Anal. Chem. 1904, 56, 2791-2797
Registry No. Co, 7440-48-4;In, 7440-74-6; Ga, 7440-55-3; Mo, 7439-98-7; Al, 7429-90-5.
LITERATURE CITED (1) Ens, W.; Standing, K. G.; Westmore, J. 6.; Ogllvie, K. K.; Nemer, M. J. Anal. Chem. 1982, 5 4 , 960-9136, (2) Glbbs, R. A.; Holland, S. P.; Foley, K. E.; Garrison, B. J.; Winograd, N. J . Chem. Phys. 1982, 7 6 , 684-695. (3) Wlttmaack, K. Appl. Phys. Lett. 1978, 2 9 , 552-554. (4) Wittmaack, K.; Clegg, J. B. Appl. Phys. Lett. 1980, 378, 265-287. (5) Clegg, J. 6.; Scott, G. B.; Hallals, J.; Mlrcea-Roussel, A. J . Appl. Phys. 1981, 52. 1110-1112. (6) Pellln, M. J.; Wright, R. B.; Gruen, D. M. J . Chem. Phys. 1981, 7 4 , 6448-6457. (7) Yu, M. L.; Grischkowsky, D.; Balant, A. C. Phys. Rev. Lett. 1982, 48, 427-430. (8) Honlg, R. E. "Advances In Mass Spectrometry"; Pergamon Press: New York, 1962; Vol. 2. (9) Coburn, J. W.; Kay, E. Appl. Phys. Lett. 1971, 19, 350-352. (10) Oechsner, H.; Oerhard, W. Surf. Sci. 1974, 44, 480-488. (11) Winograd, N.; Baxter, J. P.; Klmock, F. M. Chem. Phys. Lett. 1982, 8 8 , 581-584. (12) Klmock, F. M.; Baxter, J. P.; Winograd, N. Surf. Sci. 1983, 124, L41L46. (13) Hurst, G. S.; Payne, M. G.; Kramer, S. D.; Young, J. P. Rev. Mod. PhyS. 1979, 5 1 , 767-819. (14) Kobrin, P. H.; Baxter, J. P.; Winograd, N., unpublished work. (15) Parks, J. E.; Schmltt, H. W.; Hurst, G. S.;Fairbank, W. M. SPIE27th Ann. Tech. Symp. Proc., 27th 1883. (16) Fassett, J. D.; Travis, J. C.; Moore, L. J.; Lytle, F. E. Anal. Chem. 1983, 5 5 , 765-770. (17) Donohue, D. L.; Young, J. P.; Smith, D. H. Int. J . Mass Spectrom. Ion PhyS. 1982, 4 3 , 293-307.
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(18) Miller, C. M.; Nogar, N. S. Anal. Chem. 1983, 5 5 , 1606-1608. (19) Carter, G.; Colligon, J. S. "Ion Bombardment of Solids"; American Elsevler: New York, 1968; Chapter 7. (20) Coburn, J. W. Thin Solid Films 1979, 6 4 , 371-382. (21) Coburn, J. W. J . Vac. Sci. Techno/. 1976, 13, 1037-1044. (22) Oechsner, H.; Schoof, H.; Stumpe, E. Surf. Sci. 1978, 7 6 , 343-354. (23) Wright, R. B.; Pellin, M. J.; Gruen, D. M.; Young, C. E. Nucl. Instrum. Methods 1980, 170, 295-302. (24) Pellin, M. J.; Young, C. E.; Calaway, W. F.; Gruen, D. M. Surf. Sci., in press. (25) Wlttmaack, K. Nucl. Instrum. Methods 1980, 168, 343-356. (26) Fassett, J. D.;Moore, L. J.; Travis, J. C.; Lytle, F. E. Int. J . Mass Spectrom. Ion Procs. 1983, 5 4 , 201-218. (27) Onderdelinden, D. Can J . Phys. 1988, 46, 739-745. (28) Thompson, M. W. Phllos. Mag. 1968, 78, 377-414. (29) Morellac, J.; Normand, D.; Petite, G. Adv. At. Mol. Phys. 1982, 18, 97- 164. (30) Young, J. P.; Donohue, D. L. Anal. Chem. 1983, 5 5 , 88-91. (31) Mamyrln, B. A.; Karataev, V. I.; Shmlkk, D. V.; Zagulin, V. A. Sov. Phys.-JETP (Engl. Transl.) 1973, 3 7 , 45-48. (32) Becker, C. H.; Glllen, K. T. Anal. Chem. 1984, 9 , 1671-1674. (33) Mayo, S.; Lucatorto, T. 6.; Luther, G. G. Anal. Chem. 1982, 5 4 , 553-556.
