Anal. Chem. 1986, 58,1675-1680
Therefore, ammonia CI is the method of choice for determining the extent of labeling of compounds of this type. It is important to note that the problems associated with using E1 for determining isotopic enrichment were obvious since essentially pure labeled compounds were used. However, if this were a metabolism study in which the extent of label enrichment was small (typically 95% of the total ion current is carried in the protonated molecule and the ammonium adduct. However, CI alone is insufficient to determine the position of the label. While ammonia CI was useful for such studies of 13C-and l80-labeled compounds, this may not be true for deuterated sugars, since H/D scrambling between the analyte and CI reagent may occur.
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ACKNOWLEDGMENT The authors wish to thank Brian Musselman for helpful discussions, and E. Clark, Jr., for making available the labeled compounds. , Registry NO. "0, 14797-71-8; 13C, 14762-74-4; permethylsorbitol, 20746-36-5; [ l-'80]permethylsorbitol, 101860-67-7; [6'80]permethylsorbito1, 101977-06-4; [l-13C]permethylsorbitol, 101916-21-6.
LITERATURE CITED (1) Finlayson, P. J.; Christopher, R. K.; Duffield, A. M. Biomed. Mass
Spectrom. 1980, 7 , 450. (2) Robinson, J. R.; Staratt, A. N.; Schlahetka, E. E. Biomed. Mass Spectrom. 1978, 5 , 648. (3) Robert, J. J.; Beaufrere, E.; Koziet, J. Diabetes 1985, 54, 67. (4) Gambert, P.; Lallemant. C.; Archambault, A. J . Chromatogr. 1979, 162, 1. ( 5 ) Lindberg, C.; Johnsson, S.; Hedner, P.; Gustaffasson, A. Clin. Chem. (Wlnston-Salem, N . C . ) 1982, 28, 174. (6) Wilson, D. M.; Burllngame, A. L.; Cronholm, T.; Sjovall, J. Biochem. Biophys Res. Commun 1974, 56, 828. (7) Golovkina, L. S.; Yul'fson. N. S.; Chizhov, 0. S. Zh. Org. Khim. 1868. 4, 737. (8)Caprlolll, R. M.; Rittenberg, D. Biochemistry 1969, 8, 3375. (9) Caprblli, R. M.; Seiffert, W. E., Jr. Biochim. Biophys. Acta 1973, 297, 213. (10) Clark, E., Jr.; Ph.D. Thesis, Michigan State University, 1985. (11) Weinhouse, S.; Medes. G.; Floyd, N. F. J. Biol. Chem. 1946, 166, 691. (12) Wilson, M. S.; Dzidic, I.; McCloskey. J. A. Biochim. Biophys. Acta 1971, 240, 623. (13) Murata. T.; Takahashi, S.; Takeda, T. Anal. Chem. 1975, 4 7 , 573. (14) Houghton, E.; Teale, P.;Dumasia, M. C.; Welby, J. K. Biomed. Mass Spectrom. 1982, 9 , 459. (15) Horton, D.; Wander, J. D. Carbohydr. Res. 1984, 36, 75. (16) Hogg, A. M.;Nagabhushan. T. L. Tetrahedron Left. 1972, 4 7 , 4827. (17) DeJong, E. G.; Heerma, W.; Scherer, C. Biomed. Mass Spectrom. 1979, 6 , 242. (18) Dougherty, R. C.; Roberts, J. D. J. Org. Chem. 1974, 39, 451. (19) Reinhold, V. N.; Carr, S. A. Mass Spectrom. Rev. 1983, 2 , 157. (20) Kassel, D. E.; Martin, M.; Schall, W.; Sweeley, C. C. Biomed. Mass Spectrom., in press.
.
.
RECEIVED for review December 4,1985. Accepted February 14, 1986. This work was made possible through the financial support of the National Institutes of Health (NIH Grant No. RR00480-16).
Ionization Probability Variations due to Matrix in Ion Microscopic Analysis of Plastic-Embedded and Ashed Biological Specimens J. T. Brenna' and G. H. Morrison* Department of Chemistry, Baker Laboratory, Cornel1 University, Ithaca, New York 14853-1301 Matrix effects on ionization probabliltles have been investlgated for ion imaging of piastk-embedded and ashed bioiogical thin sections. Practical ion yleid (7) maps were constructed by udng Be, introduced by quantitative ion impiantation, as a reference element. The 7 maps show that variations In Be signal are less than 1% In plastic-embedded tissue and of the order of a few percent in ashed tissue. These results, when extended to physiologically Important elements, indicate that matrix effects are of relatively small importance In quantltatlve analyses of these samples. Spedflc methods for the conversion of lon intensltlesto elemental concentration for each sample type are discussed. Present address:
Endicott. NY 13760.
