Depth Profiling Brain Tissue Sections with a 40 ... - ACS Publications

Feb 16, 2008 - Emrys A. Jones ,* Nicholas P. Lockyer , and John C. Vickerman. Surface Analysis Research Centre, Manchester Interdisciplinary Biocentre...
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Anal. Chem. 2008, 80, 2125-2132

Depth Profiling Brain Tissue Sections with a 40 keV C60+ Primary Ion Beam Emrys A. Jones,* Nicholas P. Lockyer, and John C. Vickerman

Surface Analysis Research Centre, Manchester Interdisciplinary Biocentre, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, United Kingdom In this paper, the effect of prolonged C60+ primary ion bombardment on the chemical information available from a section of rat brain is discussed. Initial attempts demonstrate the rapid loss of molecular signal from the bombarded area with both C60+ and Au+ used as a monatomic comparison. However, the nature of this signal disappearance is shown to be different. Analysis of the C60+ data indicates a correlation between signal loss and the appearance of sodium and potassium adducts of phosphate and protein fragments; this is supported by model systems. By using an ammonium formate wash to reduce the salt levels within the tissue this effect is removed, allowing the chemistry of the tissue section to be better probed. Results collected from multiple sections suggest that at room temperature under vacuum conditions there is a migration of lipids to the surface of the tissue. Three-dimensional (3D) imaging is used to demonstrate that once these lipids are removed other species, such as proteins, are uncovered. By depth profiling the sample in a frozen state, the degree and importance of lipid migration to the observed localization of native compounds is assessed. This investigation into the behavior of biological tissue under high C60+ fluxes not only allows an evaluation of the potential accuracy of 3D SIMS mapping of important biological molecules but also demonstrates the possibility of using ion doses beyond the traditional “static limit” to provide higher secondary ion yields that could lead to greater detection limits and smaller useful lateral resolution within such analyses. Imaging mass spectrometry is a rapidly developing area of research, with the main focus of the various techniques being biological applications. Matrix-assisted laser desorption ionization (MALDI),1-3 secondary ion mass spectrometry (SIMS),4-6 and more recently desorption electrospray ionization (DESI)7 have all * To whom correspondence should be addressed. E-mail: emrys.jones@ manchester.ac.uk. (1) Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69, 4751-4760. (2) Rohner, T. C.; Staab, D.; Stoeckli, M. Mech. Ageing Dev. 2005, 126, 177185. (3) Reyzer, M. L.; Caprioli, R. M. Curr. Opin. Chem. Biol. 2007, 11, 29-35. (4) Ostrowski, S. G.; Van Bell, C. T.; Winograd, N.; Ewing, A. G. Science 2004, 305, 71-73. (5) Touboul, D.; Roy, S.; Germain, D. P.; Chaminade, P.; Brunelle, A.; Lapre´vote, O. Int. J. Mass Spectrom. 2007, 260, 158-165. (6) Sjo ¨vall, P.; Lausmaa, J.; Johansson, B.; Andersson, M. Anal. Chem. 2004, 76, 4271-4278. 10.1021/ac702127q CCC: $40.75 Published on Web 02/16/2008

