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Improved Molecular Imaging in Rodent Brain with Timeof-Flight Secondary Ion Mass Spectrometry using Gas Cluster Ion Beams and Reactive Vapour Exposure Tina Bernadette Angerer, Masoumeh Pour Dowlatshahi, Per Malmberg, and John Stephen Fletcher Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504774y • Publication Date (Web): 23 Mar 2015 Downloaded from http://pubs.acs.org on March 31, 2015
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Analytical Chemistry
Improved Molecular Imaging in Rodent Brain with Time-of-Flight Secondary Ion Mass Spectrometry using Gas Cluster Ion Beams and Reactive Vapour Exposure 1
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Tina B. Angerer, Masoumeh Dowlatshahi Pour, Per Malmberg, John S. Fletcher
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1. Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden 2. Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg, Sweden *Corresponding author e-mail:
[email protected] Abstract Imaging mass spectrometry has shown to be a valuable method in medical research and can be performed using different instrumentation and sample preparation methods, each one with specific advantages and drawbacks. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has the advantage of high spatial resolution imaging but is often restricted to low mass molecular signals and can be very sensitive to sample preparation artefacts. In this report we demonstrate the advantages of using gas cluster ion beams (GCIBs) in combination with trifluoracetic acid (TFA) vapour exposure for the imaging of lipids in mouse brain sections. There is an optimum exposure to TFA that is beneficial for increasing high mass signal as well as producing signal from previously unobserved species in the mass spectrum. Cholesterol enrichment and crystallisation on the sample surface is removed by TFA exposure uncovering a wider range of lipid species in the white matter regions of the tissue greatly expanding the chemical coverage and the potential application of ToF-SIMS imaging in neurological studies. 40 keV Ar4000 + in combination with TFA treatment facilitates high resolution, high mass imaging closing the gap between ToF-SIMS and MALDI.
Introduction There is a demand for the imaging of native and exogenous compounds in biological specimen with cellular and sub-cellular resolution in order to understand disease progression, drug action and fundamental biological processes. Imaging mass spectrometry is a promising means of meeting this demand. The use of imaging mass spectrometry for biological and medical analysis is rapidly expanding due to the technique detecting a wide range of molecular species with high chemical specificity without the need to introduce exogenous labels1-3 For the analysis of tissue samples such as rodent tissue sections or human biopsy samples the most common imaging mass spectrometry approaches are matrix assisted laser desorption ionisation (MALDI) and secondary ion mass spectrometry (SIMS), both often coupled to a time-of-flight (ToF) mass analyser.4-7 The two techniques can provide complementary information with MALDI capable of generating intact high mass species including peptides and proteins while SIMS can be used to detect low molecular weight compounds, commonly m/z 700) are in yellow. All data was acquired using 40 keV Ar4000 +, primary ion dose density 7 × 1011 ions/cm2, positive ion mode. Spectra were summed up from a 3.75 × 105 µm2 size region. Having identified the regions of major compositional variation identified in the PCA analysis, white (WM) and grey matter (GM) and the granular area (GA) between the two, spectra can be extracted from these regions for samples exposed to different amounts of TFA. For each treatment (control, 15, 30 and 60 minutes TFA exposure) positive ion spectra from each area of interest originating from a region the size of 3.75 × 105 µm2 were extracted and compared in Figure 3. For clearest visualisation the TFA 60 spectra are displayed in 2 parts (blue m/z 100-700 and yellow m/z 700-2000) so as not to be obscured by the other spectra displayed. In the figure it is evident that TFA exposure leads to increased signal beyond what can be explained by any slight variations in primary ion current. This effect was more pronounced for the 30 minute TFA exposure than 15 minutes. 60 minutes exposure produced greatly enhanced signals from low mass ions and little if any enhancement of signal >700 Da. indicating that TFA exposure improves signal only within a certain time frame/exposure regime. Very similar results were obtained using the Bi3+ source of the TOF.SIMS 5 (data not shown), indicating that the signal enhancing effect of TFA is independent of the primary ion species used. Reproducibility was checked several weeks later on new sections of mouse brain and similar enhancement was observed for separate exposures (supplementary information (Suppl. Figure 2). Looking at the signal from each area in detail, it is clear that in the grey matter regions, after 30 minutes of TFA exposure (Figure 3 a), overall lipid signal (m/z 600-800) as well as signal from lipid
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Analytical Chemistry
