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Anal. Chem. 1990, 62, 2122-2130 Meyer. M. C.; Guttman, D. E. J. fharm. Scl. 1988, 57(6),895-918. Banker, G. S.;Rhodes. C. T. Modern fharmaceufks; Marcel Dekker: New York, 1979;pp 202-207. Oester, Y. T.; Keresztes-Nagy, S.;Mais, R. F.; Beckkel, J.; Zaroslinski, J. F. J. fharm. Scl. 1978, 65(11),1673-1677. Sebille. 8.; Thaud, N.; Tillement, P. J. Chromafogr. 1978, 167,
159- 170. Wilting, J.; VanderGiesen, W. F.; Janssen, L. H. M.; Weideman, M. M.; Otagiri, M.; Perrin, J. H. J. Biol. Chem. 1980, 255 (7),3032-3037. Veronlch, K.: White, G.; Kapoor, A. J. fharm. Sci. 1979, 68 (12),
15 15- 1518. Sundlow, G.; Brikett, D. J.; Wade, N. N. Clln. Exp. fharmacol. Physiol. 1975, 2 , 129-140. Holford, N. H. G. Clin. fharmacoklnet. 1988, 7 7 , 483-504. Chu, Y.-Q.; Wainer, I. W. Pharm. Res. 1988, 5(10),680-683. Connors. K. A. Blndlng Consfanfs: The Measurements of Molecular Complex Stability; John Wiiey 8. Sons: New York, 1987;p 315. Chignell, C. F. Drug Fate and Metabolism, Methods and Techniques; Marcel Dekker: New York, 1977;Chapter 5. Cooper, P. F.; Wood, G. C. J. Pharm. fharmacol. 1988, 20, Suppi.
150s-156s. Hummel, J. P.; Dreyer, W. J. Biochim. Biophys. Acta 1982, 6 3 ,
530-532. Johnson, M. L.; Frasier, S. G. I n Methods in Enzymology; Academic: New York, 1985,Vol. 117,Chapter 16, p 325. Pinkerton. T. C.; Hagestam, I. H. Anal. Chem. 1985, 5 7 , 1757-1763. Cook, S. E.; Pinkerton, T. C. J. Chromafogr. 1988, 368, 233-248. Pinkerton, T. C.; Miller, T. D.; Cook, S.E.; Perry, J. A.; Rateike, J. D.; Szczerba. T. J. BloChromafogr. 1988, 7 (2),96-105. Dawson, C. M.; Wang. T. W.; Rainbow, S.J.; Tickner, T. R. Ann. Clln. Blochem. 1988, 25,661-667. Toshimitsu, N.; Takeda, N.; Tatematsu, A.; Maeda, K. Clin. Chem. 1988, 34 ( I l),2264-2267.
(22) Atherton, N. D. Clin. Chem. 1988, 35(6),975-978. (23) Tamlsier-Karolak, L.; Farinotti, R.; Bossant, M. J.; Dauphin, A. J. fharm. Clin. 1988, 7(4),543-556. (24) Oshima, T.; Johno, I.; Hasegawa, T.; Kitazawa, S. J. Llq. Chromatogr. 1988, 1 1 (16),3457-3470. (25) Nakagawa, T.; Shibukawa, A.; Shimono, N.; Kawashlma, T.; Tanaka, H. J. Chromafogr. 1987, 420, 297-311. (26) Shlbukawa, A.: Nakagawa, T.; Nishimura, N.; Miyake, M.; Tanaka, H. Chem. fharm. Bull. 1989, 37(3). 702-706. (27)Shlbukawa, A.; Nago, M.; Kuroda, Y.; Nakagawa, T. Anal. Chem. 1990, 62, 712-716. (28) Pinkerton, T. c.; Miller, T. D.; Janls. L. J. Anal. Chem. 1989, 67, 1171-1174. (29) Shibukawa, A.; Nakagawa, T.; Miyake, M.; Tanaka, H. Chem. fharm, Bull. 1988, 36 (5),1930-1933. (30) Shibukawa, A.; Nakagawa, T.; Mlyake, M.; Nlshimura, N.; Tanaka, H. Chem. fharm. Bull. 1989, 37(5), 1311-1115. (31) Ohshima, T.; Johno, I.: Hasegawa, T.; Kitazawa, S. J . fharm. Sci. 1990, 79 (l),77-81. (32) Yamaoka, K.; Tanlgawara, Y.; Nakagawa, T.; Uno, T. J. fharm. Dyn. 1981, 4 , 879-885. (33) Soites, L.; Bree, F.; Sebille, 6.; Tillement, J. P.; Durisva, M.; Trnovec, T. Biochem. fharm. 1985, 34 (2),4331-4334. (34) Sun. S. F.; Kuo. S . W.; Nash, R. A. J . Chromatcgr. 1984, 288, 377-388. (35) Sun, S. F.; Wong, F. Chromafcgraph/a 1985, 20 (E),495-499. (36) Stevens, F. J. Blophys. J . 1989, 55, 1155-1167. (37) Rosenthal, H. Anal. Biochem. 1987, 2 0 , 525-532. (38) Zlerler, K. Trends Biochem. Sci. 1989, 14, 314-317. (39) Fairclough, G. F.; Fruton. J. S . Biochemistry 1988, 5. 673-683.