© 2008 American Chemical Society

been used to various degrees to probe the chemical composition of the surfaces of tissues and cells. These three main techniques are complimentary on many fronts; SIMS, especially the dynamicSIMS (d-SIMS) approach, can offer the greatest spatial resolution with images of chromosomes being achieved8 and subcellular probing being demonstrated by various groups.9,10 The chemical information that is accessible with d-SIMS is limited to elements or small molecules (e.g., CN-). Static SIMS, the spatial resolution of which is typically an order of magnitude less, can obtain molecular signals from a range of biomolecules in the m/z 1001000 range, such as lipids, vitamins, and pharmaceuticals. MALDI imaging, which has to date made the greatest impact in biological imaging, cannot compete with SIMS for spatial resolution. Typical laser spot sizes, restricted by the diffraction limit, are in the region of 100 µm. This has been improved upon by using specialized optics11 or alternatively by decoupling the desorption from the detection with a position-sensitive detector;12 however, these approaches are as yet not fully developed or widespread, and in any case the spatial resolution relies largely upon the size of the crystals formed during matrix deposition making it a critical factor. The major advantage of MALDI is its ability to desorb molecules of much larger masses than SIMS with proteins and peptides detectable from tissue sections and cell layers. Although matrix clusters and native compounds may cause isobaric interferences in the low mass region, recent uses of the MS/MS approach have allowed MALDI imaging to be applied to themappingofpharmaceuticalcompoundswithintissuesections,13-15 expanding the applications of this technique. The third technique, DESI, is still in its infancy although it has the two major advantages of being able to be carried out under atmospheric conditions and without any sample preparation; (7) Wiseman, J. M.; Ifa, D. R.; Song, Q. Y.; Cooks, R. G. Angew. Chem., Int. Ed. 2006, 45, 7188-7192. (8) Strissel, P. L; Strick, R.; Gavrilov, K. L.; Levi-Setti, R. Appl. Surf. Sci. 2004, 231-232, 485-489. (9) Kleinfeld, A. M.; Kampf, J. P.; Lechene, C. J. Am. Soc. Mass Spectrom. 2004, 15, 1572-1580. (10) Chandra, S. Eur. J. Cell Biol. 2005, 84, 783-797. (11) Spengler, B.; Hubert, M. J. Am. Soc. Mass Spectrom. 2002, 13, 735-748. (12) Luxembourg, S. L.; Mize, T. H.; McDonnell, L. A.; Heeren, R. M. A. Anal. Chem. 2004, 76, 5339-5344. (13) Khatib-Shahidi, S.; Andersson, M.; Herman, J. L.; Gillespie, T. A.; Caprioli, R. M. Anal. Chem. 2006, 78, 6448-6456. (14) Hsieh, Y.; Casale, R.; Fukuda, E.; Chen, J. W.; Knemeyer, I.; Wingate, J.; Morrison, R.; Korfmacher, W. Rapid Commun. Mass Spectrom. 2006, 20, 965-972. (15) Stoeckli, M.; Staab, D.; Schweitzer A. Int. J. Mass Spectrom. 2007, 260, 195-202.

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though as it relies upon a stream of liquid droplets it suffers from limited spatial resolution and high sample consumption. Within static SIMS analysis the spatial resolution is partially determined by the achievable spot size of the beam, but for molecular species the useful lateral resolution is mainly constrained by the static limit that restricts the proportion of the surface monolayer analyzed to no more than 1% and by the low efficiency of formation of secondary ions. Unless a given compound is the main constituent of the surface, or has a very high ionization probability, there will be very few molecular ions produced from a given pixel, a number that diminishes to practically zero when submicrometer imaging is required. This limitation of minimum useful spatial resolution by molecular damage of the analyzed surface also has implications for analysis away from the surface. Etching and depth profiling are common in other surface techniques; an etch step may be used to clean contaminants from the sample, whereas depth profiling allows the distribution of given chemicals to be followed as a function of distance from the surface. These are well-established within the world of d-SIMS but historically impossible in static SIMS as the ion fluxes required to remove a contaminant layer, or to remove successive sample layers, would destroy the molecular information in the layers below. It is here that the polyatomic ion beams that have made such an impact into the world of SIMS are changing the paradigm. Significant mechanistic differences in the sputter yield compared to monatomic ion beams mean that the damage produced on impact is restricted to the upper monolayers of the surface. When this is coupled to the high sputter yields from the sample due to the polyatomic impact, the majority of damage may be removed during the event leaving unaffected material to be sampled by subsequent impacts. This low damage accumulation has been demonstrated in a range of organic materials by a range of cluster ions; however, the commercially available SF5+ 16 and C60+ 17 sources are those that are being applied most routinely. Many examples have demonstrated the ability of these ion beams to depth profile organic films,18-21 and as is the nature of analytical science in the current climate, medical and biological applications appear to be the ultimate goal. It is clear that although molecular depth profiling of samples such as drug delivery systems22 provide useful information, there is a large momentum toward coupling the unique “nondamaging” sputter characteristics of the polyatomic ions beams to the well-established imaging capabilities of the SIMS technique. An early instance of this is the work of Gillen et al. who demonstrated the three-dimensional (3D) imaging of pharmaceuticals within a poly(lactic) acid matrix.23 This approach has recently been applied to the more complex system of a (16) Kotter, F.; Benninghoven, A. Appl. Surf. Sci. 1998, 133, 47-57. (17) Weibel, D.; Wong, S.; Lockyer, N. P.; Blenkinsopp, P.; Hill, R.; Vickerman, J. C. Anal. Chem. 2003, 75, 1754-1764. (18) Cheng, J.; Kozole, J.; Hengstebeck, R.; Winograd, N. J. Am. Soc. Mass Spectrom. 2007, 18, 406-412. (19) Fletcher, J. S.; Conlan, X. A.; Jones, E. A.; Biddulph, G.; Lockyer, N. P.; Vickerman, J. C. Anal. Chem. 2006, 78, 1827-1831. (20) Mahoney, C. M.; Roberson, S. V.; Gillen, G. Anal. Chem. 2004, 76, 31993207. (21) Wagner, M. S.; Gillen, G. Appl. Surf. Sci. 2004, 231-232, 169-173. (22) Mahoney, C. M.; Roberson, S.; Gillen, G. Appl. Surf. Sci. 2004, 231-232, 174-178. (23) Gillen, G.; Fahey, A.; Wagner, M.; Mahoney, C. M. Appl. Surf. Sci. 2006, 252, 6537-6541.

