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Organic Ion Imaging Using Tandem Mass Spectrometry P.J. Todd,' R. T. Short, C. C. Grimm, a n d W.M.Holland Analytical Chemistry Division, Oak Ridge National Laboratory, P. 0. Box 2008, Oak Ridge, Tennessee 37831 -6365
S . P. Markey National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892
A trlplequadrupole mass spectrometer has been Iflerfaced wtth a wlde-angle secondary Ionmicroprobe. The comblnatlon prmtts acquknion of data necessary to determine the dbtrlbutlon of targeted organic analytes even in the presence of ov.rwh.hrlngirobarlc Interference. Micrographs generated from secondary ion intensity alone are compared to those gonerated Wng secondary Ionization wtth tandem mass spoctromotry (MWMS), both for image referenceand to show the Improvement In Image quality that can be attained when MS/MS k employed. Inhomogeneousmlxtures of glycerol, KCI, and asparagineon l-cmdlameter aluminumtargets were used to demonstrate the Instrument's sekctlvity. Secondary ions generated from samples of thk system Include iwbarlc rssCI+ hpianted from the prhnary ion beam, the 41K+-glyceroi adduct, and protonated asparagine.
INTRODUCTION For many years secondary ion mass spectrometry (SIMS) has been used with microprobes to produce elemental maps (ion micrographs) from inorganic samples.' With the microprobe, a focused primary ion beam of some kiloelectronvolt energy is rastered across the sample. Images are produced that represent the intensity of mass-selected secondary ions emitted as a function of position of the primary ion beam. The significance of the images depends on whether selected ions are representative of one specific element or compound and whether or not the intensity of those ions bears some relationship to the relative concentration of the targeted analyte. When analytes are mixed with other compounds or elements that yield secondary ions of the same mass, the value of the image is compromised. For example, organic compounds often yield both intense structurally characteristic secondary ions, and abundant nonspecific secondary Organic secondary ion images reported to date have been obtained in very special or limited circumstances, such as from grids,4*5chromatographicmaterial: and glycerol.' While these images of organic substrates do not yield useful analytical information, the cited reports represent important milestones in the development of organic ion imaging. The purpose of this paper is to show that tandem mass spectrometry (MSIMS)can be used in combinationwith SIMS
* Address correspondence to Peter J. Todd,P.O. Box 2008, Bldg. 5510, MS-6365, Oak Ridge National Laboratory,Oak Ridge, Tennessee 378316365. (1) Benninghoven, A.; Rudenauer, F. G.;Werner, H. W. Secondary i o n Mass Spectrometry; Wiley: New York, 1987; pp 912-937. (2) Field, F. H. J. Phys. Chem. 1982, 86, 5115. (3) Cole, R. B.; Guenat, C.; Hass, J. R.; Linton, R. W. Anal. Chem. 1987,59, 1930. (4) Gillen, G. L.; Simons, D. S.; Williams, P. Anal. Chem. 1990, 62, 2122.
(5)Cole, R. B.; Guenat, C.; Hass, J. R.; Linton, R. W. Anal. Chem. 1987,59, 1930. (6) DiDonato, G. G.; Busch, K. L. Anal. Chem. 1987,59, 3231. (7) Grimm,C. C.;Short, R. T.;Todd,P. J. J. A m . SOC.Mass Spectrom. 1991, 2, 362.