RECEIVED for review March 6, 1990. Accepted July 11,1990.
Molecular Ion Imaging and Dynamic Secondary Ion Mass Spectrometry of Organic Compounds Greg Gillen* a n d David S. Simons
Center for Analytical Chemistry, National Institute of Standards and Technology, Gaithersburg, Maryland 20899
Peter Williams Department of Chemistry, Arizona State University, Tempe, Arizona 85287
An ion microscope equipped with a reslstive anode encoder imaglng system has been used to acquire molecular secondary ion images, with lateral resolutions on the order of 1 pm, from several quaternary ammonium salts, an amino acid, and a polynuclear aromatlc hydrocarbon which were deposHed onto copper transmlsaion electron microscope grids. Ail images were generated by using the secondary ion signal of the parent molecular species. The variation of parent and fragment moiecuiar ion signals with primary ion dose indicates that, for many bulk organic compounds, bombardment-induced fragmentation of parent molecules saturates at prlmary ion doses of (1-8) X 10'' ions/cm2. Subsequent ion impacts cause lmle further accumulation of damage in the sample, and intact parent molecular ions are sputtered even after prolonged ion bombardment (i.e. primary ion doses >1 X 10'' ions/cm2). This saturation process allows molecular images to be obtained at high primary ion doses and allows depth profiles to be obtained from slmple mdecuiar solld/metai test structures.
INTRODUCTION In recent years there has been intense interest, both from analytical and fundamental standpoints, in the application of secondary ion mass spectrometry (SIMS) to the analysis
of organic molecules. This interest followed the demonstrations by Benninghoven, Cooks, and their co-workers in 1976-1977 that kiloelectronvolt ion bombardment could eject intact parent molecular ions from compounds deposited onto metal surfaces (1,2). This technique has proven particularly useful for analytical and structural studies of involatile, thermally labile, organic compounds that were difficult to analyze by electron impact mass spectrometry. With the secondary ion imaging capabilities found in many secondary ion mass spectrometers, it should be possible to generate molecular secondary ion images using the same techniques developed for imaging elemental species (3-5). Imaging of molecular species in biological materials might have a major impact on a wide variety of studies of molecular processes in biological systems where previously only gross assays were possible. Progress toward this goal has recently been reported (6-14). A major difficulty in generating molecular secondary ion images using conventional (high ion dose) SIMS is that each primary ion, while desorbing several atoms or molecules from the surface layer, penetrates below the surface undergoing numerous collisions that can rupture bonds in many more molecules. Intuitively, it might be expected that after the outer few monolayers of the sample have been removed by sputtering, few intact molecules would remain below the sputtered surface for a depth on the order of the primary ion
0003-2700/90/0362-2122$02.50/00 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 19, OCTOBER 1, 1990
range, 10-20 nm for normally incident 10-keV Ar+ bombardment. Molecules lying below this damaged layer would, in turn, be expected to be fragmented by the penetrating primary ions before they are exposed by the continuing erosion of the sputtered surface. Experimental studies of amino acids deposited as multilayers (several molecular layers) on metal substrates have supported this hypothesis, indicating that subsurface molecular layers do not contribute significantly to the parent ion signal (15). T o avoid the effects of primary ion beam damage, the majority of organic SIMS experiments have been conducted under "static" conditions, where the cumulative primary ion dose experienced by the sample during an analysis is limited to -1 X 1013 ions/cm2, a t which point roughly 10% of the outer monolayer of the sample has been damaged (1). Reduction of the cumulative primary ion dose to this level assures a high probability that each primary ion will impact an area of the sample that has not been damaged by previous ion impacts, resulting in mass spectra that are more readily interpretable in terms of the original molecular composition of the sample. However, signal intensities are low because a very limited number of analyte molecules are sampled. Such an approach may produce insufficient signal for molecular ion imaging a t high spatial resolutions of dilute species, species with low secondary ion yields, or species with large primary beam-induced damage cross sections. Also, the lack of significant erosion of the sample surface precludes the acquisition of indepth compositional information. Although the static SIMS approach is generally assumed to be a prerequisite for all organic SIMS studies, there is increasing evidence that many organic solids may be more resistant to primary ion beam damage than previously expected. For example, dilution of various organic compounds in solid ammonium chloride is stated to result in enhanced molecular secondary ion signals, less fragmentation, and increased sample longevity, compared with the undiluted compounds (16, 17). Similar advantages were obtained by sputtering compounds from frozen aqueous solutions, a matrix chosen as a model for frozen hydrated biological tissue (18, 19). Gillen et al. found that frozen solutions (1 X M) of quaternary ammonium salts exhibited high and stable molecular ion signals, suitable for imaging, after primary ion doses exceeding 1 X 1015ions/cm2, and that ionization efficiencies from this system were several orders of magnitude higher than for neat compounds (18,19). Extrapolation of the parent ion signal-primary ion dose curve indicates that intact parent molecular ion signals would have been observed a t doses greater than 1 x 10'' ions/cm2, corresponding to an erosion depth of -1 pm. Molecular ion emission at high primary ion doses has also been