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single cell, where lipid distributions were imaged in three dimensions.24 Many of the early investigations into the potential of polyatomic ion beams for biological tissue analysis have been carried out on rodent brain sections,6,25,26 and this has allowed a comparison to be made between ion beams in different laboratories and on different instruments. Following this trend, Debois et al. use this sample to ascertain whether 10 keV C60+ can be used to successfully depth profile tissue sections.27 Initially, it may be expected that the amount of variation in the z-axis of a tissue section measuring tens of micrometers will be minimal when the organ itself measures in the centimeters; nevertheless, this direction of study is important. By studying these sample types much can be learned about the behavior of biological samples under polyatomic bombardment, the aim being to create general rules for the SIMS analysis of biological systems. Additionally, if low levels of surface damage do result, then the static limit can be abandoned and increased spatial resolution and limits of detection become possible because of the greater number of molecules available for analysis. The work referred to above used a 10 keV C60+ beam to sputter the sample and a Bi3+ LMIG source to carry out the acquisition. The conclusion was that the experiment could not be satisfactorily conducted under these conditions. It has recently been shown that within certain sample types the sputter yield at 10 keV may not be large enough to reach a “steady state”, a concept elegantly described in the work of Cheng et al.28 It is our intention therefore to investigate the depth profiling of tissue sections using a 40 keV C60+ source,19 in an attempt to evaluate its potential for such applications and to further aid in the discussions relating to this rapidly expanding field. EXPERIMENTAL SECTION Materials and Sample Preparation. The standard materials used in these experiments were obtained from Sigma (Poole, U.K.) and analyzed without further purification unless stated. The rat brain sections were provided by GlaxoSmithKline (Harlow, U.K.). Following sacrifice of the rat the brain was removed, snap-frozen in liquid nitrogen cooled isopentane, and sectioned immediately on a cryo-microtome at a thickness of 12 µm. Following thaw mounting onto stainless steel shards the samples were stored at -80 °C until analysis. Unless stated, the samples were allowed to warm to room temperature in a desiccator for 1 h and then introduced into the TOF-SIMS instrument. Where sample washing was used, this step was introduced prior to the desiccation and consisted of two 30 s washes in a 0.15 M solution of ammonium formate, with a fresh solution used for each wash. Standard films of phospholipid were prepared by spin casting the material onto cleaned silicon substrates from chloroform solutions at a concentration of 0.01 M. Additional experiments were carried out where (24) Fletcher, J. S.; Lockyer, N. P.; Vaidyanathan, S.; Vickerman, J. C. Anal. Chem. 2007, 79, 2199-2206. (25) Touboul, D.; Kollmer, F.; Niehuis, E.; Brunelle, A.; Lapre´vote, O. J. Am. Soc. Mass Spectrom. 2005, 16, 1608-1618. (26) Jones, E. A.; Lockyer, N. P.; Vickerman, J. C. Int. J. Mass Spectrom. 2007, 260, 146-157. (27) Debois, D.; Brunelle, A.; Laprevote, O. Int. J. Mass Spectrom. 2007, 260, 115-120. (28) Cheng, J.; Wucher, A.; Winograd, N. J. Phys. Chem. B 2006, 110, 83298336.