0003-2700/92/0384-187 1$03.00/0
to produce meaningful images representative of specific organic analyte distributions. Tandem mass spectrometry provides a dimension of secondary ion analysis which may be more discriminating than increased mass resolution. The utility of MSIMS is widely recognized in many different contexts.8~9We use the intensity of unique ions in the MSI MS spectra of respective isobaric organic secondary ions to determine the spatial distribution of targeted analytes. In short, we use MSIMS to reduce isobaric chemical noise and, from the intensity of the refined signal, produce images. In a previous paper? we described a wide-angle secondary ion microprobe source and data system and showed that, with a single-quadrupole mass analyzer, secondary ions from a large (1-cm-diameter) sample could be transmitted with efficiency greater than 50%. The mass-resolved secondary ions can be used to produce an ion image. With the system, large samples can be viewed in entirety, and distributions of very dilute analytes can be measured. These features are likely to be important for analysis of biological tissue samples of a size comparable to those analyzed by autoradiography.10 Analysis of large biological samples is the raison d'etre for this instrument development project. In this paper, we describe the addition of second and third quadrupoles to the instrument. The QQQ instrument configuration is now well accepted for organic compound characterization.llJ2 In light of this, replacing a single quadrupole with a triple quadrupole may seem a trivial enterprise. However,optimizing the system for high transmission by using advanced ion optical design, verifying the high-transmission characteristics, and validating the capability of MSIMS for compositional mapping are not trivial. Furthermore, obtaining images based on MSIMS has presented new challenges with regard to the formidable volume of data required to produce images. For example,each MSIMS image representa about 1 order of magnitude more data (160 OOO bytes) than is required to print all the characters of this paper. The combination of secondary ion mass spectromety (SIMS) with tandem mass spectrometry (MSIMS) presents a new challenge for nomenclature.13J4 We refer to the instrument as a secondary ion mass spectrometrylmass spectrometry system or SIMSIMS, consistent with conventional terminology. All ions created as a result of primary ion impact with the sample are denoted as secondary ions. When an ionic species is selected for collision and fragmentation, the ionic species is referred to as either a secondary ion or as (8) McLafferty, F. W., Ed. Tandem Mass Spectrometry (MsIMS); Wiley: New York, 1983. (9) Busch, K.; Glish, G.L.; McLuckey, S. Mass SpectrometrylMass Spectrometry; VCH Publishers: Deerfield, FL, 1988. (10) Herkenham, M.; Little, M. D.; Bankiewicz, K.; Yang, S.-c.;Markey, s. P.; Johannessen, J. N. Neuroscience 1991, 40,133. (11) McGilvery, D. C.; Morrision, J. D. i n t . J. Mass Spectrom. i o n Phys. 1978, 28, 81. (12) Yost, R.; Enke, C. G. Anal. Chem. 1979, 51, 1251A. (13) Price, P. J. A m . SOC.Mass Spectrom. 1991, 2, 336. (14) Glish, G. L. J. Am. SOC.Mass Spectrom. 1991, 2, 349.
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a progenitor, consistent with IUPAC definiti011s.l~ T h e singular term descendent is used to represent ionic products of a common structure produced by collision and fragmentation of progenitor ions, rather than the recommended plural term13 progeny. While the term descendent ion is not specifically approved by IUPAC, like progenitor, descendent is a singular noun, is grammatically correct, and maintains t h e connotation of familial relationship between t h e respective ion structures.
EXPERIMENTAL SECTION Instrumentation. As described earlier: the microprobe consisted of a Cs+primary ion gun, objective with raster capability, wide-angle secondary ion source, quadrupole mass filter, and continuous dynode detector. This system was modified by addition of second and third 5/s-in. (1.585-cm) quadrupole assemblies (Extrel) to produce an MS/MS instrument of QQQ configuration. As before, control and data acquisition are performed by an 80286-based PC, using software developed a t ORNL. Secondary ion images reported here are based on the intensity of mass-resolved secondary ions. SIMS/MS images are created from the measured intensity of selected descendent ions produced by collisional excitation and dissociation from massresolved secondary ions. The original continuous dynode electron multiplier was replaced with a postanalysis acceleration detector (PAD) (Phrasor Scientific, Duarte, CA). A gas inlet was attached to the vacuum housing of the second quadrupole to allow introduction of collision gas. One end of polyethylene tubing is attached to the quadrupole housing via O-ring fitting (Cajon Ultratorr) and to the internal quadrupole mount. The other end of the tubing is attached to a manifold via a needle valve. The manifold includes a regulated source of inert gas and a valved roughing pumping system. Baffles are located at either end of the second quadrupole, between the quadrupole housing and the vacuum housing. These baffles, reduce the pumping load on the two turbomolecular pumps located below the secondary ion source and beside the third quadrupole. The arrangement allows a reasonably high collision gas pressure in the second quadrupole. We found that the baffles significantly reduce the likelihood of arcing in the detector during collisioninduced MS/MS experiments. In an earlier report, we demonstrated that the ion beam exiting a quadrupole mass filter was largely a function of the beam parameters at the entrance to the quadrupole.16 Our concern at that time was to maintain as small an ion beam diameter as possible so that the ion beam could be focused by electrostatic lenses subsequent to the quadrupole. Electrostatic lenses suffer large spherical aberrations, and we had planned to focus the beam onto a solid-state surface for performing collisional excitation of selected ions.17-lQ We found that regardless of the conditions employed, we were unable to measure descendent ion efficiencies greater than 1 % using a variety of metal and semiconductor surfaces.20 Consequently, the use of solid targets for collisional excitation was abandoned. Maintaining a small ion beam diameter a t the exit of the first quadrupole is also important for high ion transmission through triple-quadrupole MS/MS systems. Although rf quadrupoles do not have first-order focusing properties, transmission losses of ions between quadrupoles are believed t o arise from fringe field effects.21 The radial (perpendicular to the mean ion axis) field along the central axis of a quadrupole mass filter is zero and (15) Irving, H. M. N. H.; Freiser, H.; West, T. S. Compendium of Analytical Nomenclature, Definitive Rules; Pergamon: Oxford, U . K . , 1978. (16)Short, R.T.; Grim, C. C.; Todd, P. J. J.Am. SOC.Mass Spectrom. 1991,2, 226. (17)Bier, M.E.; Amy, J. W.; Cooks, R. G.; Syka, J. E. P.; Ceja, P.; Stafford, G. Int. J. Mass Spectrom. Ion Processes 1987,77, 31. (18)Aberth, W. Anal. Chem. 1990,62,609. (19)Williams, E. R.;Henry, K. D.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. F. J. Am. SOC.Mass Spectrom. 1990,1 , 413. (20)Short, R. T.;Todd, P. J.; Grirnm, C. C. Proc. Ann. Conf. Mass Spectrom. Allied Topics, 39th 1991,825. (21)Dawson, P.H.In Quadrupole Mass Spectrometry; Dawson, P. H., Ed.; Elsevier: Amsterdam, 1976;p 31.