RECEIVED for review April 30, 1984. Accepted July 23, 1984. The authors are grateful for the financial support of the National Science Foundation (Grant No. CHE 81-08382),the Office of Naval Research (Grant No. N00014-83-K-0052),the Air Force Office of Scientific Research (Grant No. AFOSR82-0057), and the donors of the Petroleum Research Fund, administered by the American Chemical Society.
Low Temperature Ashing Preconcentration for Elemental Localization in Biological Soft Tissues by Ion Microscopy J. T. Brenna and G. H.Morrison* Department of Chemistry, Cornell University, Ithaca, New York 14853
Low temperature oxygen plasma ashlng (LTA) was Investigated as a preconcentration method for major and trace eiementai iocaiiratlon in biological soft tissue sections. I t was found that LTA pretreatment provides satisfactory preservation of elemental morphology. Experiments with fabricated standards show that LTA enhances elemental sensitlvlties 30 to 1500-fold dependlng on the element. Copper and aluminum ion micrographs, whlch are unobtainable in intact plastic Sections, were generated from ashed sections of intestine taken from normal healthy mice. These data suggest a unique applicability of LTA in ion microscopical studies of trace e i e ment dlstrlbutlon in bloiogical samples.
The elemental microcharacterization of thin-sectioned biological tissue is a subject of intense interest. Ion microscopy via secondary ion mass spectrometry (SIMS) (1) has been shown to be a useful tool for this purpose. Among its advantages as an analytical technique are high sensitivity and the ability to distinguish isotopes of the same element. Major elemental constituents of tissue such as Na, K, Ca, Mg, and C1 are routinely localized by SIMS (I,2); however, studies on transition metals and other minor elements have generally been limited to cases in which the target element concentration has been artifically raised to toxicological or pharmacological levels. These trace elements at their ambient levels are of sufficiently low concentration and ionization probability as 0003-2700/64/0356-279 1$0 1.50/0
to preclude imaging from intact resin embedded thin sections. Low temperature oxygen plasma ashing (LTA) is a wellknown and well-characterized technique used for the preconcentration of inorganic constituents from organic material (3-6). LTA treatment consists of exposing an organic sample to a stream of oxygen excited by radio frequency to the singlet state (02, A): and free atoms (0,3P) which react with and remove organic material (C, H, N) at relatively low temperatures (7, 8). In high doses, LTA is known to completely remove organic material while giving quantitative retention for most elements with no detectable contamination (4,9,10). For resin embedded biological thin sections mounted on smooth surfaces, LTA treatment in sufficient doses produces ash patterns (spodograms) of high morphological integrity (3, 11-14). Ion microscopy applied to the determination of elemental distributions in ashed sections has not previously been investigated. Thus, the purpose of this study was to characterize the usefulness of LTA pretreatment for the ion microscopic localization of biologically important trace elements at their normal levels in thin tissue sections. Mouse intestine prepared by use of conventional fixation procedures and embedment in plastic served as a model system. The data from this study indicate signal enhancements of a minimum of 30-fold for Ca to 1500-fold for Co are obtained upon ashing. The absence of serious spectral interferences at masses 63,65, and 27 allows the direct imaging of Cu and A1 at their physiological concentrations. 0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
h a s h e d I n t e s t i n e (t)
Table I. Conditions for Low Temperature Ashing instrument forward power reflected power O2flow rate chamber 02 pressure ashing time ashing temperature
Tracerlab Model LTA-BOO 50 W 0.2 w 50 mL/min 0.2 torr