Figure 1. Depth profiling of a section of rat brain using 40 keV C60+. The region chosen was the corpus callosum. The data demonstrate the rapid loss of characteristic organic signal (a) with m/z 44 the immonium ion of alanine, m/z 369 and 385 from cholesterol, and m/z 184 from the PC headgroup all decreasing into the noise in the first few etches. This is accompanied by an increase in the intensity of inorganic species (b) as assigned in Table 1; their dominance above m/z 50 is demonstrated along with the large contribution of Na+ and K+ in (c), and the dramatic rise in the intensity of the anions CN- and CNO- which are part of these complexes is shown in (d).

sodium chloride was added to the solution such that its concentration was 20% by weight prior to spin casting. For the bovine serum experiments, the film was produced by using the dried droplet technique from a 0.01 M aqueous solution. The desalination was carried out using C18 Millipore “Ziptips”, used as stated by the manufacturer for the cleanup of proteins. Instrumentation. All experiments were carried out on the BioTOF-SIMS instrument as described elsewhere.29 Briefly, primary ion bombardment leads to the desorption of secondary ions from the surface under analysis. These secondary ions are accelerated to a reflectron-type time-of-flight (TOF) mass analyzer by applying a 2.5. kV extraction pulse and detected using a multichannel plate detector with 21 keV post acceleration. The analysis stage of the Bio-TOF-SIMS instrument is equipped with a cold stage that, by the use of liquid nitrogen cooled nitrogen gas, was taken to and held at -120 °C. Atomic force microscopy (AFM) analysis was carried using a NanoPics 2100 (Seiko Instruments). Depth Profiling. The Bio-TOF-SIMS instrument has been set up to run automated depth-profiling experiments, the process of which has been described in a recent publication.24 In each of the depth profiles the area that is etched with the dc beam is (29) Braun, R. M.; Blenkinsopp, P.; Mullock, S. J.; Corlett, C.; Willey, K. F.; Vickerman, J. C.; Winograd, N. Rapid Commun. Mass Spectrom. 1998, 12, 1246-1252.

significantly (usually 4 times) larger than the area that is analyzed, in order to minimize any crater edge effects. In order to avoid sample charging, a low-energy electron beam (25 eV) was unblanked during the etching; the severity of the damage caused by the electrons is considered negligible with regards to the amount of material removed by the dc C60+ beam. The sample was held at ground potential during etching cycles. Imaging. The 3D imaging was carried out using the same process as described for the depth profiling; however, images of 30 shots per pixel, with 256 × 256 pixels covering a 500 µm2 area, were collected between the etch steps in place of the normal spectra. The images were processed with MATLAB (TheMathWorksInc.) and AVS Express (Advanced Visual Systems Inc.). Briefly, MATLAB routines written in-house were used to normalize each of the separate layers for a given ion or selection of ions to the total ion count of that layer. The output of this procedure was then read into the AVS Express visualization software. The images produced have been downsized by a factor of 5 in each of the three dimensions, for ease of manipulation and viewing, and plotted as isosurface plots, with the transparency level set for optimum visualization of the distribution of the ion(s) of interest. As this is a subjective procedure, the images are presented to illustrate general trends and are not used to define relative concentrations of molecules. Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

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Table 1. Proposed Structures of the Adduct Ions That Increase in Intensity under Prolonged Bombardment of Biological Tissue with C60+ proposed ion

m/z 88 104 120 141 157 197 213

2Na+

(CNO)- or Na+ K+ (CN)2K+ (CN)- or Na+ K+ (CNO)2K+ (CNO)2K+ (PO2)- or Na+ K+ (PO3)2K+ (PO3)3K+ (HPO3)2- or Na+ 2K+ (HPO4)23K+ (HPO4)2-