Table I. Instrumental Parameters and Sampling Conditions parameter value primary ion species cs+ 6 keV primary ion kinetic energy 13 nA primary ion current 100 pm primary ion spot diameter primary ion current density 130 pA/cm2 1.2 x 1013 ions/cm2 primary ion dose per image 1cmXlcm raster size secondary ion energy 10 eV collision energy 40 eV collision pressure Torr PAD dynode potential -6000 v -1200 v multiplier potential 50 kHz data acquisition rate 128 data points per pixel 100pm X 100pm pixel size 4 x 109 A-' gain factor nearly zero near the axis. Fringing fields a t the entrance and exit of quadrupoles similarly vanish near the mean ion axis. When a small beam diameter is maintained at the entrance of the first quadrupole, a small beam enters the second quadrupole, etc. and a high ion transmission should be attained. T o test this argument, the secondary ion source was modified to generate ions created by electron impact ionization of acetone. SIMS is inappropriate for measurement of transmission because each sample is an integral part of the ion optics of the system; replacing samples can cause a greater change in detected ion current than changing components, and secondary ion intensity Furthermore, the kinetic energy disis time-dependent.2*3~22-24 tribution of polyatomic secondary ions1 is of the order of a few electronvolts, similar to the kinetic energy distribution of ion beams formed by electron impact ionization of molecules. Atomic secondary ions have a much broader kinetic energy distribution than do polyatomic secondary ions; ions formed by electron impact ionization cannot be used as a legitimate substitute for atomic secondary ions. Under various electron-impact ion source conditions, ion current for a constant pressure of acetone was measured after transmission through the ion source and one quadrupole. The transmission of this system is known to be near 100% in the rf-only mode and 50% in the mass-selective mode.' The second and third quadrupoles were installed, and ion current was measured again with the second and third quadrupoles in the rf-only mode. Detected intensity was identical within experimental error to that measured after transmission through one quadrupole. When the first and third quadrupoles were operated each with unit mass resolution, a transmission of 25% was measured. As is the general case for triple-quadrupole mass spectrometers,the total current of detected descendent ions under collision-MS/MS conditions was found to equal the intensity of progenitor ions lost to collision and fragmentation. Table I lists the operational parameters pertinent to the generation of ion images for this paper. The intensities of all images and Cartesian plots have been normalized to a common gain factor, with electronic baseline subtracted. Division of the indicated intensity by the gain factor will yield the current in amperes a t the output of the electron multiplier detector. It is possible to compare the intensities among images on a common basis. Two points, however, must be taken into account when intensities of descendant ion images are compared to those of secondary ions. First, absolute secondary ion (and therefore descendant ion) intensities vary with time, because of evaporation and damage from the primary ion beam. Typically, analyte secondary ion emission from glycerol decreases with increased exposure to the primary ion beam. Secondly, operating quadrupole 3 in the mass-selective mode results in a 50% decrease in overall transmission of secondary ions to the detector. Conse(22)Todd, P. J.; Groenewold, G. J. Anal. Chem. 1986,58, 895. (23)Gale, P. J.; Bentz, B. L.; Chait, B. T.; Field, F. H.; Cotter, R. J. Anal. Chem. 1986,58,1070. (24)Wong, S.S.;Rollgen, F. W. Nucl. Instrum. Methods 1986, €314, 436.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992
quently, even with 100%conversion efficiency of secondary ions to descendant ions, a descendant ion image has at most, an intensity half that of the secondary ion image. Samples. Glycerol (Aldrich Gold Label) was obtained commercially, degassed as described previou~ly,~~ and maintained under low vacuum (ca. Torr). Asparagine hydrate (Sigma) and reagent grade potassium chloride (Baker) were purchased commercially and used without further purification. Solutions of KCl were prepared by dissolving KCl in glycerol. Solutions of asparagine in glycerol were prepared using glycerol which had been acidified with 3 N HC1. After preparation, solutions were again degassed and maintained under vacuum. The sample introduction system of the mass spectrometer allows indexed loading of up to four samples of 1-cm diameter simultaneously. While the images described below are from a single loading, similar results were obtained with other sample loadings. With the droplet method used, reproducing identical images from different samples is virtually impossible, mainly because each droplet has a slightly different shape. Subject to this limitation, the results reported here have been reproduced. Image quality was checked and calibrated using grids,as described earlier, and samples were viewed continuously using a television camera and monitor. Aluminum foil was placed over a copper sample target into which either two holes of 4-mmdiameter or one hole of 9.5-mm diameter had been drilled. Slight depressions (about 1mm deep) were made in the foil at the location for each sample, to keep the liquid samplesfrom flowing. Liquid sampleswere placed in these depressions, so that the menisci were approximately at the same height as the plane of the target because sample height and topography can influence the transmission of secondary ions. Single crystals of asparagine hydrate when used were loaded from a sample of stock with tweezers.