4h -80 OC
Table 11. Ion Microscope Instrumental Parameters instrument primary ions primary ion energy primary ion current primary ion current density primary ion beam size primary ion raster size transfer optics detection mode
CAMECA IMS-3f ion microanalyzer
ia
o*+
5.5 keV (16.0 keV for PO,-) 1 nA to 2 NA 1X to 2 X A/cm2
26
38
48
5a
66
76
ea
SE
im
Mass 1x1E7Tb
Ashed I n t e s t i n e ( t )
-
100 pm2 250 X 250 to 400 X 400 gm2 150 and 400 pm imaged field
microchannel plate/photographic film
imaging studies mass resolution field aperture contrast diaphragm aperture
300 1800 km 150 Nm
high resolution studies 8000 750 wm
Mass
400 wm
EXPERIMENTAL SECTION All reagents were made up with deionized distilled water. Normal healthy mice were sacrificed via cervical dislocation. Samples of intestine were excised, fixed in 5 % glutaraldehyde in phosphate buffer (pH 7.0-7.4) for 1 h, and postfixed in 1% Os04 in buffer solution for an additional hour. The specimens were then dehydrated in graded acetone solutions and embedded in Spurr resin. Half micrometer sections were cut on a LKB Ultrotome I11 ultramicrotome with glass knives onto deionized distilled water troughs and transferred to 99.999% gold (Materials Research Corp.) coated polished silicon wafers for ashing. A high-purity gold substrate was used to maximize surface cleanliness and minimize molecular interferences which would arise from a lower atomic weight substrate such as Si (Si+,Si2+,SiO+, etc.). Mass scans showed this surface to be free from major impurities. To ensure complete ashing, sections were exposed to the oxygen plasma for 4 h at the conditions given in Table I. Ash residues were analyzed on a CAMECA IMS-3f ion microanalyzer operated with an 02+ primary beam under instrumental parameters given in Table 11. Primary ion currents were varied according to the analyte of interest; major elements (e.g., Ca) required 1-30 nA while trace elements required 1.0-2.0 p A for optimal imaging. Ash residues were also studied for molecular interferences at nominal masses for trace elements. High mass resolution data were recorded on photographic film and approximately quantified by microdensitometry on a Joyce Loebl Model 6 microdensitometer. Secondary ion intensity enhancement experiments were performed with pure Spurr resin doped with elements of interest. Separate plastic blocks were doped for each element by dissolving a specific quantity of a compound containing the element of interest in propylene oxide. Compounds were chosen for maximum solubility in plastic monomer and minimum solubility in water. The doped solution was then mixed with one component of the Spurr resin and the propylene oxide was evaporated with a stream of dry nitrogen. The other plastic components were then added to the doped component and the mixture was cured in the usual way (70 "C, 12 h). Samples of each block were wet ashed with nitric acid in Teflon-lined acid digestion bombs (Parr). The final elemental concentration in each block was then determined by atomic absorption spectrophotometry (AA). AA sample size requirements dictate that only two separate determinations may be made from a single block. It was found that for Co and Mn these two determinations agreed to within 1%;therefore one
hashed Intestine (-) CN
Mass Ashed I n t e s t i n e ( - )
Mass Nominal mass resolution secondary ion mass spectra of unashed and ashed intestine sections: (a) positive secondary ions from unashed intestine obtained with 450-nA primary beam current; (b) positive secondary ions from ashed intestine obtained with 5-nA primary beam current (note comparable elemental signals using greatly reduced primary ion beam current);(c)negative secondary ions from unashed intestine; (d) negative secondary ions from ashed intestine (note decrease in organic peaks and increase in 0, PO2, and PO3, peaks in ashed vs. unashed spectra). Figure 1.
determination was made on the remaining blocks.