RESULTS AND DISCUSSION Depth Profiling Untreated Tissue. The initial attempts to demonstrate stable molecular signal from the surface of a rat brain section under large primary ion doses of C60+ are presented in Figure 1. The majority of secondary ion signals that are detected from these sample types are from the various lipids, in both positive and negative ion modes. Cholesterol, phospholipids, sphingomyelin, sulfatides, and fragments of these molecules dominate the spectra above m/z 200.30 Below this value the spectrum contains a plethora of peaks relating to organic fragments and peaks from PO4- and native salts. Proteins, which are the second most abundant group of biomolecules present in tissue, are not detectable by their molecular ion with SIMS due to their size; however, their presence is indicated by characteristic immonium ion peaks typically ranging from m/z 30 to m/z 159 in the spectrum. When the behavior of the secondary ions from these species is followed during a depth profile, as shown in Figure 1a, it is clear that there is a sudden decrease in the intensity of these peaks. The figure plots ions for cholesterol (m/z 369 and 385), a common phospholipid headgroup ion at m/z 184, and the immonium ion of alanine at m/z 44. The ability to confidently identify their presence in the spectra is lost following the first few etches of 1 × 1013 ions/cm2 each for the 40 keV analysis. Optical inspection of the crater and the absence of any chemical evidence of substrate peaks suggests that the sample has not been fully consumed; thus, the loss of secondary ion peaks must be attributed to damage or disappearance of the molecules of interest or a change in the chemistry of the surface which prevents the formation of the secondary ions. When the spectra from this end point are analyzed, the initial observation is that the spectrum is dominated by peaks from sodium and potassium cations in the positive spectra, and CN- and CNO- in the negative spectra, as is indicated in Figure 1, parts c and d; the peaks from CN- and CNO- increase significantly more than other small organic fragments such as C- and C2- as shown in Figure 1d. Additionally, in the positive ion spectrum the remainder of the peaks above m/z 50 are at m/z values that were not significant in the initial spectrum (Figure 1, parts b and c). Through the analysis of these peaks they appear to strongly match the predicted ion distribution if the main negative ions were doubly cationized with either two Na+, two K+, or one of each, as shown in Figure 1c. Structural assignments are suggested in Table 1. Previous work has been carried out on brain tissue under prolonged bombardment with various ion beams both polyatomic (30) Touboul, D.; Halgand, F.; Brunelle, A.; Kersting, R.; Tallarek, E.; Hagenhoff, B.; Lapre´vote, O. Anal. Chem. 2004, 76, 1550-1559.

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and monatomic, and work from our laboratory has compared C60+ to Au+.26 From this data it was shown that there was a rapid decrease in signal from the Au+ beam that was attributed to surface damage, in line with many other studies demonstrating the difference in damage accumulation between monatomic and polyatomic projectiles. On further analysis of this data, the decrease in the intensity of the molecular ion species is not accompanied by an increase in the “salt adduct” peaks identified from the C60+ study. The data from Au3+ demonstrates a similar outcome, the loss of peaks of interest without the appearance of new ion species within the spectrum. This suggests that the mechanism of signal loss is different under Aun+ bombardment as compared to C60+. This apparent formation (or increase in abundance) of new species under C60+ bombardment is reminiscent of the work by Van Stipdonk et al.31 who demonstrated that C60+ created a much larger proportion of the rearrangement product ion (NaF)F- from a BF4-Na+ surface than Cs+ or Csn+ primary ions. It may be the case that in the presence of native sodium and potassium ions, a C60+ impact upon the constituents of biological tissue could lead to recombination and rearrangements in the surface. This effect could be largely exclusive to polyatomic impacts due to the different physics involved with these ions when compared to monatomic ions, with greater degrees of energy transfer and subsurface collisions.32 Model Systems. In order to assess the importance of this salt effect, two model systems were created. The first was a comparison of films of the protein bovine serum albumin (BSA) on silicon, depth profiled with C60+ first as supplied, which contained a significant level of sodium ions in the positive spectrum, and then following desalination with C18-packed pipet tips. The second systems consisted of films of the much-studied phospholipid DPPC, which has been shown to provide a stable molecular ion signal under C60+ bombardment.17 Two different amounts of NaCl were added to the solution prior to film deposition, and their behavior under C60+ depth profiling was compared. Analysis of the protein film demonstrates a marked difference between the samples with and without the salt present. Within the first spectrum of the analyses, the sodium peak in the initial sample contributes 10% to the total spectrum, whereas the value is only 0.9% in the desalinated protein. This leads to a clear difference in the behavior of the two surfaces under C60+ bombardment as shown in Figure 2. The peaks from the immonium ions of the protein rapidly decrease in intensity when there is a high presence of sodium in the spectrum (Figure 2a), with adduct peaks attributed to [2Na+(CN)-]+ at m/z 72 and [2Na+(CNO)-]+ at m/z 88 increasing in intensity. Following a fluence of 1 × 1014 ions/cm2 these are the only significant peaks present in the spectrum above m/z 50 (inset of Figure 2a). When the BSA is desalinated using Ziptips the results of the depthprofiling experiment are very different. Following an initial period of change, the organic signal is maintained all the way to the substrate (Figure 2b), and there is very little evidence of the peaks at m/z 72 and m/z 88. In order to investigate whether similar effects are seen with another group of biomolecules, films of the phospholipid DPPC (31) Van Stipdonk, M. J.; Santiago, V.; Schweikert, E. A. J. Mass Spectrom. 1999, 34, 554-562. (32) Czerwins´ki, B.; Samson, R.; Garrison, B. J.; Winograd, N.; Postawa, Z. Vacuum, 2006, 81, 167-173.