IBM Corp., Systems Technology Division,
Secondary ion mass spectrometry (SIMS) is a routine tool for elemental analysis of many types of solid specimens. The technique is particularly useful for trace elements and can achieve high spatial resolution, both laterally (1)and in-depth (2). However, for quantitative work, SIMS analyses suffer from a number of artifacts that affect measured elemental intensities. Among these, the variation of ionization probability of ejected secondary species due to changes in local chemical environment is frequently the most severe problem to overcome. This parameter may vary over 5 orders of magnitude for a single element in different matrices, the SIMS matrix effect, and different elements in the same matrix (3). Further, this behavior is difficult to catalog as a change in matrix and may produce changes in ionization probabilities that vary in direction as well as magnitude. Despite continuing advances in the theoretical treatment of secondary ion
0003-2700/86/0358-1675$01.50/00 1986 American Chemical Society
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emission, models have failed to reliably predict ionization probabilities, and therefore elemental concentrations, from secondary ion intensities. As a result, quantitative analysis has been most accurately achieved by empirical calibration methods, where external as well as internal standards (3-6) are used. It is in these studies that the variations in elemental sensitivities have been determined. Typically, elemental secondary ion intensities are determined in samples of uniform matrix in the probe mode of analysis. With this information and a knowledge of bulk elemental concentration, instrumental transmission, and in some cases other information such as sputtering yield ionization probabilities may be obtained. By use of this procedure, studies of matrix systems to determine the effects of varying matrix element concentration on measured secondary ion yields require a separate specimen for each set of concentration levels of interest. For specimens heterogeneous on the micrometer scale, this is often impractical or impossible, since the precise composition a t any position in the specimen may be unknown and accessible only with great difficulty, if a t all. As a result, secondary ion imaging (ion microscopy ( 2 ) )of solid surfaces has been done almost exclusively without consideration to matrix effects. Lateral localization of elements in biological tissue is often accomplished by ion microscopy. Most of these studies have been qualitative in nature, primarily due to SIMS artifacts and the difficulties of determining the extent of matrix effects. Although variations in ionization probability in biological samples have been investigated for dried bulk standard reference materials (7), no studies have been reported to data for localization studies. Recent work (8) has shown that low-temperature ashing (LTA) by exposure of plastic-embedded thin sections to an oxygen plasma prior to SIMS analysis substantially increases the detectability of positive secondary ions, while leaving elemental morphology intact. We have constructed practical ion yield maps in order to investigate matrix effects for both intact and plastic-embedded biological specimens. In this report, we detail the experimental protocol for practical ion yield map construction and the results for these specific specimens and their implications for quantitative analysis of biological samples. The method is generally applicable to arbitrary heterogeneous specimens, without a priori detailed information about the composition of the matrix. The practical ion yield, 7, is defined as the ratio of the number of M+ ions detected to the number of M atoms sputtered from the sample 7
= M+ ions detected/M atoms sputtered = pq (1)
where 7 may be broken down into two factors, the ionization probability, 8, and the instrumental transmission, q. 8 is defined as the probability that an element will be sputtered as a particular charged species, either molecular or monatomic, rather than as a neutral or other charged species. Given identical instrumental conditions (i.e., identical q’s), a comparison of S’S from two different samples is a direct comparison of the p’s. In an ion image each picture element (pixel) may be considered to be a separate sample analyzed under identical circumstances. Thus, a comparison of the S’S from pixel to pixel is a comparison of the p’s in the matrix represented by each pixel. s maps are constructed to evaluate local changes in 0,as follows. An element sensitive to matrix effects but absent from the sample is uniformly introduced by quantitative ion implantation (3, 5 , 9, 10). The sample is then analyzed by ion microscopy, and the implanted element is imaged throughout the entire depth of the implant in an image depth profile ( 1 2 ) . The collected images are then summed to form a map of the total implant signal. Since the lateral concentration of the implanted species is known and constant throughout the section, any variation in signal intensity must
Table I. Ion Microanalyzer Instrumental Parameters primary ions primary ion energy primary ion current primary ion current density primary ion beam size primary ion raster area transfer optics detection mode contrast aperture
o*+
5.5 keV 50-200 nA 0.5-2.0 mA/cm2 100 pm2
250 Mmz 150-pm field of view
microchannelplate/fluorescent screen or faraday cup 150 pm diameter
be due to changes in ionization probability from pixel to pixel. The integration of the signal is necessary, since variations in sample composition cause variations in implant range (10). In addition, the uncertainty in the mass of analyte sampled due to pixel to pixel differential etch rates is eliminated. A number of factors are important in the choice of the reference element for ion implantation. The ideal element would (1)be easy to generate in a sufficiently intense beam of appropriate energy in an ion implanter, (2) be sensitive to matrix effects, (3) not damage the section (i.e., presputter), (4) be absent in the section, and (5) not suffer from serious mass spectral interferences during analysis. Beryllium is one of the few choices that satisfies all of these criteria for these samples. For reasons to be discussed later, energies of the order of 20 keV or less are required to produce an implant of sufficiently shallow range. At these low energies, monatomic Be beams can be produced with reasonable intensity. Be signals have been shown to be sensitive to matrix effects in studies of well-defined materials (12-14). Since Be is a light ion (implanted as Be+), it causes negligible sputter damage to the sample during implantation. Normal biological tissue is almost devoid of Be as it is one of the most toxic elements. Lastly, it does not suffer from serious spectral interferences as organic and oxide molecular ions, which are of most concern in these samples, all occur a t higher masses. For all these reasons it is an excellent choice for use as a reference element.