RESULTS AND DISCUSSION Mass Spectra. Nominal mass spectra of unashed and ashed tissue sections are shown in Figure 1. Signals were corrected for the dead time of the pulse counting system (70
ANALYTICAL CKMISTRY. VOL. 56. NO. 14, DECEMBER 1984
ns). All spectra were obtained from similar regions in consecutive 0.5 pm thick sections from the enme tissue sample. The sensitivity to all peaks was substantially enhancsd in the ashed section. A 450-nA beam was required to generate the spectra for the unashed sections; 5 nA was used for the ashed sections. Examination of the positive secondary ion spectra reveals that the C and N signals are greatly attenuated in the ashed section relative to the unasbed section, as expected. The negative secondary ion spectrum for ashed tiasue also shows attenuation of these peaks and organic molecular ion peaks (C2,CN, etc.) relative to the unashed negative spectrum. In addition, the negative secondary ion spectrum of the ashed section shows prominant peaks a t masses 63 and 79, which are not nearly as intense in the unashed spectrum. These peaks correspond to PO; and PO3-, respectively and their enhancement by ashing is due to the highly oxygenated form of P on the substrate surface. Major Element Imaging. Calcium and magnesium were used as test elements to determine whether gross elemental redistribution occurs during the ashing process. Calcium was chosen because it is a major element whose distribution in intact. plastic sections is well-defined. I t is found in high concentration in nuclei and membranes and, to a lesser extent in cytoplasm. The magnesium distribution in these samples is leas well-defined due to difficulty of imaging this element, even with large beam currents (e.& 2 pA). Ion micrographs of Ca and Mg in ashed sections obtained with a 30-nA primary beam are shown in Figure 2. Brush border, muscle fibers, individual cells,nuclei, and intranuclear detail are clearly discerned. The Mg micrcgraph shows musde fibers and intranuclear detail, but no emission from the brush border. Close examination of the micrographs reveals that features of 1 pm diameter are resolved. This indicates that satisfadory preservation of elemental morphology is obtained after ashing a t the spacial resolution of the ion microscope. Image Evolution. Calcium was used as a model element to investigate image evolution in ashed samples. Figure 3 shows four images taken a t consecutive times after the initiation of sputtering. An image is immediately apparent at the onset of sputtering. (This is in contrast to plastic sections which exhibit a delay time before producing a signal.) This image is generally a high intensity, low contrast image aa is shown in Figure 3% Very little intratieaue detail is discernible. Within a short period of time, regions of lower ash content disappear, leaving only regions of higher and intermediate levels as in Figure 3b.c. Features such as nuclei, bruah border, and muscle fibers appear, as well aa a fold which waa present in the unashed section, but not visible in the fmt micrograph. In the late stages, only regions of very high ash content are left, as shown in Figure 3d. Now, only nuclei, muscle fibers, and the fold, which contains about twice as much ash aa the area immediately surrounding it, are left. This image evolution phenomenon may be explained most concisely by means of a schematic. In frame i. Figure 4, the sample consists of a plastic thin section with three anal* concentrations located in three stripes. The concentrations in stripesa and c are identical while the concentration in stripe b is substantially less. When this intact section is analyzed by the ion microscope, the three stripes are imaged with intensities directly related to their concentrations (frame ii). When this sample is ashed, however, the situation is different. Now,the surface concentration of the three stripes is identical (frame iii) and initially ion microscopy shows the regions to be indistinguishable (frame iv). After a short time, the s h e d material in the stripe of lower concentration,b, has completely been removed and the image appears as in frame v. As time goes on, the stripes of higher concentration, a and c, will be removed.
bl
27S3
. .. .. . -!.