Figure 2. C60+ depth profiling of a bovine serum albumin (BSA) layer: (a) prior to desalination, the immonium ions at m/z 44, 70, and 110 decrease rapidly with the spectrum dominated by sodium adducts at m/z 72 and 88 (inset in panel a) following 1 × 1014 ions/cm2, whereas following desalination (b) the immonium ions are detected through to the appearance of the substrate.

Figure 3. Effect of sodium chloride on the depth profiling of DPPC with C60+: (a) the pure DPPC shows a good signal stability of the fragment ion m/z 184 while the sodium adduct ions (b) are insignificant in the spectrum and do not increase. This is contrasted by the sample where NaCl is added (c); there is an initial large decrease in the m/z 184 signal which is complimented by an increase in the adduct ions m/z 109 and m/z 143 (d).

were depth profiled with and without NaCl added to the solution prior to film deposition, such that the samples contained 0% and 20% NaCl by weight of solid. As with the protein sample, and in accordance with previously reported results,17 the DPPC film reaches a steady state under C60+ bombardment where molecular information is obtained under continuous bombardment when no

salts are present (Figure 3a). The main PC headgroup ion, at m/z 184, decreases to 80% of the initial value before reaching a plateau. This is in contrast to the sample where the salt was added, which demonstrates a much greater decrease in this characteristic ion. Interestingly, the smaller fragment of the PC headgroup at m/z 86 behaves similarly in both samples, decreasing to 60% of its initial Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

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Figure 4. Depth profiling of a tissue section following ammonium formate wash: (a) the signal from m/z 44, alanine immonium ion, and m/z 70, proline, are stable through the sample; (b) this is in contrast to the cholesterol ion at m/z 369, while there appear to be two different regimes in the behavior of the m/z 184 ion. This is supported by 3D imaging experiments which show the first 70 layers of a depth profile through a white/gray matter boundary region. (c) An AFM analysis of the sputtered tissue region allowing the crater depth to be measured; this was shown to be 4 µm. (d) The cholesterol signal at m/z 369 is confined to the upper layers of the white matter only; (e) the m/z 184 ion is initially intense in the gray matter but then decreases to nothing, while its intensity increases in the white matter region. The signal from the immonium ions (f) is only detected from the gray matter region and appears to increase following the first few etches. The circle at the top right corner allows the orientation of all three ion images to be compared; rotation was required to best demonstrate the ion signal distributions.

value by the time the steady state is reached. From analysis of the spectra, this fragment contributes a higher percentage of the total DPPC-related ions in the sample with added salt, suggesting a difference between the two surfaces; however, more work is required to fully understand the mechanisms involved. As with the previous examples there is an appearance in the spectrum of peaks that relate to cationized fragments of the molecule, in this case at m/z 109 and 143, which have been attributed to [2Na+PO2-]+ and [2Na+H2PO4-]+. When the same samples are bombarded with Au+ (results not shown) as has been demonstrated with the tissue section, there is a loss of molecular signal; however, this is due to damage accumulation as opposed to surface modification. The mechanism behind this chemical modification under C60+ bombardment is not understood; nevertheless it clearly appears to be a significant effect and something that must be taken into account when planning any experiments in biological samples with the C60+ beam. At this point it must be remembered that native salts are problematic in other forms of mass spectrometry; electrospray and MALDI analysis of peptide samples are also hindered by the presence of native salts and other contaminants.33,34 In these cases the compound of interest (e.g., the peptides) can be isolated by extraction; this cannot be done to this degree with direct analysis; however, there are approaches that may allow the effect to be reduced. (33) Schwartz, S. A.; Reyzer, M. L.; Caprioli, R. M. J. Mass Spectrom. 2003, 38, 699-708. (34) Tang, L.; Kebarle, P. P. Anal. Chem. 1991, 63, 2709-2080.