observed in polymer systems. In particular, characteristic fragment molecular ion signals have been observed after prolonged ion bombardment in high sputter yield (20) fluorinated polymers such as poly(tetrafluoroethylene) (PTFE)(21,22). Preliminary studies have indicated that the molecular cation signals from many organic salt compounds exhibit similar stability at high primary ion doses (19, 23). If intact molecular ions could be sampled while sputtering to a depth of 1 pm below the original surface, the total detectable molecular ion signal could be more than 3 orders of magnitude greater than the signal from the surface monolayer alone. Such an increase could make molecular ion imaging with micrometer lateral resolution feasible, even for dilute species, or species with low secondary ion yields. Also, the capability to sample intact molecules from subsurface layers may allow molecular depth profiling analysis. The terms "dynamic" or 'high dose" SIMS have been applied to SIMS analyses using high primary ion doses in which
2123
the surface is appreciably eroded. In this paper we demonstrate that high spatial resolution molecular ion images, and molecular ion depth profiles, can be obtained under high dose SIMS conditions from model systems consisting of several types of compounds separately deposited onto copper grids or metal surfaces. Various compounds have been studied including quaternary ammonium salts, an amino acid, and a polynuclear aromatic hydrocarbon. Primary beam-induced damage in these materials has been studied by determining the rate of variation of fragment and molecular ion signals with primary ion dose and energy.
EXPERIMENTAL SECTION The 1-methyl-4-phenylpyridinium (MPP) salt used in this study was obtained from S. Markey at the National Institutes of Health. Other compounds were purchased from Sigma Chemical Corp. All compounds were used as received without further purification. Model systems for the molecular imaging studies were prepared by depositing the compound of interest from saturated aqueous (amino acid), ethanolic (quaternary ammonium salts), or methylene chloride (polynuclear aromatic hydrocarbon) solutions onto copper TEM grids. The grids were mounted with silver paint over - 2 mm diameter holes drilled in a brass block. Samples were deposited on the grids using a microsyringe with a typical loading of 2-10 pL. For primary ion beam damage studies, compounds were deposited from solution onto tantalum or brass substrates which had been roughened with 600 grit sandpaper to improve wetting of the substrate by the analyte solution. Multiple depositions were used to make thick films. Prior to the deposition of the solution, the substrates were heated in a 135 "C oven for 1-2 min to enhance evaporation of the solvent. A Cameca IMS-3F ion microscope was used for acquisition of all molecular ion images and for damage studies. In the IMS-3F, an energetic primary ion beam (Ar+ in our studies) strikes the sample, which is maintained at a potential of 4.5 kV and is positioned 4.5 mm from a grounded extraction electrode. By adjustment of the primary ion beam accelerating potential, the ion impact energy could be varied from 2.0 to 10.5 keV. For imaging studies, an impact energy of 8.0 keV was used. The optic axis of the primary ion column intersects the sample at an angle of 30' to the sample normal. The primary beam is deflected to larger impact angles by the field near the sample, resulting in an impact angle which decreases with increasing primary voltage from 64' at 2.0-keV impact to 37O at 10.5-keV impact (for positive secondary ions) (24). The focused beam was rastered over an area of either 250 X 250 pm2 or 500 X 500 Nm*. The mass range of this double focusing instrument, as presently configured, is 340 amu. The instrument was equipped with a resistive anode encoder (RAE) imaging system (Charles Evans and Associates, Redwood City, CA). This position-sensitive,pulse-counting detector, which can detect and calculate the has been previously described (W), position of single ion arrivals. For most images, secondary ion signals were collected for 10-30 s. Digitized molecular ion images were initially stored on an IBM-AT computer. The image files were later transferred to a Macintosh I1 computer and were displayed in a gray scale format using a public domain image processing program written by Wayne Rasband at the National Institutes of Mental Health. The gray scale for each image is scaled from zero to the maximum pixel value in the image field. The intensity of each pixel in the image corresponds to the total number of molecular secondary ions collected from that position. The presence of the analyk compound on the test grid was verified by observation, in the mass spectrum, of the parent and fragment molecular ion isotopic patterns that are characteristic of the compound. An adjacent area of the grid was then used for generation of molecular ion images. The mass spectra shown in the results section are typical of those acquired while generating molecular ion images. Each image was acquired with primary ion current densities of 1-5 pA/cm2. Thus,during a typical image acquisition time of 10-30 s (sample dependent), the sample was subjected to a cumulative primary ion dose of 6 X l O l 3 to 9 X 1014 ions/cm2. To study primary ion beam-induced damage, various parent
2124 * ANALYTICAL CHEMISTRY. VOL. 62. NO. 19. OCTOBER 1. 1990
30000
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Flgure 1. (a)Mass spectrum and (b) molecular secondary ion image of the neurotransminer acetylcholine bromae deposited onto a copper TEM gra. The Wge was generated by using the secondary ion signal Of the parent cation at 146 am". Grid bar spacing in the image is 25 r m . Maximum pixel value is 21 1 counts,
Figure 2. (a)Mass spectrum and (b) molecular secondary ion image 01 t h e germicide benzalkonium chloride deposited onto a copper TEM grid. The imge was generated by using the Secondary ion signal 01 me parent cation at 304 amu. Wki bar spacing in the image is 25 rm.
