The electron line positions are tabulated in order of decreasing binding energy. The intensity of the individual electron lines was obtained from single scan spectra. In each of these spectra, the most intense lines are the oxygen 1s at 532 eV, the nitrogen 1s at 400 eV and the carbon
1s at 285 eV. Low intensity lines due to chloride ion 198.1 eV, sulfate ion 168.5 eV, organic disulfide 163 eV, phosphate 133.2 eV, and sodium ion 63 eV (Auger KLL 122 270 eV) are present in the spectra obtained from the unwashed cells. In the spectra from the cells washed with isotonic sodium chloride, the line due to chloride ion increases in intensity while the electron lines due to the sulfate and phosphate decrease in intensity. All lines due to those ions except phosphate are essentially absent in the spectra of the cells washed with distilled water. Presumably, the ions, proteins, and amino acids present in the growth medium adsorbed on the surface of the cells would be removed in varying amounts by washing with isotonic sodium chloride solution or distilled water. It is possible that washing with water could rupture the cells and expose interior components of the cells. There were residual lines due to organic disulfide and phosphate. These lines were assumed to be inherent to the surface structure of the cell. The intensities of the photoelectron lines can be converted to approximate atom ratios using appropriate elemental sensitivity factor (45). The surface atom ratios are usually accurate within 5% with organic substrates (53) and the instrument used for these studies. Due to the uncertainty associated with the carbon line intensity, the oxygen Is and nitrogen Is line intensities are believed to be the most reliable parameters to characterize the surface of the cells. These line intensities and the oxygen atom to nitrogen atom ratio are presented in Table 11. The oxygen to nitrogen atom ratio varied between 2.78 and 1.95 for these three specimens. The line intensities from normal and transformed cells (unfixed preparations on polystyrene) are presented in Table 11. Wide scan spectra from those specimens contained lines due to oxygen, nitrogen, carbon, sodium, and phosphorus. The oxygen to nitrogen atom ratio was 2 to 1within experimental error for these two specimens. This atom ratio was not changed due to viral transformation. Preliminary Results on Fixed Cells. The wide scan spectra of a sample of fiied cells is shown in Figure 1spectrum d, and the CNO line intensities and line positions are given in Table 11. Several lines were present in the spectrum of the fixed cells that were absent in the spectra of the unfixed cells. These new lines were tin (496 eV, 2 p3: 488 eV, 3s); lead (144 and 139 eV, 4f7);silicon (154 and 103 eV, 2s and 2p); and gold (86.5 and 83.0 eV, 4 f 7 p 4f5& The silicon lines resulted from the exposed substrate, and the gold lines originated from the sample mount surface not covered by the glass disk containing the cells. Tin and lead were found in varying amounts in the fixed cell samples and not in the fresh unfixed cells. These elements were apparently introduced from the reagents and solvents used in the fixing procedure. Results on Fixed Normal and Transformed Cells. A number of normal and transformed cell preparations were surface analyzed to see if reproducible differences could be measured. These cell specimens were all grown on glass substrates and chemically fixed and critical-point dried prior to analysis. Wide scan spectra indicated the presence of varying amounts of tin and lead in these preparations. Figure 2 illustrates the single scan electron lines obtained from normal cells (specimen 7, Table 11). Table I11 contains data from two different preparations of cells. For each preparation, normal and transformed cells were analyzed. The oxygen to nitrogen atom ratio for normal cells grown at different times was fairly reproducible. This ratio was 2.6 for specimen No. 7 and 2.5 for specimen No. 9. The corresponding ratio for the transformed cells was 2.0 for specimen No. 8 and 2.2 for specimen No. 11. These data would suggest that the oxygen to nitrogen ratio on the surface decreases slightly when the cells are ANALYTICAL CHEMISTRY, VOL. 49, NO. 9, AUGUST 1977
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-
ASH TIME
100
(min)
eV
2.5
1.5
0.5
eV eV
Flgure 2.