RESULTS AND DISCUSSION There are two requirements necessary to obtain the distribution of a targeted compound in a given sample using SIMSIMS. First is that the compound must exhibit a characteristic secondary ion. Whether or not this criterion can be met can be determined from a SIMS or FAB spectrum of the isolated compound. Second, there must exist a t least one descendent ion of reasonable intensity in the MS/MS spectrum of the selected secondary ion that is unique to the progenitor. This condition depends upon the matrix and other analytes present in the sample. While "reasonable" can be quantified for inorganic secondary ion micrographs by a specified number of counts per pixel, the same is not necessarily true for analytes in a complex mixture such as a biological tissue sample. A series of MSIMS spectra are required to verify the criterion of reasonable signal, because there may be interference even in the descendent ion signal. Suitable linear combinations of descendent ion intensities characteristic of both target and matrix ions could, in principle, be used to mitigate the second requirement. However, a %ormal" MSIMS image for us consists of 40 000 picture elements or pixels. The algebraic manipulation and data acquisition necessary to perform data reduction on 40 000 MSIMS spectra is presently beyond our capabilities. Results from various mixtures of glycerol, KC1, and asparagine demonstrate the essential features of SIMSIMS. It is anticipated that the same procedures can be applied to imaging other organic samples, although the nature of each analyte and matrix may require special handling and preparation procedures. For example, with biological samples of 1-2 cm in diameter, we have found sample charging to be a significant problem in that it causes image distortion. It is always necessary to validate images generated by any technique. Direct visual images are not reliable for the samples employed here because the solutions used are equally transparent to visible light and they tend to flow. However, the fidelity of secondary ion images generated by the mi-
R. A.
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I I t Y . . ,
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miz Flgure 1. Secondary ion mass spectra of glycerol solutionscontaining (a) KCI and (b) asparagine. The reference of the (linear) relative abundance scale Is defined by the most intense peak In the mass spectrum as 100.
croprobe has been verified by using metal grids of known dimensions.' The validity of secondary ion images produced with this instrument can thus be used to validate SIMSIMS images if they are obtained using identical primary and secondary source conditions. It is only necessary that the reference ions used togenerate reference secondary ion images be free from interference. Figure 1 shows mass spectra of glycerol solutions of (a) KCl and (b) asparagine. Both spectra show a significant secondary ion at rnlz 133. For Figure la, rnlz 133corresponds to the adduct of glycerol and the minor isotope of the potassium ion, 41K+ (41K/39K= 0.0739). By inspection of the m/z 131 and 133 intensity ratios in Figure 1, it is clear that a contribution to the intensity at rnlz 133 comes from other sources, mainly 133Cs+,but also from random fragmentation of various glycerol ions. There is, as well, a contribution to the rnlz 133intensity from the39K+-glycero1-180 adduct. For Figure lb, rnlz 133is a major ion, especially in relation to rnlz 131, and corresponds to protonated asparagine. Figure 2 shows the rnlz 133 MSIMS spectra of (a) the secondary K+-glycerol adduct and (b) secondary protonated asparagine. The former shows a predominant descendent ion a rnlz 41, an expected result, consistent with the MSIMS spectrum of the secondary 39K+-glyceroladduct ion at rnlz 131. The descendent ion observed at mlz 39 arises from the 39K+-glycerol-180 contribution to rnlz 133. The ratio of the descendent ion intensities, [mlz 391/[mlz 411, is consistent with the relative contributions of '80 and 41K to rnlz 133 on the basis of the known natural isotope ratios of oxygen and potassium, and the respective number of each atom in the K+-glycerol adduct. The MSIMS spectrum of protonated asparagine is far more complex, with the most prominent descendent peak in the spectrum at mlz 74. This ion corresponds to MH+ - NHzC(0)CH3,i.e., loss from the protonated molecule of the R group that defines asparagine. The mlz 74 ion is reasonably abundant, clearly characteristic of protonated asparagine, and does not suffer interference from descendent ions of the 4*K+-glyceroladduct ion. By use of descendent rnlz 74 as a representative of asparagine and descendent mlz 41 as a
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(a) ASPARAGINE
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mi.? 133 Secondary
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mlz Flpure 2. MSlMS spectra of mlz 133 secondaty bns from (a)glyceroiic KCI and (b) glycarollc asparagine solutions. The reference of the (Ilnear)relative abundance scale is defined by the precursor peak
intenskj as
0
100.