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Tissue Depth Profiling Following Salt Removal. In light of the identification of native salts as a possible cause of the loss of molecular ion signal from tissue sections under C60+ bombardment, methods of salt removal from the literature were applied to the tissue sections prior to carrying out the depth-profiling experiment. Of the two reported washing techniques, 70% ethanol35 and ammonium formate,6 the latter was the method that yielded the most satisfactory results. As Figure 4 shows, the molecular ion signal from the surface does not disappear completely as in Figure 1, and the contribution of the “inorganic” fragment ions within the spectrum even after a relatively large ion dose is not significant. As the levels of inorganic salts within biological samples are approximately 2% by weight of the total wet weight, then it is clear that there does not need to be a large concentration to have a significant effect upon the success of the experiment. By employing an ammonium formate wash, the tissue section could be depth profiled all the way through to the steel substrate with characteristic molecular ions detected through the whole sample, as shown in Figure 4, parts a and b, with the Fe+ ion detected at m/z 56 once the substrate was reached. The AFM image in Figure 4c shows the crater formed that was measured at 4 µm depth; the discrepancy between measured and initial thickness is due to shrinkage upon dehydration. With the use of this measure of sample thickness it can be estimated that 160 nm3 of material is removed for each C60+ impact, which is similar (35) Amini, A.; Dormady, S. J.; Riggs, L.; Regnier, F. E. J. Chromatogr., A 2000, 894, 345-355.

Figure 5. Depth profiling of a tissue section held at -120 °C: (a) ions relating to cholesterol (m/z 369) and phopholipids (m/z 104, 184, and 760); (b) immonium ions characteristic of proteins (m/z 44, 70, 84, and 110) and a nonspecific organic fragment at m/z 43. In contrast to the depth profiles of tissue at room temperature, the signal from cholesterol does not rapidly decrease.

to values reported for trehalose (20 keV C60+) of 180 nm2 per impact36 and Irganox 1010 (30 keV C60+) of 250 nm3 per impact.37 Although there is molecular signal detected from surface to substrate, the chemistry detected varies with respect to distance from the surface. This is shown in Figure 4, parts a and b, and supported by the 3D images in Figure 4d-f. The images are from an area that covers a boundary between the white and gray matter of the rat brain, and the difference in the behavior of the two areas is clearly shown. The white matter identified by its initial high concentration of cholesterol (Figure 4d) quickly changes to a region of the tissue that yields a high signal from the m/z 184 ion (Figure 4e). The peaks attributed to immonium ions from protein are not detected strongly from this region at any depth (Figure 4f), which may reflect the high lipid content of the myelin sheaths that dominate this domain. The gray matter region, which is predominantly cell bodies, yields an initially high signal from the m/z 184 ion that signifies the presence of the phosphotydylcholine headgroup, the most efficiently detected ion from phospholipids in the positive ion spectrum. Following the initial etches this signal decreases significantly and is replaced by ions from proteins which are initially low in intensity as shown in Figure 4f. But why should a sample that contains structures visible to the eye in the x and y directions vary so rapidly in the z direction under molecular depthprofiling analysis? It has been suggested previously that there is molecular migration within the tissue sections at room temperature under the vacuum conditions of the instrument38 and that when the sample is warmed up from -100 °C to room temperature the molecules that are detected vary greatly in intensity. If this redistribution of molecules occurs within the vacuum, then it may be expected that this movement of compounds is restricted if the sample is held at -120 °C. Figure 5 demonstrates that this is indeed the case; the signal from cholesterol is much lower than is seen at room temperature; however, the intensity of the signal does not vary as the dose increases, which is in stark contrast to the room-temperature samples seen in both Figures 1 and 4. (36) Cheng, J.; Winograd, N. Anal. Chem. 2005, 77, 3651-3659. (37) Shard, A. G.; Brewer, P. J.; Green, F. M.; Gilmore, I. S. Surf. Interface Anal. 2007, 39, 294-298. (38) Sjovall, P.; Johansson, B.; Lausmaa, J. Appl. Surf. Sci. 2006. 252, 69666974.