dimers (m/z 1400-1600) increased and enhancement was more pronounced for [M+H]+ ions as indicated by the ratios of the list of identified peaks in Table 1. Compared to the control no completely new peaks were observed in the mass spectrum. From the images in Figure 2 it is evident that the granular area is much more pronounced in TFA exposed sections. The spectral comparison (Figure 3 b) shows that additional to the overall signal enhancement a number of new peaks were being detect which were weak or non-existent in the control spectrum. This is true for all TFA exposure times with the most pronounced effect after 30 minutes. The new peaks up to m/z 900 can be mainly assigned to different galactoceramides. A further series of, as yet, unidentified peaks in the range of m/z 900-1300 are also observed. These peaks appear at m/z M.9 (where M is the integer mass, e.g. m/z 1072.8943 in Table 1). Phospholipid species in this region of the mass spectrum would be expected to appear at m/z M.7 (e.g. MIPC(t18:0/24:0) with molecular weight 1071.7198) while previously reported ganglioside related peaks should appear at m/z M.5-M.7.32,33Figure 4 e, f and k, l shows single ion images of those newly uncovered peaks and their distribution in treated/untreated tissue. The only significant reduction in signal following TFA exposure was for cholesterol (Figure 4 g, m). This partially explains the drastic change in the spectrum (Figure 3 c) in the white matter. The control spectrum mainly consists of 2 intensive peaks at m/z 369.35 (cholesterol [M+H-H2O]+) and m/z 1160.07 (cholesterol 3-mer [3M+H]+). With the cholesterol removed many other signals are being uncovered which can be assigned to different PC species and ceramides. This effect on cholesterol was also clearly observed in depth profiles obtained using Bi3+ as analysis gun and C60++ as a sputter source as can be seen in Suppl. Figure 3. Many of the unidentified peaks are evenly distributed or at least appear in both white matter and granular area but do not appear at all in the grey matter. We are therefore confident, that we see real brain chemistry and not artefacts of the TFA exposure we introduced into the sample. In the peak list, peaks that appear in white matter and granular area/multiple areas as shown in Figure 4 c, f and h are marked with an asterisk.
Figure 4. RGB overlays of a 30 minute TFA exposed rodent brain (a) and control brain (b) (red m/z 570.53, green m/z 866.65 and blue m/z 1096.90 for TFA and m/z 369.35 for the control section). Below is a comparison of single ion images for 30 minute TFA exposure (c - h), control brain (I - n) of
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m/z 886.65 (c + i), m/z 1494.16 (d + j), m/z 1096.90 (e + k), m/z 1116.90 (f + l), m/z 369.35 (g + m) and m/z 798.54 (h + n). Peak identities can be found in the peak list in Table 1. All data was acquired using 40 keV Ar4000+ primary ion dose density 7 × 1011 ions/cm2 in positive ion mode. Image area of 4.8 × 4.8 mm2 comprising 192 × 192 pixels. In negative ion mode the effect of TFA on the sample is less pronounced than in positive ion mode. TFA has similar effects on the spectra (see Suppl. Figure 4 and Suppl. Table 1), little change after 15 minutes TFA exposure, slight enhancement for most peaks but, in contrast to positive mode, also suppression after 30 minutes and smaller mass fragments and decreased high mass signal after 60 minutes. In the grey matter the combination of our high energy gas cluster ion beam with TFA treatment generates clear peaks with masses up to 1800 Da. These can be assigned to different ganglioside species. The biggest changes can be observed in the white matter and granular area between mass 1000 and 1300 Da. Similar to positive ion mode new peaks appear that are not present in the control spectra at all so they appear to be generated due to reaction with the TFA. Also the cholesterol signal from the white matter is reduced. Unlike positive ion mode analysis the granular area is visible on the control sections rather than being uncovered by the treatment. In positive ion mode the increase in signal can be explained by the introduction of additional protons into the sample provided by the TFA. Because of those additional protons in negative ion mode, we were expecting an overall decrease in signal but that is not the case. Improvements in secondary ion signal of intact molecular species has direct implications for high resolution molecular imaging. Beams made of big gas cluster ions have the advantage of generating increased signal from larger molecular ions but most of the time such beams are hard to focus and with argon the ionisation efficiency is poor compared to e.g. water clusters.34 As we very recently demonstrated,21 higher energies gas cluster ions increase the ionisation efficiency for secondary ions as well as improving the focus of the beam but for higher mass species image resolution was still signal limited. High energy GCIBs in combination with TFA treatment makes this possible, as shown in Figure 5, top. Line scans of m/z 906.63 (C24-OH Sulfatide, C48H92SNO12) on the interface of white matter and granular area (Figure 5, bottom) show that we can achieve spatial resolutions of