EXPERIMENTAL SECTION Mouse intestine prepared as previously described (8)and embedded in epoxy resin was used for investigation. Sections of 0.5 pm thickness were mounted on a smooth, high-puritygold surface for ashing. One-half micrometer sections were used as this thickness is optimal for sensitivity and preservation of ash morphology. One micrometer thin sections were used for study of intact plastic sections, in order to establish implant range and allow imaging of other elements after analysis of the implant. These sections were mounted on high-purity semiconductorgrade polished silicon wafers. The sections were transferred to an Accelerators, Inc., 300R ion implanter for quantitative ion implantation. A mass-filtered 20-keV Be+ beam generated from a cold cathode source by PF5sputtering of solid Be0 was implanted into the sections. Beam current was kept low (99.9% of the Be signal resides in the top 0.5 pm of the seetion, and therefore entirely within the 0.5-pm section to be ashed. In addition. the tail of the Be signal extending past the implant range reveals a background signal a t mass 9 of about 10 counts/s. which is a negligible contribution to the total signal. Be+ was imaged and its signal was collected on videotape. A slow sputter rate was used in each case in order to stay
Flgure 2. (a. top) T& map from intact plasticambedded section. In ttSsue regions. Be emission is homogeneous and greater than that in pure plastic regions. (b. bottom) Calcium ion micrograph, recorded aner U?s data faUm map were obtained, l a aid in ldentitying smxhrai featues: N. nuclei 01 columnar epilhellai cells: C. cylopksm: 8. brush border. which is a dense membranous tissue boundary: M,muscle fiber region. which runs up the center of the viilus: P. piastic resin devoid
of tissue.
Table 11. Practical Ion Yields in Intact Resin-Ernbsddsd Sections section resin tissue
7
(3.0 (4.1
* SD.
* 0.1) x 10-5
0.1) x 10-5
'Standard deviation. within the dynamic range of the system. In the case of plastic the sputter rate was about 2 nm/s. A total of approximately 6000 frames were collected on videotape, a t the rate of 30 framesls for the intact sections and 20000 frames for the ashed sections. Subsequently, the entire series of frames is calibrated and summed for each sample to yield r maps. Seven r maps were constructed for each treatment, intact plastic and ashed. A typical r a map of an intact resin-embedded section is shown in Figure 2a (top). An accompanying Ca ion micrograph of the same section obtained after the Be images were acquired is presented in Figure 2b (bottom) for aid in identifying microfeatures. The Be signal for villi is clearly more intense than that from the surrounding plastic, which is devoid of tissue. The average ion yields for the tissue and surrounding plastic calculated from all the replicates are presented in Table 11. Comparison of these values indicates that Be sensitivity in tissue is 37% greater than in plastic. Ion yields from nuclei, cytoplasm, and the Ca emissive regions of muscle fibers (which may represent nuclei of muscle cells) were determined by digitally overlaying the Ca map on the ra map ( 1 7 ) to precisely establish their location on the T& map. Analyses of these data did not indicate statistically different ion yield means for any of these structures.
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Table 111. Practical Ion Yield in Ashed Sections section
resin tissue (a")
cytoplasm nuclei
muscle
7
f
SD"
(6.9 i 0.2) X (7.6 i 0.2) X (7.4 i 0.1) x (7.9 i 0.2) x (7.9 i 0.2) x
IO4 IO4 10.' 10.' 10-4
.Standard deviation.