characteristicof the K+-glyceroladduct, the second criterion necessary to use MS/MS to map the distributions of both species in a heterogeneous sample can be met. Single drops from each of the glycerol solutions of KCI and asparagine were deposited on the sample target as shown schematicallyin Figure 3a. While recognizing that secondary ion imaging,with or without MSIMS, is a form of semiquantitatiue analysis, we do not report on quantitative aspeds here. Quantitative analysis by SIMS, whether inorganic or organic, is a complex subject, mainly because of ion optical and matrix effects. With inorganic samples, for example, use of internal standards is required for quantitation. With organicsamplw,suchas thesolutionsinvestigatedhere,effects of samplelmatrix evaporation, sputtering, and irreversible chemical reactionwouldneed to be determined tocharacterize the quantitative capabilities of SIMS/MS. Except where indicated in the text, we have assumed that a greater intensity of diagnostic secondary or descendent ion corresponds to a greater concentration of targeted analyte. Studies of secondary ion intensity as a function of concentration*m indicate this is a safe assumption provided that the matrix is not changed. Because secondary ion yields differ among compounds, comparisons of concentrations of different compounds based on the intensity of their respective secondary ions is not valid. Figure 3b shows the image generated from the intensity of secondary ion m/z 133 under static SIMS conditions, Le., where the primary ion dose is below lOI4 ions/cm2. This image indicatessecondary emission of mlz 133only from thesample and mainly from the region occupied by asparagine solution. Figure 3c shows a similar image, but taken after the primary dose has exceeded l O I 4 ions/cm2,and the sample moved to center of the field of view. In contrast to the image generated under static SIMS conditbns, the most notable feature in (25) Todd. P. J. J. Am. Soe. M u s Speetrom. 1991, 2, 33.
(26) Barber,M.;Bordoli, R.S.;Elliott, G. J.; Sedgwiek,R.D.; Tyler, A. N.J. Chem. Soc., Faraday ‘ h w . 1 1983, 79,1249. (27) Leibrnan,C.P.; Todd, P. J.; Mamantov, G. Org. Mass Speetrom. 1988, 23,634. (28) Brenna, J. T.: Morrison, G. H.And. Chem. 1986,58,1675-1680.
miz 133 Secondarj
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0 Reference schematic (a) lor SIMSIMS Images shown in Figures 3-7 and secondary mlz 133 Image (b). taken under static SIMS conditions. The white distance marker in the lower right corresponds to a distance of 1 mm. The gray scale Indicated to ttm mht 01 (b) is used throughout the paper. wlth white corresponding to ttm most Intense current measured at some pixel in me image and black corresponding to the electronic baseline. The number at the top of each gray scale corresponds to the electron multiplier output current in amps, multiplied by same amplifier gain factor for all images. The image shown in (c)is the mlz 133 image taken from the sample after primary ion erpcwre above the dose limn (ca. lor static SIMS). Inadditbntosewndaryemlssbnofimplanted ‘33Cs+fromthealumlnum target, secondary ion emission from the glycerol solution of asparagine is reduced in (c) compared to (b). Flgure 3.