l i
+.. I
Fbun 2. Ion micrographs obtained with 30-nA primary ion beam
cunent. (a) Calcium: brush bwder, individual celis. muscle flberr. wdd and Inbanuckr detal are ckarty discembb. mid of vlew Is 300 pm diameter. (b) Magneslum: lndivkdual ceils, muscie fibers, nuclei. and intracellular detail Is resoved. field of view is 150 pm diameter. The situation is complicated by differing quantities of ash in each microlocation on the substrate surface. This situation is represented graphidly in Figure 5. In this sample (frame i) the concentrationof the target element, represented by "X", is uniform throughout. Here there is another element "0" present whose oxidized forms are not volatile (i.e., will remain after ashing), which exists in identid concentrations in stripes a and e, and is lower in b. Direct sputtering of the plastic sample will yield a uniform X image (frameii). However, when ashing pretreatment is administered, the target element X may be considered to exist in a matrix of 0 (frame iii). The concentration of x , and hence the surface concentration of X that
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ANALYTICAL CHEMISTRY. VOL. 56. NO. 14. DECEMBER 1984
Flpxa 9. Calcium ion images taken at consecutive times to illustrate image evolution. (a) lnnlally image Is high intensky. low Mntrasl. Later. (b) and (c). features appear as regions 01 lower ash content sputter through. (d) Finally only regions of high ash content are len.
the ion beam w,is dependent on the quantity of 0 present in each microlocation in each stripe. Frame iv illustrates the x image which will occur upon sputtering this ashed sample, assuming a uniform sputtering yield. The stripe of lower 0 concentration, b, will be more intense than stripes a and e. After sputtering for some time, a brief crossover will occur when the image intensity will be uniform (frame v). During this time region h will have partially sputtered through. Shortly thereafter, region b will disappear leaving only regions a and c. In ashed tissue sections, regions of high ash content such as memhranea and nuclei will sputter more slowly than regions of low ash content such as cytnplasm. For example, calcium in identical concentrations in cytoplasm and nucleus will
initially appear to be more highly concentrated in the c y b plasm, since the surface concentration will be higher in this region due to the lower abundance of ash. The cytoplasmic image would dissipate more rapidly than the nuclear image, which would linger for a greater time to allow ash to erode away. The ion micrographs of ashed tissue that have been shown thus far were taken a t an intermediate stage in the sputtering proeeas. For qualitative investigations, this stage yields the most information about elemental distribution. Trace Element Imaging. Molecular interferences are generally of negligible intensity when analyzing biological samples for major elements. However, in the concentration range of trace elements molecular ions would be expected to significantly interfere with analyte detection in the low res-
ANALYTICAL CEMISTRY. VOL. 56. NO. 14, C€couBo;1 1984
-4. Schmlmtk~tbot~hum8hdMdllllhed modsl symems: 0 Sample is a p(astic section vl(h elanent"X" h Uwesfipes,otccmcmbntbnrsnkhga=c > b . ODteclafmtybb shormmethee sflpss ma@ wnh intsnafllss ~ l t o a w c ce&atbn. (I LTA ) of pla& seclhm. Matdx is nmoved and " X is lea on sufam. Q Analysis ot ash residue WaaW shorn Unnam sutam CmCBnbatlm ot "X". (v) After splneme for s ~ n tme. e "X" h sMpe b has sputtered away and smpes ori@nally01 p n t e r "X" mnmnbatbn are left.
*MI
~ r n f 3 I1
.:.:.:.:.:......b .....:.:. r:.:.:
a
b
?
,
o
b . (11) DhKt analysls of " X " ybb ullform slgMI. (ill)LTA yblds sample where "X" and "(0)" coaxist on substrate swiam. Now. mnmnbalbn of "X" In a "mslrlx" 01 "(0)"in each strlpe is b > a = c. (iv)Analysis ot "X" inlaafty shows highu "X" wnmnkatbn h sfipe b. assurdng u M c m spunerlng yields. As ash spmas. sfipe b wlth lowest ash qtmnlny WM spUner a X a @ taste81 and a crossova will occur (v) whore slpnak horn each s m p are IdmUcaI (at rmS poht b wil be parlhlly sputtered through). (vl) Fhally b Is removed leaving only smpes a and c.