However, it appears that there is still heterogeneity with respect to depth as a decrease in phospholipid signal is complemented by an increase in peaks relating to protein immonium ions. As the depth profiling with imaging experiment is a lengthy process which may take over 2 days with the current instrumentation to carry out, this kind of experiment on a cold stage is impractical. However, with at least two groups looking to couple dc beam sputtering to orthogonally placed mass analyzers, the length of these experiments may be significantly decreased, and hence, it is important that the behavior of these surfaces is understood prior to the use of these new ion probe instruments. CONCLUSIONS Here we have shown that it is possible to create and detect characteristic ions from various biological groups from the whole depth of a rat brain tissue section. The initial attempts on untreated sections appear to be thwarted by the native salt levels of the tissue, a problem that was solved by introducing a washing step into the procedure. The presence of high salt concentrations is typically problematic in mass spectrometry with great care taken to remove buffer salts and potential salt contamination prior to the analysis of biological samples. It is not surprising, therefore, that strong sodium and potassium signals from the tissue samples lead to disruption of the depth-profiling experiment. By removing the salts it was possible to follow the molecular signatures of lipids and proteins through the whole thickness of the sample, where significant unexpected partitioning of the various compounds appeared which varied in both the white and gray matter. Analysis of the sample on a cold stage demonstrated that the distribution of the most mobile compound, cholesterol, was virtually constant with respect to depth. This suggests that perhaps the ideal approach is to analyze biological samples in a frozen hydrated state. This has the dual benefits of avoiding molecular redistribution during the freeze-drying step and also when in vacuum during analysis, while ensuring the sample is analyzed in as native a state as possible. The presence of water molecules may also be a good source of protons thus aiding ionization.39 As these two points indicate, sample preparation and analytical conditions are every(39) Conlan, X. A.; Lockyer, P. N.; Vickerman, J. C. Rapid Commun. Mass Spectrom. 2006, 20, 1327-1334.

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thing in these direct analyses of biological samples. Through the careful investigation of the processes and the optimization of the protocols, the desired goals can be achieved and the significance of results obtained will increase as a greater knowledge of the system can be demonstrated. From this investigation it is clear that the depth profiling of tissue sections is not straightforward and that many experimental and environmental considerations need to be taken into account. However, we believe that this body of work demonstrates that meaningful profiles of biological tissue can be achieved if effects such as native salts and lipid redistribution are controlled. This conclusion presents a more positive outlook to this area of research than that presented by Debois et al.27 In this paper the authors conclude that the lipids are segregated to the upper few hundred nanometers and that a meaningful depth profile is not possible. In agreement with other work, we have shown in this paper that molecular migration can be controlled by altering the sample temperature, although at the cost of increasing the complexity of the experiment. The other significant consideration is the energy of the primary ion beam used in the depth-profiling study. Studies by Shard et al.37 have demonstrated that on certain samples there is a threshold energy below which C60+ ion impacts do not remove enough material to allow a steady state to be achieved. From the sputter yield comparisons presented in the discussion section of this paper, the volume removed per ion on a tissue section is lower than some of the model systems analyzed, even at the higher energies used here. If tissue sections present a surface of low sputter yield, it may be that the 10 keV impacts used by Debois et al. were not sufficiently energetic to allow a successful depth profile. To be able to obtain molecular information from beyond the surface of a range of biological samples with SIMS will lead to many exciting possibilities. First it will allow the distribution of a

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given species to be determined in three dimensions; the obvious target system in this case are single cells, and work to this effect is beginning to appear. It is clear that to be able to accurately plot a depth scale to a 3D image, in the same manner as the xand y-axes are labeled, then an understanding of the relative sputter yields of the various components must be quantified. This is work that is on going on model systems and will eventually allow the accurate use of the acquired data to create 3D representations of the analyzed system. Other samples, such as brain sections, which may be treated as heterogeneous in the z-axis when its scale is compared to that of the x and y dimensions and, possibly more importantly, the whole length of the tissue it is derived from, can still benefit from these advances as the number of molecules accessible to the analysis is greatly increased. ACKNOWLEDGMENT We are very grateful to Alex Henderson and John Fletcher at the University of Manchester for providing the MATLAB routines for the treatment of the data and in sharing their expertise with regards to image processing. We also thank Hailey Cordingley, Martin Vigeonhart, and Andy Organ at GSK, Harlow, for the supply of tissue sections and helpful discussions. The funding for this project was very kindly provided by the Engineering and Physical Sciences Research Council Life Sciences Interface Programme (ESPRC-LSI). This article is based on a paper presented at the 44th IUVSTA Workshop, Sputtering and Ion Emission by Cluster Ion Beams, Scotland, U.K., April 23-27, 2007.

Received for review December 19, 2007. AC702127Q

October

16,

2007.

Accepted