and fragment molecular ion signals were monitored as a function of time (dose). To verify that the molecular ion signals at high primary ion doses were not being obscured by background peaks, mass spectra were obtained at the end of each measurement. In these experiments, it was necessary to determine the primary ion current and raster size accurately. The primary beam Faraday cup on the Cameca IMS-3F has been observed to read primary ion currents that may be an order of magnitude higher than the true value (26). This results from incomplete suppression of secondary electron emission. In our damage experiments, we have used a Faraday cup built into a brass block sample holder. Before each run, the primary ion current collected in this Faraday cup, biased at +I8 V, was measured with a picoammeter. The sample high voltage was disconnected for this measurement. Variations in current during the run were monitored with the standard Cameca IMS-3F Faraday cup and were typically no greater than 3%. The raster size was calibrated by imaging the AI+ secondary ion signal from a Cu/AI test grid structure
the secondary ion signal of the parent molecular cation (146 amu) which was sputtered from the bromide salt. The spacing between adjacent grid bars is 25 rm. Figure 2a shows the mass spectrum and Figure 2b the molecular ion image of the germicide benzalkonium chloride deposited onto a copper TEM grid. The image was generated by using the secondary ion signal of the parent cation (304 amu) which was sputtered from the chloride salt. The spacing between adjacent grid bars is 25 rm. Figure 3a shows the mass spectrum and Figure 3b the molecular ion image of the 1-methyl-4-phenylpyridinium (MPP)+cation (170 m u ) , sputtered from the iodide salt. The sample was deposited onto a copper TEM grid which was sitting on a brass substrate. The diameter of the square field of view in the image, in which the RAE was gated to a square active area, is 50 rm, demonstrating the high lateral resolution of this imaging system. Imaging studies of (MPP)+are of some physiological importance because this compound has been implicated as an active agent in the development of druginduced Parkinson's disease (27,28). The compounds used to generate the images in Figures 1-3 were chosen because they contain quaternary ammonium groups, which give high secondary ion yields and make this class of compounds readily detectable. However, we have found that we are able to acquire molecular ion images for
RESULTS AND DISCUSSION Molecular Secondary Ion Imaging. Figure l a shows the mass spectrum and Figure l b a molecular ion image of the neurotransmitter acetylcholine deposited onto a copper TEM grid. As is typical for a SIMS analysis of quaternary ammonium salts, most of the fragment ions observed in the maw spectrum can be explained by simple neutral losses from the even-electron parent ions. The image was generated by using
Maximum pixel value is 39 counts.
ANALYTICAL CHEMISTRY, VOL. 62, NO. 19, OCTOBER 1. 1990
2125
PHENYLALANINE 170
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Flgure 3. (a) Mass spectrum and (b) molecular secondary ion image of I-methyl-4phenylpyridine iodlde deposited onto a copper TEM grid. The image was generated by using the secondary ion signal of the parent cation at 170 am". Grid bar spacing in the image is 25 pm, Maximum pixel value is 25 counts.