olution mode of operation. Thus,the ertent of interferenoea a t nominal mass resolution was BBseased before attempting to image trace elements in ash reaiduea. H e h mass resolution data for selected masees are given in Table 111. In addition to the masses shown, masses 52 (Cr) and 92 (Mo)were investigated and found to give barely detectable signals. The data in Table 111 show that nAl. "Cu. and @ T uare the only trace elements which can reliably be imaged in the low maea resolution mode (MIAM = 300). Since the interfering molecular ions consist of elements which are ubiquitous in biological systems, they would be expected to be of importance in all LTA-SIMS investigations of biological samples. High mass resolution or secondary ion energy discrimination might be used to overcome contaminating cluster ions; however, a t the low concentrations of trace elements the concomitant decrease in intensity of the analyte peak would rule out imeping. Figure 6m-c shows ion micrographs of ku,nAl, and PO;. mpectively. In the case of the Cu and AI micrographs, a primary ion beam m o t of 2 rrA wan necBBBary for optimal imaging. At this N m n t , the ash lasted for only about 6 s of sputtering. For POs. a beam a m e n t of only 5 nA was neceeaary for an optimal image. Since all of the ash is likely to be saturated with oxygen, this micrograph would be expected to be a p o d reflection of P distribution in ashed seetions. In thin perticular micrcgraph, two stages of sputtering are shown.
27W
2796
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
mass
species
approx intens
27
27A1 26MgH
0.086
3000
66Mn KO
1.000 0.438
2700
56Fe
1.000 1.488 0.134
2500 1400
55 56
MgOz
1.000 0.244 0.088
3800 3200
59
1.000 15.6 29.9 6.0
2900 2100 1500
~
~
0
69c~ 43ca0
"CaOH ?
a
63
63Cua
1.000
65
66cua
1.000
z
E
6sNi 2
d
MIAM
Si2 4
--. I
1.000
4wa0
58
Unashed Fe S t d
1 ~ 1 8 ~
Table 111. High Mass Resolution Data at Selected Masses for Positive Secondary Ions in Ashed Intestine
I/ I08
288
388
Time Tb
408
is,)
A s h e d Fe S t d
No detectable interferences. 5
The villus on the left was presputtered for a time and is thus in a later imaging stage. The villus on the right shows strong emission from nuclei, brush border, and cytoplasm. The presputtered villus shows high concentrations of P in nuclei and muscle fibers only. The Cu image shows a uniform emission from the columnar epithelial cells and the muscle fibers running up the center of the villi. The A1 image is less intense than the Cu image and shows more detail in the region between muscle fibers and epithelial cells. At this juncture it should be emphasized that these micrographs may not reflect physiological distributions of these elements due to the nature of the sample preparatory regime. Enhancement Studies. The extent to which LTA pretreatment enhances elemental signal was investigated by using Spurr resin doped with selected elements a t concentrations of about 100 ppm. Signal enhancement is a product of two major factors in ashed vs. unashed sections: (a) matrix removal (i.e., preconcentration) and (b) increased ionization probability due to surface saturation with oxygen. The degree of signal improvement was assessed by following signal vs. sputtering time in ashed and unashed 0.5 pm doped thin sections under identical analytical conditions. Figure 7 shows typical depth profiles of unashed (a) and ashed (b) sections. The element shown here is Fe although the shapes of the curves are typical for all elements. The unashed section shows a short rise time to a steady-state signal. The depth profile of the ashed section shows a brief rise to a peak followed by a rapid dropoff. A single convenient parameter for describing enhancement may be constructed from these plots. We define the signal
18
15
28
Time ( s . ) Figure 7. Depth profiles of irondoped plastic sections: (a) unashed and (b) ashed.