Elemental electron lines from normal cells. Sample 7, Table
I
0.0 138
133
eV
Table 111. Electron Line Intensities and Atom Ratios for Chemically Fixed and Critically Point Dried Normal and Transformed Cells Core Sample level 7
c 1s
0 1s N Is 8
Si 2p c 1s 0 Is N 1s
9
10
11
12
Si 2p
c 1s
0 Is N Is Si 2p c 1s 0 1s N Is Si 2p
c Is
0 Is N 1s Si 2p c Is 0 Is N 1s Si 2p
Oxygen/ Intensity Corrected nitrogen (1000 counts/s) intensity atom ratio 75 53.5 85 2.6 14 53.3 1.5 (0 Is, 9.52) 72.5 58.5 76 2.0 15.7 37.5 3.0 61.5 45 78 2.5 12 28.5 0.7 (0 Is, 4.5) 31 27.8 16.8 4 9.5 0.4 (0 Is, 2.56) 55 43.5 59 11 2.2 2.0 (0 Is, 12.7) 26.2 26 16.8 32.3 2.72 5 11.9
transformed. In order to reduce the contribution of the glass substrate signals to the signal from the cells, grazing angle measurements were made on these specimens. The relative carbon, oxygen, and nitrogen signal intensities changed, and the silicon signal was virtually absent in the grazing angle spectra. The oxygen to nitrogen atom ratio for sample No. 9 increased from 2.5 to 2.9 for sample No. 10 for grazing angle measurements. This ratio for the transformed cells changed from 2.2 for sample No. 11 to 2.72 for sample No. 12 from grazing angle measurements. This ratio increased in both cases for the grazing angle data; however, the magnitude of the increase was much greater for the transformed cells. This change was in opposition to the trend observed from flat measurements. This difference may reflect differences in the outer few monolayers only. Depth Profiling and Ash Analysis of Normal and Transformed Cells. Depth Profile Analysis. Conventional composition profile studies on metal and refractory surfaces are usally accomplished by combining Auger spectroscopy for analysis and argon ion bombardment to etch away surface layers (30,54-56). XPS was used in this study to accomplish surface analysis and low temperature oxygen plasma etching to gently remove organic material from the surface of the cells. 1294
ANALYTICAL CHEMISTRY, VOL. 49, NO. 9, AUGUST 1977
dB
, 3'5 c---L-___ 138
133
eV
Phosphorus 2p electron line intensity as a function of ash time from normal cells. Sample 12, Table I Flgure 3.
Oxygen plasma etching was selected to remove the outer surface of the cells for several reasons. Argon ion etching has been found to alter the surface of oxides causing surface decomposition (57, 58). The damaging effect of argon ion bombardment of systems of lower thermal stability, such as biological samples or polymer samples, is much more severe (59). Oxygen plasma ashing has been used to oxidize and volatilize organic substances in applications where thermal decomposition and fine structure disruption or damage is to be avoided. The applications of this technique for sample preparation in analytical applications and for sample treatment for fine structure studies have recently been reviewed (36, 60, 61). An important question remains concerning the rate of oxidation and the uniformity of the removal of the outer surface (36). Scanning electron micrographs of the surface of cells after exposure to an oxygen plasma for varying periods of time support uniform oxidation at short ash times, but not at long ash times. Nonuniform surface removal usually occurs between 5 and 10 min ashing time (manuscript in preparation). Under similar conditions, Thomas, Millard, and Scherrer have estimated the rate of oxygen plasma oxidation of an organic substrate to be approximately 9 nm/min by measuring the dimensional changes of polystyrene beads after varying periods of exposure (35). This rate was determined only for polystyrene, and some uncertainty exists in estimating a rate of surface removal for the cell system studied here; however, 9 nm/min will be used as an approximate number. In order to obtain a composition profile analysis for various elements as a function of depth into the surface of the cells, the cells were subjected to a low temperature oxygen plasma for varying periods of time, and the electron lines measured on the surface over the region 550 to 50 eV. To minimize the nonuniform oxidation of the outer surface, the cell surface was ashed for half-minute intervals for no longer than 4.5 min total ashing time for composition profile analysis. The phosphorus 2p electron line intensity as a function of ash time for normal cells is given in Figure 3. The electron line intensity data for phosphorus, silicon, and sodium at 1-min ashing intervals up to 4.5 minutes for normal and transformed cells are given in Table IV. Glass contains sodium, and the contribution of the sodium intensity from the exposed glass substrate has been estimated and substracted from the intensity. The Na/Si intensity ratio is measured from glass and the ratio used to calculate the Na intensity from any given Si intensity. The sodium intensity data presented in Table IV have been adjusted in this way. A plot of the elemental composition as a function of plasma oxygen ash time yields a composition profile curve. The
Table IV. Phosphorus 2p, Silicon 2p, and Na Auger (KLL 1 2 2 ) Line Intensities from Normal and Transformed Cells as a Function of Oxygen Plasma Oxidationa Transformed Normal Na KLL Plasma Na KLL p 2P Si 2p corrected exposure p 2P Si 2p corrected intensity intensity intensity intensity intensity time, min intensity 0.5 3.3 3.05 0.0 0.56 1.5 1.85 0.7 6 9.87 0.5 1.05 2.5 5.41 1.1 8 10.3 1.5 1.3 4.0 6.55 1.3 9.4 10.88 2.5 1.74 4.5 8.53 1.6 10 12.3 3.5 1.8 6.0 9.67 2.0 11.0 13.67 4.5 2.6 9.0 11.36 a Intensity is in units of 1000 counts/s.