any compound from which an identifiable mass spectrum can be generated. Figure 4a shows the mass spectrum and Figure 4h the molecular ion image of the amino acid phenylalanine generated using the secondary ion signal of the protonated molecular ion (M + HI+ at 166 amu. The compound was deposited from aqueous solution (pH 5) onto a copper TEM grid. The spacing between adjacent grid bars in this image is 100 pm. Figure 5a shows the mass spectrum and Figure 5h the molecular ion image of the polynuclear aromatic hydrocarbon benzo[ghi]perylene deposited onto a copper TEM grid. The image was generated by using the radical parent molecular ion a t 276 amu. The spacing between adjacent grid bars is 25 pm. Each of the molecular ion images shown in this section was obtained under Ar+ bombardment with an impact energy of 8.0 keV, which would be typical for elemental SIMS depth profiling analysis. Under these experimental conditions, the primary impact energy and angle are such that the nuclear stopping is near maximum so that sputter yields and hence sensitivity are high (for elemental targets). However, for moleular ion sputtering, these conditions may not be optimal for minimizing primary ion beam damage and thereby producing the highest yield of intact parent molecular ions. During the acquisition of the molecular ion images from these
,
:I
-
image
me amino acid phenylalanine deposaed onto a copper TEM grid The image was generated by using the secondary ion signal of the prctonated molecular ion at 166 am". Grid bar spacing in the image is 100 pm. Maximum pixel value is 25 counts. 01
grid structures, secondary ion signals appeared to decay in some tens of seconds. Also, the primary ion dose required to generate an image exceeded "static" SIMS primary ion doses. For compounds such as quaternary ammonium salts, which give high parent ion signals, short integration times could he used, and the primary ion dose accumulated by the sample would typically he on the order of -6 X l O I 3 ions/cm2. For compounds with lower parent molecular ion signals, such as phenylalanine and henzo[ghi]perylene, longer integration times were required, and the accumulated primary ion dose was as high as 9 X IO" ions/cm2. While the signal collected by the integrating resistive anode encoder system was sufficient to generate images from all of the compounds used in this study, it is not clear whether this would be the case for more dilute species, species with larger damage cross sections (Le. higher molecular weight species), or species with low secondary ion yields. Studies of P r i m a r y Ion Beam Damage. T o study primary ion beam damage of organic compounds, we monitored the signals of parent and fragment ions as a function of primary ion dose. For example, the data for the parent molecular cation of acetylcholine a t 146 amu and the CH3CO+ fragment ion a t 43 amu are shown in Figure 6. These ions were generated by sputtering acetylcholine bromide with 10.5-keV Art ions a t an impact angle of 37' to the sample
2126 a
ANALYTICAL CHEMISTRY, VOL. 62, NO. 19. OCTOBER 1. 1990
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Flgure 5 . (a) Mass spectrum and (b) moiecular secondary ion image of ltw polynuclear aromatic hydrOcarbOn benzo[ghi]peryienedeposited onto a copper TEM gid. The image was generated by using the secondary ion sQnal of the radical mlecular ion at 276 amu. Grd t a r spacing is 25 pm. Maximum pixel value is 13 counts.
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5e+15 8e+l5 te+16 Primary Ion Dose (ionslcm ') Figure 6. Decay of the parent (146 amu) and fragment (43 amu. C,H@+) molecular ion signals of acetylcholine bromide on tamalum as a function 01 primary ion dose. The samole was bombarded with 10.5-keV Ar' primary ions 0
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normal. The secondary ion signal of the parent molecular cation initially decays rapidly with increasing primary ion dose. At a dose of approximately (5-8) X 10" ions/cm*, the slope of the decay curve changed significantly, and intact parent
molecular cations continued to be observed up to a dose of 1 x 1016 ions/cm2. The variation in the major fragment ion signals of acetylcholine at 43.58, and 87 amu (see mass spectrum, Figure la), with increasing primary ion dose, resembled the variation in signal for the parent molecular ion. However, as a general rule, and as shown in Figure 6 for the 43 amu fragment, the fragment ion intensities decreased less with increasing primary ion dose than did the parent ion signal. Influence of Experimental Parameters on Molecular Ion Signal Decay. The primary ion impact energy and angle had a pronounced effect on the signal decays of both parent and fragment molecular ion signals of acetylcholine. In Figure 7 are plotted the decays of the parent molecular ion signal of acetylcholine for primary Ar+ ion impact energies of 2.5, 8.0, and 10.5 keV, which correspond to impact angles with respect to the surface normal of Bo,39". and 37O, respectively. In the low dose region, the initially greater parent ion signals at higher primary impact energies are consistent with the increase in sputter yield with increasing primary impact energy, as determined by elemental SIMS experiments (29). Also, the greater rate of signal decay with increasing impact energy appears to be consistent with Benninghoven's ohservation that damage cross sections for molecular fragmentation increase with increasing impact energy (30). If this is true, then the fragment ion signals should increase relative to the parent ion signals for higher impact energies. In Figure 8 are plotted the ratios of the CH,CO+ fragment (43 amu) to the parent molecular cation (146 amu), as a function of increasing primary ion dose, for the same primary impact energies and angles used to generate the data in Figure 7. Comparing the three curves, it can be seen that the highest ratios are obtained for the greater impact energies. Interestingly, as the primary ion dose is increased, the ratios reach a saturation point after
ANALYTICAL CHEMISTRY, VOL. 62, NO. 19, OCTOBER 1, 1990
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Flgure 10. Decay of the molecular ion of methylene blue (284 amu) as a function of primary ion dose for various Ar+ primary ion impact energies.