enhancement factor (SEF) as the ratio of the maximum signal obtained from the ashed section per arbitrary time unit to the maximum signal obtained from the unashed section per same time unit. A time unit of 0.2 was chosen for convenience. The SEF is then calculated by dividing the maximum signal (in counts) during 0.2 s for the ashed section by the average 0.2-9 signal from the steady-state region of the unashed section
This factor would be expected to be a composite of enhancement due to both sources and is of general use for determining LTA/SIMS detection limits for specific elements. SEF's for selected elements are shown in Table IV along with their first ionization potentials (IP's) and oxide standard free energies of formation (15,16).The magnitude of the SEF for each element appears to be directly related to the first ionization potential of the element and stability of its bonds to oxygen. A linear least-squares fit to a plot of SEF vs. IP has a positive slope and yields a correlation coefficient ( r 2 ) of 0.80. This trend is consistent with trends found in SIMS analyses of dopants and matrix elements in defined semiconductor materials, where it was determined that changes in matrices affects ion yields more severely for elements of higher IP (17). Values of SEF which appear anomalously low based on their IP may be so due to an increase in their probability of being sputtered as an oxide molecular rather than an atomic species because of increased surface 0 con-
Table IV. Signal Enhancement Factors (SEF's)for Selected Elements element
Na A1
Ca Mn
cu
dopant
ambient Al(aca~)~ Ca(a~ac)~ M~z(CO)~O Cu(acac)z
Fe
Fe(CO),
co
C02(C0)8
concn in resin: ppm
-
mass resolution
1st IP*
AGr' (M-0)"
SEFd
17
nominal
nominal nominal 4000 1500
5.1 6.0 6.1 7.4 7.7
nominal
7.9
84 -1577 -604 -363 -129 -246
150
100
5000
7.9
-217
62 47 69 49 173
60 30
110 830 1400 1500
By atomic absorption spectrophotometry. First ionization potential in electronvolts. Free energy of formation of the monoxide (A1203 standard deviation. eAcetoacetonate.
for AI) in J/kmol. d + l O %
ANALYTICAL CHEMISTRY, VOL. 56,
centration. These elements, Ca, Al, and Mn all have high oxide bond energies relative to the other elements studied, which may give them a propensity to forming highly stable gas-phase oxides. Reexamination of the positive secondary ion bar graph data (Figure la and Figure lb) reveals that the Ca/CaOH (masses 40 and 57) ratio reduces from 25 to 4 in unashed vs. ashed sections, suggesting that the probability of ejection of this element as an oxide is greatly increased. LTA has been evaluated as a method of sample preconcentration for electron probe microanalysis and particle-induced X-ray emission (PIXE) (18-22). LTA was found to decrease detection limits by a factor of 2-4 times for electron probe analysis (18). For PIXE analysis of freeze-dried blood serum, ashing produced a decrease in detection limits of 2-3 times with respect to original wet weight (22). For both methods, increases in sensitivity have been ascribed to decrease in the bremsstrahlung background radiation due to the decrease in matrix material in the ashed samples (18, 22). The dramatic LTA induced increase in detectability for SIMS relative to X-ray methods may be accounted for in terms of the enhancement mechanisms already discussed for each technique. In the X-ray methods, every atom through the volume of the plastic section is subject to excitation. After ashing the same number of analyte atoms are available for excitation, but the interfering interactions of the exciting species with matrix are reduced, thereby reducing background and detection limits. However, since the measured X-rays are a result of inner shell transitions, detection limits are largely unaffected by the chemical form of the analyte. In SIMS, only the top layers of the plastic section are sampled at a time, so only a small fraction of the total inorganic mass is subject to detection. Ashing substantially increases the surface concentration and thus allows for a far greater fraction of analyte to be sampled. In addition, the highly oxidized chemical matrix increases elemental sensitivity per se.
CONCLUSION The data presented here indicate that LTA pretreatment is an effective method for substantially enhancing SIMS detectability for elements which form stable positive secondary ions, while satisfactorily preserving elemental morphology present in the original plastic section. LTA allows the imaging of species which are difficult or impossible to image in untreated sections. The high mass resolution results show that only a few minor elements may be imaged in the nominal mass resolution mode. However, in samples more rigorously prepared for elemental localization studies, e.g., freeze substitution or cry0 methods which give far higher retention of most elements than aqueous methods (used here), minor elemental concentrations may be at sufficient levels for high mass res-
NO. 14, DECEMBER 1984 2797
olution or energy discrimination to be used to remove contaminating peaks. The interpretation of LTA-SIMS images is not completely straightforward, as borne out by the image evolution data. Consideration must be given to the stage of sputtering at which a particular image is acquired. Because of this limitation, quantitative analysis of these images is not yet possible. Efforts toward this goal are ongoing in this laboratory.