la
ETCH DEPTH
R
-
Balb 3T3 A31 HYF MSV/ MLV Balb 3T3 A31 HYF
U
0
-2 5
2 0
b
15
3
*
3
10
0
2
v)
0
os
L
0
I
I
I
1
2
3
4
a
OXYGEN PLASMA ASH TIME (MINUTES)
Phosphorus composition profile for normal (A) and transformed
Figure 4.
cells C hi0
A s h Time (min)
75
60
~
50
550 eV
Flgure 5.
cells
Wide scan spectra as a function of ash time for transformed
phosphorus composition profile curve for normal and transformed cells for ash times up to 4.5 min is given in Figure 4. Ash time in minutes can be converted to an approximate etch depth using the conversion factor of 9 nm/min. As can be seen from Figure 4,the composition profile for the two types of cells is fairly similar up to ash times for 4.5 min. The phosphorus concentration increases linearly with time during the first 5 minutes of ashing. This is most reasonably in-
20 40 OXYGEN ASH TIME ( M i n )
60
Flgure 6. (a) Silicon 2p electron substrate intensity as a function of ash time and (b) phosphorus 2p electron intensity as a function of ash time
terpreted as a uniform distribution of phosphorus in the region extending from the surface of the cell to well within the interior. A phosphorus rich or depleted region would presumably result in a change in the slope of the curve. Ashing. The results of plasma ashing of biological cells is of interest even under nonuniform conditions when the interpretation of concentration changes in terms of depth profiles is not applicable. Plasma ashing of biological samples is a method of choice for sample preparation when removal of organic components is desired, and retention of elements normally lost during high temperature incineration is of interest. As ashing proceeds and the organic components are lost, the inorganic constituents are concentrated in the form of an ash. The appearance of elements not necessarily on the surface of cells and their buildup in the ash can be followed by analyzing the surface of the ashed sample at varying periods of ashing time. A series of wide scan electron spectra as a function of ash time is presented in Figure 5 for transformed cells. Some lines of interest and the approximate position in units of electron volts for the stronger binding energy lines are as follows: 0 1s 531, N 1s 400, Ca 2p 348, K 2p 292, C 1s 285, Na KLL 263, S 2p 168, P 2p 134, and Si 2p 103. The silicon lines are from the substrate. The individual phosphorus 2p line intensity as a function of ash time up to 60 min is given in Figure 6a for normal and transformed cells. As the organic components of the cells are lost, the concentration of the inorganic components, such as phosphorus will increase in ANALYTICAL CHEMISTRY, VOL. 49,
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concentration until all of the carbon containing component is lost. I t appears that after 60 min the concentration of phosphorus on the surface of the ash is beginning to reach a plateau. Some estimation of the coverage of the glass substrate by cells and the rate of oxidation of the cells by the oxygen plasma can be obtained from the intensity of the silicon 2p electrons from the glass substrate. The initial silicon 2p intensity is around 2000 counts/s. The silicon 2p electron intensity from a clean 6.35-mm diameter glass disk is around 20000 counts/s. Assuming that the intensity of the 2p electron signal is inversely proportional to the degree of surface coverage, the culture cells cover about 90% of the glass surface initially. The increase in the silicon 2p signal intensity upon oxygen plasma treatment is related to the rate of oxidation of the cell on the substrate by plasma oxidation. In Figure 6a the silicon 2p electron intensity is plotted as a function of oxygen plasma ash time for the normal and transformed colls. The Si 2p signal increases sharply during the first 10 min of ashing and then increases slowly and steadily. The thin outer portion of the cell near the edge is apparently rapidly oxidized, thereby exposing the substrate. After oxidiation of this thin exterior portion, the substrate is exposed more slowly because of coverage by thicker portion of the cell.
ACKNOWLEDGMENT We thank Agatha Wang for her assistance in fixing the cells and Hisao Yokota for culturing the cells. M. Millard especially thanks Richard S. Thomas and Rene Scherrer for their helpful discussion and critical review of the manuscript.
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6.
RECEIVED for review February 11, 1977. Accepted May 13, 1977. This research was supported in part by NIH Grant No. NCI-1-RO-1-CA 14828 and the U S . Energy Resources and Development Administration. Reference to a company or product name does not imply approval or recommendation of that product by the U S . Department of Agriculture to the exclusion of others that may be suitable.