which there appears to be little additional accumulation of damage (fragment species) in the material. In contrast to the behavior in the low dose regime, a t primary ion doses greater than (5-8) x 1014 ions/cm2, the initial positions of the curves shown in Figure 7 are reversed, with the lower primary ion impact energies giving the highest yield of intact parent molecules. The order of magnitude differences in signal for these curves are not easily rationalized on the basis of sputter yield variations. The data in Figure 8 would suggest that the higher parent ion signals a t lower primary impact energies may result from the larger number of intact parent molecules available to be sputtered as determined from the lower fragment/parent ion ratio. It should be noted that while the 43 amu fragment ion was used in these studies, very similar behavior was also observed for both the 58 and 87 amu fragment ions of acetylcholine. The primary ion species also influenced the molecular signal-primary ion dose behavior of organic compounds. In Figure 9 are plotted the signal decay curves for the parent ion of acetylcholine (146 amu) and the CH,CO+ fragment ion (43 amu) under Xe+ and Ar+ primary ion bombardment a t an impact energy of 8.0 keV. As would be expected from the sputter yield increase, the more massive Xe+ primary ion gives higher parent ion signals in the low dose regime. Parent ion signals in the high dose regime are also significantly higher. This enhancement appears to be related not only to an increase in sputter yield but also to a reduction in the level of damage accumulation as shown by the lower 43/146 signal ratio for the Xe+ data. We have also explored the effect of primary ion current density on the observed decay curves. For the parent ion of acetylcholine, four analyses were conducted with Ar+ bombardment a t an impact energy of 8 keV. Using the same rastered area, primary ion currents of 1,5,10, and 20 nA were used to generate four experimental curves. When the resulting curves were plotted as a function of increasing primary ion dose, they were superimposed, indicating that the damage as a function of primary ion dose is insensitive to the primary ion current used in the analysis. Higher primary ion current densities would be useful in such applications as depth profiling. Damage Behavior of Other Compounds. The general shape of the decay curves and the variations with primary ion impact energy described above for the parent and fragment ions of acetylcholine have been observed for all the compounds used in the imaging portion of this study. This includes compounds that are desorbed as preformed ions, protonated species, and radical ions. However, we should note that, for the amino acid sample, identification of parent ion species
a t high primary ion doses has proven problematic because of the very low secondary ion signals observed under our experimental conditions. In addition to the compounds studied here, parent ion emission a t high primary doses has also been observed for several other compounds including tetralkylammonium bromide salts (tetraethyl-tetrapentyl), organic dyes such as methylene blue and methyl red, and neuroactive compounds such as muscarine and neostigmine. As an example of the behavior of another type of compound, Figure 10 shows the parent ion signal variation with primary ion dose for the parent cation of methylene blue (284 amu) as a function of primary ion impact energy (angle). These data are shown for lower total primary ion doses so that the transitional behavior of the signal decay is more apparent. Also, to reduce the influence of coverage variations, the count rates for each curve have been normalized to the same initial value. In comparison to acetylcholine, slopes of the initial signal decay are greater and the molecular ion signal in the high dose region is lower. Additionally, the saturation point is reached at a lower dose (1 X 1014 ions/cm2) than it is for acetylcholine ((5-8) X 1014ions/cm2). This is typical of most compounds with higher molecular weights and may reflect the higher damage cross sections for these organic compounds. (Damage cross sections for the parent molecule are calculated from the characteristic exponential decay in parent ion signal as a function of primary ion dose. The damage cross section, u, in cm2, is defined as u = (In (Io)- In ( I # ) ) / $where , I is the secondary ion signal and $ is the primary ion dose in ions/ cm2.) In fact, acetylcholine may represent a somewhat atypical example of damage behavior because it has significantly smaller damage cross sections in the low dose region (varying cm2 for the data shown in Figure 7) than from (1 to 3) x does methylene blue (varying from (2 to 6) x cm2 for the data shown in Figure 10). The values for methylene blue are more typical of other compounds we have examined. Model for Damage Accumulation in Molecular Solids. The data presented above demonstrate that, in the samples we have examined, parent molecular ion emission is observed a t very high primary ion doses. The data also indicate that the rates of signal decay differ significantly in the low dose (1 x 1015 ions/cm2) regions. The beginning of the low dose region corresponds to primary ion doses which would typically be encountered in a static SIMS experiment. In this case, where it is possible to sample molecules from undamaged regions of the surface, the intensity of molecular ion emission as a function of impact energy is qualitatively consistent with the influence of energy (and impact angle) on sputter yields. Similarly, at somewhat higher doses, the differences in rates of signal decay are
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 19, OCTOBER 1, 1990
Table I. Sputter Yields of Molecular Solids molecular solid Teflon
sputter yield 100 CF2 molecules/ion
(300 atoms/ion] acetylcholine 30-40 molecules/ion [780 atoms/ion] water ice 40 molecules/ion [120 atoms/ion] ammonium 34 molecules/ion chloride [204 atoms/ion] frozen 47 molecules/ion dvcerol 1658 atoms/ionl
experimental conditions
ref
1.0 keV Art, 0'
20
14.5 keV Cs+, 25'
32
10.0 keV Art, 45O
33
8.0 keV Art, 39O
34
10.0 keV Art, 45O
34