The ash matrix has been shown to greatly enhance seeondary ion intensities relative to intact plastic. The possibility then exists that Be, which was not detected at significant levels in the plastic sections, may be detectable in the ashed specimens. Any such signal would affect the accuracy of the analysis. Before implanted sections were analyzed, unimplanted sections were ashed and analyzed for signal a t mass 9. These analyses revealed no significant mass 9 background. Figure 3a (top) is a 7% map produced from an ashed section. Since no ash remains after analysis to generate a Ca map for comparison, a dark-field reflected light micrograph of an ashed serial section is presented in Figure 3b (bottom) for comparison. This I map reveals a greater relative variability in Be ion yield within tissue than is the case for plastic-embedded tissue. In addition, the average ion yield in tissue is greater than that in plastic alone. Table 111 gives the average ion yields for tissue and plastic, as well as various microfeatures within the tissue. The data show that the relative variability between tissue and plastic and among microfeatures exists to a greater extent than in unashed material, but the range of this variability is smaller than in the intact plastic-embedded case.
DISCUSSION
Flgure 3. (a. top) map from ash& plastic-embedded section. Be emission within tissue is more heterogeneous than in the intact plastic-embedded case: however. the range over the entire map is smaller (see Table 111). (b. bottom) Dark field reflected light micrograph of a section similar to that in a, presented tor comparison.
Quantitative ion implantation has been applied previously to characterize analytical parameters in a single study of SIMS analysis of biological tissue (20). In this work, the implanted element, boron, was used as an internal reference element to calibrate the effects of differential sampling volume. In this method, matrix effects on ionization probabilities are not addressed, and, in fact, might have an adverse effect on the analysis. In addition, the method requires implantation for each replicate sample. The present method does not address the prohlem of differential sampling volume, hut rather is designed to remove it from consideration in order to study matrix effects directly. This method is to be used as a screening tool, in order to a s s e s the extent to which matrix composition variations affect ionizstion probabilities in various specimen types. As such, it need only he applied to cases where matrix composition may change between specimens. The data from the intact section indicate that variability in matrix composition within tissue is negligible with respect to influencing Be ion yield, but that Be ion yield variations from tissue to resin are significant. The ashed sections show variability within the tissue; however, the range of variability is much smaller than in the intact resin case since the greatest ratio of ion yields here is r(nucleus)/r(resin) = 1.14. This increase in relative variability within tissue indicates that the ashing treatment concentrates the inorganic constituents of the section sufficiently such that they beeome matrix elements. The decreased range of variability may be explained by the fact that surfaces saturated with oxygen are known to be less susceptible to matrix effects (21). These data apply rigorously only to the case of Be analysis in plastic or ash matrices. However, to the extent that effects of matrix on Be signal can be applied to other elements, the data suggest that specific methods are appropriate for correcting SIMS artifacts in quantitative analysis of these samples. For plastic-embedded sections, variations in ionization
probability in tissue appear to he negligihle. Given this ohservation and assuming the initial section is planar, the most serious sample-dependent artifact that occurs during SIMS analysis is uncertainty in analyzed volume, which occurs due to differential sputtering. T h e first-order correction for differential sputtering described by Patkin e t al. (22) effectively compensates for this phenomenon. It is executed by collecting an image of the desired analyte ion, then constructing a 'burn-through" map by subsequently imaging a substrate element in an image depth profile and determining the time of appearance of suhstrate in each pixel. The appearance time is directly related to sputter rate and can be used to correct the analyte map for variation in analyzed volume. The method intrinsically corrects for all phenomena that affect the signal of all species in the same direction and magnitude, such as nonuniform primary heam density. With a corrected image. a single calihration factor may he applied for conversion to concentration. Ashed sections poae a different challenge for quantification. The image evolution phenomenon ohserved with these samples makes snapshot ion images of them highly qualitative in nature. The image a t any stage in the sputtering process depends on the total ash and total analyte present through the thickness of the section (see ref 8 for detailed discussion). An appropriate solution to this prohlem is to acquire analyte images through the entire section and integrate the signal in a manner analogous to the T maps. These elemental maps will reflect concentration more significantly homogenized in depth than corresponding plastic sections. since a single image of a plastic section will sample 2-20 nm in depth. This situation is not particularly serious however; with the spatial resolution of the present instrument a t ahout 0.5 fim such a map would represent elemental concentration in cuhes (voxels) 0.5 pm on a side. The integration pnredure combined with
ANALYTICAL CHEMISTRY. VOL. 58. NO. 8. JULY 1986
w
e 4.
ashed thin
Integrated Calcium map horn a
0.5-grn. carventionally fixed,
Section.
the high sensitivity of the ash matrix would also allow the use of much thinner sections, perhaps of the order of