Figure 3c is predominance of mlz 133 surrounding the droplets. Because glycerol solutions do not demonstrata appreciable changes in their mms spectra with primary ion dose, emission of implantedCs+fromthealuminum foiltarget appears to be a good measure of when the static SIMS dose limit is reached. Matrix effects such as differences in emissivity are well documented in the literature of inorganic
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m/z 133 + 41 Descendant
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Flgure 4. Reference Secondary ion image of the sample shown In Flgure 3. taken aner exposure near the static SIMS limit. using the mlz 131 ion, which arises mainly from the 39K+-giyceroladduct. The image fidelityof the instrument to produce secondary ion images was established uslng grMs.’ Because mlz 131 is a secondary ion and does not suffer Isobaric interference. this image provaes a reference for the dlstrlbutlon of emission of the “K+-gtycerol adduct and, after MSIMS, Its descendent ions. For example, by comparison to Figure 3c. mlz 131 is not emitted from the aluminum target, so that the backgroundvisible in Figure3cis notapparent,nor isthereappreciable emission characteristic from the region of the sample where asparagine solutlon was deposited. SIMS.1s,29 Furthermore, calculations used for ion implantation show that Cs+ ions are 100% implanted in the aluminum target, with a most probable depth of 35 A, under the conditions employed here; i.e. no primary Cs+ are hackscattered.30 They can only he emitted as secondary ions. Figure4is thesecondaryionmicrographofthesamesample used for Figure 3 but taken using the secondary m / z 131, which arises almost exclusively from attachment of 39K+ to glycerol. Unlike m/z 133, mlz 131 doesnotsuffer theisoharic interference from protonated asparagine and Cs+. Nor is there significant emission of mlz 131 from the aluminum target, consistent with the view that the preponderance of secondary m/r 133 from the target is Cs+. Thus, Figure 4 reflects the distribution of glycerolic KCI, and can he used as a reference image for comparison with SIMSiMS data. Note that emission from the spot occupied by the KCI solution is not homogeneous. Subsequent investigation with other glycerol solutions indicates that some droplets reproducibly show homoeeneous distribution and some remoduciblv show heterogeneous emission. For SIMS/MS images to he valid, the distribution of‘LK+glycerol secondaries determined from descendent rlKc intensity must matchthedistributiondetermineddirectlyfrom the rn z 131 secondary ions. Sincetheability of the instrument M generate valid secondary ion images has already been established,? regardless of whether the reference displays heterogeneous emission or not, all that matters is that the descendent ion image match the secondary ion image. Figure 5 is the SIMS MS micrograph taken using them r 41 descendent from m i z 133. This figure shows nearly the same distribution as the micrograph shown in Figure 4, i.e., an expected result. The fact that the descendent m l r 41 imageand thesecondarym 2131 imapecorrespondvalidates use of US MS M create images. While it may seem obvious chat the SIMS MS image should mimic the SIMS image, we (29)Patkin. A. J.: Chandra, A.; Morrison. C..H.Anal. Chem. 1982.54. 2507. (30)Zicgler. J. F.: Rimsrk. J. P.; Litrmark, U The Slopping Pouer and Rang? o t l o m tn Solido: Pergammon: New York. 1985.
Flgure 5. SIMSiMS image of the K+-glycerol adduct based on the intensity of the descendent ion mlz 41. Correlation wkh Figure 4 Is crucial. because it establishesthe fidelityof Secondary iondescendent ian images. The vertical cursor line through the image is used fw referencein a Cartesian plot of descendent ion intensity along the line. have ohserved that images can he misleading as well as informative. Consequently, it is our view that the greater the number of independent ways in which a new type of imaging can be verified, the less likely will he the misinterpretation or overinterpretation of the image. The principal advantage of using the secondary m l t 131 distribution for reference is that the samples need not he particularly welldefined, e.g. as grids, or fixed. For example, migration of KCI is evident from both the secondary m/z 131 intensity and descendent 41K+ intensity distributions. Comparison of Figures 4 and 5 can he used to obtain a semiquantitative estimate of relative MS/MS efficiencywhen the instrument is used for imaging. The maximum intensity of m/z 131 in Figure 4 is 12000. This intensity arises exclusively from the 39K+-glycerol adduct. The intensity of m/z 133 due to the “K+-glycerol adduct is approximately 7% (ratio of “K/39K) of this, or 840. In the MSIMS mode, we have a50% reductionin transmission fromoperatingquadrupole 3 in the mass-selective mode, so this intensity is reduced to 420. The maximum descendant ion intensity of m/z 41 in Figure 5 is 180, roughly 40% of the estimated “available” intensity of “K+-glycerol secondaries. This number is consistent with Figure 2a, where the m / z 41 peak is approximately 30% of the base m/z 133 peak. We attribute our relatively low MSIMS efficiency to a broken gas capillary line, which we discovered after submission of the original manuscript. Despite this instrumental flaw, the efficiency of the instrument is sufficient for purposes of demonstration. Figure 6 shows the SIMS/MS image based on the intensity of the m/z 74 descendent from secondary mlz 133. The mlz 74 descendent ion is diagnostic for asparagine. This image corresponds to the location and shape of the droplet of glycerolic asparagine as it was loaded onto the sample. I t is noteworthy that there is apparently no background signal in this image. The reason for this is that m / z 74 is an even electron ion containing one nitrogen. In contrast, the preponderance of other secondary ions a t mlz 133 in the spectrum of the asparagine solution contains non nitrogen atoms. Because descendent ions are mainly even electron ions, the MS/MS spectrum of chemical noise that arises a t m/z 133in themassspectrumoftheasparaginesolutionshould have almost no descendent ions with even mass-to-charge ratio. As a result, the SIMS/MS image of mlz 74 displays virtually no chemical background. Figure7 showsthesummedimageoftheMS1MStransitiona
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r n h 133 + 74 Descendant
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0 Figure E. SIMSIMS image of protonated asparagine based on the intensity of the mlz 74 descendent. Note the nearly complete sumression of ion intensitv from the “K+-ai~ceroladduct. Figures 5 and 6 are complementary images of t h e m/z 133 image 2 the sample.