consistent with energy-dependent variations in damage cross sections for organic molecules (30). The desorption of intact molecular ions, from surfaces irradiated with high primary ion doses, may not readily be explained on the basis of the energy dependence of sputter yields or damage cross sections. Rather, emission of intact molecules at these high doses can be described as occurring from a sputtered surface in which a saturation in the accumulation of damage (intramolecular bond breaking) has been reached. Recently, Williams and Gillen (18) suggested a qualitative explanation for this process of damage saturation in ion-bombarded molecular solids. A primary ion impacting at a few kiloelectronvolts enery on a molecular solid loses energy at a rate which varies with depth in an approximately Gaussian distribution, with the peak rate of energy loss at a depth approximately 50-70% of the mean projected range of the ion in the solid (31). The majority of the energy loss occurs in ion-atom collisions that can break molecular bonds; the average energy loss rate is some hundreds of electronvolts per nanometer. Bond breaking can result not only from the direct collision of the primary ion with the surface and subsurface molecules but also from collisions involving the energetic subsurface atoms recoiling from the primary ion collisions. The energy deposition also results in the sputtering of material from the surface region; both parent molecules and a fraction of the material damaged in the current or previous ion impacts may be sputtered. As sputtering proceeds, the fraction of damaged subsurface molecules will increase but will eventually saturate at a level inversely proportional to the sputtering yield. That is, there exists a finite probability that any given subsurface molecule will escape being damaged by the several ion impacts necessary to remove the material above it. The higher the sputtering yield, for a given primary ion penetration depth, the greater the probability of sampling an intact molecule originally located below the surface, because fewer impacts are required to remove overlying material. As listed in Table I, many molecular solids have sputter yields that may be several hundred atoms/primary ion. These values should be compared to typical sputter yields for metals of 0.1-10 atoms/ion (29). The model described above predicts that saturation in the accumulation of damage in the solid would be reached after sputter removal of a depth of material approximately equal to 2Rd where Rd is the mean depth of the damage distribution curve. The dose, Q (in ions/cm2), corresponding to removal of this depth of material may be expressed as Q = 2Rd D / Y where D is the density of the solid in molecules/cm3 and Y is the sputter yield of the compound in molecules/ion. To determine the value of Rd for an organic sample, we have used the Monte Carlo TRIM 89 code (35) to evaluate qualitatively the creation of vacancies in a target with the nominal composition and density of methylene blue (neglecting the counterion). For 8.0-keV Ar+ impacting at 39' to the sample
normal (the default values for displacement energy, 20 eV, and lattice energy, 1 eV, were used in the calculation), the mean of the damage distribution is predicted to be at a depth of 8.5 nm (about 50% of the mean projected range of the Ar' ions). Assuming the sputter yield for methylene blue is in the neighborhood of 30 molecules/ion we would predict a saturation in damage accumulation, and therefore a constant parent ion signal, at a primary ion dose of 1x 1014ions/cm2. Similarly, reducing the primary impact energy (and increasing the impact angle) results in a reduction of Rd which should lower the primary ion dose at which saturation occurs. These predictions are consistent with the data shown in Figure 10. This model also allows us to predict qualitatively the magnitude of the expected signal decay for a parent molecule. The cross section for a bond-breaking impact on a methylene blue molecule under 8.0-keV Ar+ impact can be calculated from the initial portion of the experimental curve to be -6 X cm2. To sputter through a thickness equal to 2Rd, and thus reach the saturation region, requires a primary ion dose of 1 X 1014ions/cm2. Thus, a methylene blue molecule which is situated just beyond the primary ion penetration depth will suffer on average six collisions before it is sputtered. Because the primary ion impacts are randomly distributed, there is a fiiite probability, on the order of e* or approximately 0.25% (assuming Poisson statistics), that a molecule will be exposed a t the surface and sputtered without having suffered a bond-breaking impact from a primary ion. From the data in Figure 10, it can be seen that the parent ion signal a t a dose of 1 X 1014ions/cm2 has decayed to -0.1% of the original value. Similar calculations for the other curves in Figure 10, using experimental damage cross sections and calculated values of Rd,indicate that the measured signal a t saturation is within a factor of 2-5 of that predicted on the basis of the simple damage model. For a given secondary ion yield, optimization of parent ion signals in the high-dose region requires experimental conditions that increase the sputter yield and/or decrease the ion range and the depth over which the molecules are damaged. Both of these effects can be achieved by using more massive primary ions. For a given primary species, increasing the incidence angle of the primary ions has a similar effect. In our instrument, the angle of incidence can only be increased by lowering the primary ion energy, which lowers the sputter yield, but at the same time also reduces the primary ion range and the depth over which damage is produced; Figure 10 shows that decreasing the primary ion energy causes damage to saturate a t a lower primary ion dose, resulting in a higher steady-state molecular ion signal, as this discussion would predict. It is clear that in order to take full advantage of the damage saturation effect for molecular mapping studies, it is desirable to use low primary ion impact energies and high incidence beam angles with respect to the surface normal. Such experimental conditions are not optimal for imaging using a microprobe technique; high primary energies and high angles of incidence are required to produce small diameter ion beams. However, spatial resolution in the ion microscope is not dependent on the primary beam size, and so this instrument appears well-suited to take advantage of the damage saturation effects at high angles/low energies for molecular mapping studies. Our model for the sputtering of molecular solids would suggest that no additional parent ion signal decay should occur after the saturation point. In fact, a slow continued decay is seen in all our molecular ion signal-primary ion dose curves. We believe this to result from uneven coverage of the substrate by the molecular films. In all of the films used in this study, substrate ion signals were detectable at the start of sputtering,
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ANALYTICAL CHEMISTRY, VOL. 62. NO.
le+05
19, OCTOBER 1. 1990 2129
,
0
le+17
Ze+17
3e+17
4e+17
Primary Ion Dose (ions/crn*) Figure 12. Deplh profile of a thick layer of acetylcholine bromide deposited onto tantalum sputtered by 8.0 keV AI+ ions. The parent molecular ion of acetylcholine (148 amu) and the tantalum secondary ion (181 amu) are monilored as a function of primary ion dose.