ACKNOWLEDGMENT The authors gratefully acknowledge the assistance of G. 0. Ramseyer in the early stages of this project. Registry No. Na, 7440-23-5;Al, 7429-90-5;Ca, 7440-70-2;Mn, 7439-96-5; Cu, 7440-50-8;Fe, 7439-89-6; Co, 7440-48-4; P, 772314-0; 0, 7782-44-7; 6 5 C ~14119-06-3; , PO3-, 15389-19-2. LITERATURE CITED (1) Morrison, G. H.; Slodzian, G. Anal. Chem. 1975, 4 7 , 932A-943A. (2) Spurr, A. R. Scanning 1980, 3 (2), 97-109. (3) Thomas, R. S. I n "Techniques and Applications of Plasma Chemistry"; Hollahan, J. R.. Bell, A. T., Eds.; Wley-Interscience; New York. 1974; pp 255-346. (4) Lutz, G. J.; Stemple, J. S.; Rook, H. L. J . Radoanal. Chem. 1877, 39, 277-283. (5) Sansoni, B.; Ponday, V. K. I n "Analytical Technlques for Heavy Metals in Biological Fluids"; Facchetti, Ed.; Elsevier: Amsterdam 198 1; pp 91-131. (6) Wong, S. Ph.D. Thesis, Cornell University, 1973; pp 11-14. (7) Hollahan, J. R. I n "Technlques and Applications of Plasma Chemistry"; Hollahan, J. R., Bell, A. T., Eds.; Wiley-lnterscience: New York, 1974; pp 229-254. (8) McTaggart, F. K. "Plasma Chemistry in Electrical Discharges"; Elsevier: New York, 1967. (9) Gleit, C. E.; Holland, W. D. Anal. Chem. 1862, 3 4 , 1434. (IO) Evans, C. A., Jr.; Morrison, G. H. Anal. Chem. 1988, 4 0 , 869. (11) Hohman, W. R., I n "Principles and Techniques of Electron Microscopy"; Hayat, M. A., Ed.; Van Nostrand Reinhold: New York, 1974, pp 124-158. (12) Hohman, W. R.; Schaer, H. J . CellBiol. 1872, 55, 328-354. (13) Mason, A. 2.; Nott, J. A. J. Histochem. Cytochem. 1880, 28, 1301-1311. (14) Thomas, R. S. J . Hlstochem. Cytochem. 1981, 29, 379-393. (15) Samsonov, G. V., Ed. "The Oxide Handbook, 2nd Edition"; translated by Johnston, R. K.; IFI/Plenum Data Co.: New York, 1982. (16) Weast, R. C., Ed. "CRC Handbook of Chemistry and Physics, 64th Editlon"; CRC Press: Boca Raton, FL, 1983. (17) Galuska, A. A.; Morrison, G. H. Int. J. Mass Spectrosc. Ion Roc., in press. (18) Barnard, T.; Thomas, R;,S. J. Microsc. 1878, 773 (3), 269-276. (19) Mason, A. Z., I n Biomineralization and Blological Metal Accumulation"; Westbroek, P., de Jong, E. W., Eds.; Reidel: Dordrecht, 1983. (20) Thomas, R. S.; Corlett, M. I. J. Hlstchem. Gytochem. 1981, 29 (3), 394-407. (21) Pallon, J.; Malmqvist, K. G. Nucl. Instrum. Methods 1881, 181, 71-75. (22) Lecomte, R.; Landsberger, S.; Monaro, S. Int . J . Appl. Radiat . h o t , 1982, 33, 121-125.
RECEIVED for review May 25,1984. Accepted August 24,1984. This work was supported by the National Institutes of Health and The National Science Foundation.