S u m Descendant m h 41 a n d m h 74
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PIXEL # Flgure 8. PTofile of asparagine (a) and glycerol adduct (b) along the vertical cursor indicated In Figures 5 and 6 . The wldm of the cursor Is the width of one pixel (ca.100 pm).
rn/z 133 Secondary
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0 0 Figure 7. Summed absolute intensity distributions from Figures 5 and 6. This image compares favorably with that shown in Figure 3b. Chemical noise from mlz 133 emitted from the aluminum has been
completely suppressed. The roughly equal maximum intensity of both descendent ion species is purely coincidental.
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m/z 133 m/z 74 and m/z 133 m/z 41. The summing procedureconsists of addingthe respective absolute intensities a t each pixel and then normalizing the summed intensities. Figure 7 can be compared to Figure 3c. The main difference between the two images is that, in Figure 3c, the image is dominated hymiz 133fromCs+implantedintothealuminum target and subsequently reemitted, while in Figilre 7, intensities from descendents of molecular ion m/z 133 dominate the image. Interference from isobaric inorganic secondary ions is eliminated by use of MS/MS. The same information displayed in an image can also he viewed a~ a series of ion intensity profiles. Figure 8 shows theprofilesofdescendent (a)m/z74and(h)m/z41intensitiea, respectively, along the vertical line shown in Figure 7. The purposeof the plotsis toprovidea morequantitativedepiction of the data shown in Figures 3-7 and to convey information not obvious in the image. For example, while only representing0.5% ofthedatadisplayed inFigureI,Figure8shows that K+ tends to diffuse to a greater extent than asparagine over the time frame of the experiment. Figures 3-8 provide substantial evidence of the validity andutilityofSIMS/MSfor imagingorganicsamples. Because
Figure 9. Secondary ion image based on mlr 133 lntenslly from a sample of KClIgiycerol solution onto which was placed a nyslal of asparagine.
the droplets of glycerolic solutions were at different positions within the probe’s field of view, comparison of the secondary ion and descendent ion images is straightforward. This demonstration is entirely differnt form the projected application of secondary ion descendent imaging, where the goal is todetermine thedistrihution oftargeted compoundswithin a common matrix. Within the general context of the word “matrix”,’5 a glycerolic solution of KCI is a different matrix than a glycerolic solution of asparagine. The fact that the two samples used for Figures 3-7 were only incidentally in contactmeansthatitremainstohedemonstrated that, within a common matrix, the SIMS/MS imaging approach is valid. In order to demonstrate imaging in a slightly different matrix, a crystal of asparagine was placed in a droplet of glycerolic KCI which had been deposited on the target. The secondary m/z 133 image of this sample is shown in Figure 9. The approximately round shape of the image is consistent with the actual shape of the sample. The enhanced emission from the periphery gives the image a “doughnut” shape, and there is what appears to he a further enhancement a t the right of the image. Heterogeneous emission of characteristic secondary ions from presumably homogeneous droplets is reproducible. The heterogeneous emission evident in Figure 9 appears to he a surface matrix effect peculiar to liquid samples. Matrix effects can alter the secondary emission of both characteristic and noncharacteristic ions beyond any
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mlz 41 Descendant 660
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Flgure 10. SIMSIMS image of the sample shown in Figure 9 based on the m1.z 133 41 transition. mid
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0 11. SlMS MS~mageoIlnesampesno~n nfigures9and 10. * 74 lransl on characlewlc of protonated asparagine ouring sample preparabon
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effectsattributahle toanalytpconcentration. For the purpose ofthiswork,whichis todemonstrate theutilityofSIMS MS for organic ion imaging, we simply note that matrix effects need to be considered and have been addressed in the literature of inorganic S1MS.I 21.2q Figure 10 shows them z 133 descendent ion rn z 4 1 image ofthesample,showinganintensedistributionweightedmore heavily toward the right periphery of the droplet image. A similar image (not shown) was generated using the m z 131 descendent m z 39 intensity, verifying the result shown in Figure 10. Figure 11 shows the m z 133 descendent ion m z 74 distribution from the same sample. This distribution shows enhanced emission at the location that corresponds to the central locationoftheasparaginecrystal.Minor localmaxima in the asparagine distribution are also evident, consistent with the method of depositing the asparagine crystal in the matrix, as some asparagine powder invariably falls onto the target. The also appears to be a weak signal from the periphery of thesample. This may bedueeither to migration of asparagine or some sort of chemical noise. The imponant aspect of SIMS MS is not that MS MS eliminates chemical noise but rather that MS MSsubatantiollj reduceschemical noise: this feature is clearly evident in Figure 11. Figures 9-11 demonstrate the capability of SIMS MS for imaging, which in the context of organic analysis means