Flgure 11. Molecular secondary ion images of acetylcholine bromide on a copper TEM gridlbrass substrate as a function of increasing primary ion dose (5.5-keV Art bombardment). The image was generated by using the secondary ion signal of the parent molecular ion (146 amu). Primary ion dose accumulated by the sample at the end of each image acquisition: (a) 3.3 X lo1'. (b) 5.3 X 10". (c) 1.3 X 10". and Id) 2.8 X lo" ions/cm2. indicating the presence of pinholes in the film. These pinholes were necessary to reduce the loss of molecular secondary ion signals due to charging. In our experimental curves, the slow signal decrease a t high primary ion dose appears to result from the steady enlargement of these pinholes by sputtering and the consequent decrease in the surface area of the film. Preliminary damage studies on a time-of-flight (TOF) SIMS instrument, which is more immune to charging artifacts because of the low primary ion current densities used, demonstrated that parent molecular ion signals from a several millimeters thick pellet of acetylcholine could be obtained at primary ion doses of 1 X loL9ions/cm2, with no significant decay in signal after a dose of 1 X lOI5 ions/cmz. We should also briefly comment on why this damage saturation effect has not been routinely observed in other studies. From the simple model presented above, it is clear that the sample must be of sufficient thickness to allow the saturation point to be reached. In the case of acetylcholine under IO-keV AI+ bombardment, the sample would need to have a minimum thickness of 15 nm. Assuming the sample was prepared sufficiently thick, it would then need to he bombarded with a primary ion dose exceeding 10" ions/cm2. For the majority of previously conducted damage studies on thin molecular layers on metal substrates, the samples were either too thin or the Drimarv ion dose was too low for the saturation point to have been-reached. Hieh Dose Molecular Ion Imaeine a n d Molecular Depth Profiling. The integrated molecular ion signal available to form an image can be increased significantly by using a lower primary ion impact energy. For the data displayed in Figure 7, an order of magnitude increase in integrated counts is obtained by using an impact energy of 2.5 keV instead of 10.5 keV. Similar advantages may be obtained by using Xe+ bombardment. Because of the high signal levels, molecular ion images can also be obtained a t very high primary ion doses. When compounds are imaged on grids, difficulties arise because the layer of analyte compound, which needs to be relatively thin to prevent charging effects, be completely sputter-removed during the experiment. One method to circumvent this difficulty is to place the grid on a metal
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substrate and use the space between the grid bars as wells to contain repeated evaporations of the compounds. In Figure 11 are shown four images of acetylcholine (using the 146 molecular ion) beginning a t a total primary ion dose of 3.3 x loL5ions/cm2 and ending a t a primary ion dose of 2 X lo'' ions/cm*. Each of these images was obtained with a 20-9 integration. Using the sputter yield of acetylcholine (32) to estimate its removal rate, a 20-9 integration time corresponds to acquisition of an image over a sputtered depth interval of 10 nm. The last image in the series corresponds to sampling acetylcholine molecules which were originally some 4 pm below the sample surface. If molecular ion signals persist to high primary ion doses, simple molecular depth profiles can be obtained. As a preliminary experiment, a layer of acetylcholine on tantalum metal was profiled as shown in Figure 12. The acetylcholine overlayer is clearly defined in the profile. We do not have an accurate means to determine the thickness of the layer; however, if we again use a sputter yield of 30 for acetylcholine, on the basis of the dose required to reach the maximum in the tantalum signal, the layer would again be on the order of 4 pm in thickness. The absence of an acetylcholine signal in the initial portion of the profile is attributable to charging of the sample. As the beam sputters into the acetylcholine layer, the metal underlayer begins to be exposed, charging is reduced, and molecular ion signals are observed.
CONCLUSlON We have demonstrated that it is possible to generate molecular ion images with high spatial resolution from simple model systems. Such a capability should be extendable to biological tissue. One major stumbling block to significant progress in molecular ion imaging of tissue is the destructive nature of ion bombardment which limits the number of intact analyte molecules which are sampled and thus available to form an image. A unique feature of ion bombardment of bulk organic compounds, analyzed under "dynamic" or 'high dose" SIMS conditions, is the presence of intact parent molecular ion signals a t high primary ion doses, a result of the competition between the creation of primary beam damage and its removal by sputtering. For some of the small (