m/z 133-74
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Figure 12. Profilesof selectedion intensnbsalongthecursor indicated In Figures 9-1 1. The profile of mlz 133 shown in (a)and the profile of mlz 131 shown in (b) indicate that most of the secondary mlz 133 image is from the”K+-giyceroladduct. The profileshownin (c)reflects the ratio of descendent mlr 74 ion intensity to the intensity of mlz 133 Shown in (a). determining the spatial relationship between compounds in a sample. Figure 12, however, provides a more dramatic demonstration of the power that MSIMS adds to imaging. Figure 12a shows the distribution of mlz 133 along the horizontal cursor mark shown in Figure 9. It reflects contributions from Cs+, “K+-glycerol, and protonated asparagine. Figure 12b shows the same distribution created from mlz 131 from the same sample, reflecting almost exclusively the 39K-glycerol adduct secondary ion. Figure 12c is a plot of the descendent ion m/z 14 current as a fraction of total miz 133 along the same cursor coordinates as in (a) and (b). The protonated asparagine signal-to-noise (SIN) ratio in Figure E a is, at best, about 0.3. A review of Figure 9 shows that a SIN of 0.3 corresponds to being invisible. In Figure 12c, the protonated asparagine SIN is at least 100. With respect to Figure 11,the result is that the distribution of protonated asparagine dominates the image.
CONCLUSIONS We have demonstrated the capability to obtain organic ion imagesusing a technique we refer to as SIMSIMS. Relatively simple samples have been used to test the instrumentation and verify that the instrument produces valid images. From these experiments, we have established a set of elementary criteria for the application of SIMS/MS. This set includes the requirements that the targeted analyte yield a characteristic secondary ion of reasonable intensity, that some ion descendent of the characteristic secondaryion exist, and that the descendent must be unique. Further criteria may be applicabletomorecomplexsamples,and theremay be criteria peculiar to particular analytical questionsnot addressed here. Hence the term ‘reasonable intensity” is intentionally vague. While the results presented here represent the simplest application oftandem massspectrometry tomeaswe targeted analyte distribution, an intended application of this instrument will be for analysis of biological tissue samples. It is our belief that the unique ability of tandem mass spectrometry
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for identification of related compounds,when combined with secondary ion microprobe capabilities, will allow determination of the distribution and identity of various metabolites in tissue samples. Such samples are likely to be 1-2 cm in diameter, which is why such a large field of view has been a design criterion of this instrument. Except for the limitation of the collision gas introduction system, secondary ion transmission of the instrument has been optimized to mitigate problems with the decreased secondary ion yield expected from biological matrices as compared to glycerol. The fact that SIMS/MS images could be obtained using a collision system with less than optimal performance attests to the sensitivity of the technique. Development of this instrumentation is one step toward organic ion imaging of biological samples, the success of which project will also depend on developmentof methods for mitigating sample charging and enhancing the emission of secondary ions characteristic of the targeted analytes, problems that have not been solved as of yet. In any case, the instrument and method described here should be valuable for organic surface analysis, with a unique capability to characterize the spatial distribution of
organic materials, including liquids.
ACKNOWLEDGMENT We are grateful to J. T. B r e w and W. H. Christie for helpful discussions and encouragement, to Carla Brown for her help in developing sample preparation methods, and to C. P. Leibman and L. K. Bertram for initial installation of the instrument. The instrument is presently on loan from the Biomedical and Instrumentation Engineering Program, National Center for Research Resources, NIH. This work was supported by the National Institutes of Health under Grant No. R01 GM41617 and by the U. S. Department of Energy, Office of Basic Energy Sciences, under Contract 840R21400with Martin Marietta Energy Systems, Inc. C.C.G. gratefully acknowledges receipt of a postdoctoral fellowship sponsored by Oak Ridge Associated Universities. RECEIVED for review January 9, 1992. Accepted May 14, 1992.
