MALDI Mass Spectrometry Imaging of Bioactive Lipids in Mouse Brain

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MALDI Mass Spectrometry Imaging of Bioactive Lipids in Mouse Brain with a Synapt G2#S Mass Spectrometer Operated at Elevated Pressure: Improving the Analytical Sensitivity and the Lateral Resolution to Ten Microns Hans Kettling, Simeon Vens-Cappell, Jens Soltwisch, Alexander Pirkl, Jörg Haier, Johannes Müthing, and Klaus Dreisewerd Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5017248 • Publication Date (Web): 09 Jul 2014 Downloaded from http://pubs.acs.org on July 15, 2014

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Analytical Chemistry

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MALDI Mass Spectrometry Imaging of Bioactive Lipids in Mouse Brain with a Synapt G2‐‐S Mass Spectrometer Operated at Elevated Pressure: Improving the Analytical Sensitivity and the Lateral Resolution to Ten Microns

Hans Kettling1,2, Simeon Vens‐Cappell1,2, Jens Soltwisch1, Alexander Pirkl1, Jörg Haier3, Johannes Müthing1, Klaus Dreisewerd1,2,*

1

Institute for Hygiene, University of Münster, Robert-Koch-Str. 41, 48149 Münster, Germany 2

Interdisciplinary Center for Clinical Research (IZKF) Münster, University of Münster, Domagkstr. 3, 48149 Münster, Germany

3

Comprehensive Cancer Center Münster, University Hospital Münster, Waldeyerstr. 1, 48149 Münster

* address correspondence to: Dr. Klaus Dreisewerd, [email protected]

Key words: MALDI-MS imaging, Synapt G2-S, Dithranol, Lateral resolution, Laser beam shaping, Collisional cooling

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ABSTRACT Mass spectrometers from the Synapt-G1/G2 family (Waters) are widely employed for matrixassisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). A lateral resolution of about 50 µm is typically achieved with these instruments, that is, however, below the often desired cellular resolution. Here we show the first MALDI-MSI examples demonstrating a lateral resolution of about ten microns obtained with a Synapt G2-S HDMS mass spectrometer without oversampling. This improvement became possible by laser beam shaping using a 4:1 beam expander, a circular aperture for spatial mode filtering, and by replacement of the default focusing lens. We used dithranol as an effective matrix for imaging of acidic lipids such as sulfatides, gangliosides, and phosphatidylinositols in the negative ion mode. At the same time, the matrix enables MS imaging of more basic lipids in the positive ion mode. Uniform matrix coatings with crystals having average dimensions between 0.5–3 µm were obtained upon spraying a chloroform/methanol matrix solution. Increasing the cooling gas pressure in the MALDI ion source after adding an additional gas line was furthermore found to increase the ion abundances of labile lipids such as gangliosides. The combined characteristics are demonstrated with the MALDI-MSI analysis of fine structures in coronal mouse brain slices.


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INTRODUCTION Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is an emerging technology with increasing applications in the biomedical field.1,2 A major advantage of MALDI-MSI compared to other molecular imaging techniques is the simultaneous label-free detection of a wide range of endogenous biomolecules (lipids, peptides, metabolites) directly from snap-frozen tissue slices. Development of MALDI-MSI for the analysis of lipids has recently found a particularly high interest, due to the numerous physiological roles exerted by bioactive lipids, and the potential of using altered lipid profiles for example as markers for disease states.3,4 Neutral glycosphingolipids (GSLs) such as dihexosylceramides and negatively charged gangliosides (sialylated GSLs) of the plasma membrane of eukaryotic cells, moreover, act as specific receptors for pathogenic microbes, viruses, bacteria and the toxins produced by them.5,6 Tracking the distribution of GSLs in tissue can thus potentially provide valuable insights into pathogen-mediated inflammation processes or shed light on initial infection routes. Two key issues for further advancing the method are improvement of analytical sensitivity (e.g., to enable the detection of small abundances of physiologically important phospho- and glycolipids) and improvement of the lateral resolution for achieving a cellular resolution. Both factors are critically determined by the MALDI matrix system used, especially with regard to the morphology of the matrix coating generated on the tissue slice, the efficiency of analyte extraction, and the desorption/ionization efficiency. Moreover, relatively few of the currently commonly employed MALDI matrices show a similarly high potential for imaging of more acidic and more basic lipids in the negative and positive ion modes, respectively.7 Also, as a factor complicating the handling, some particular potent matrices such as 9aminoacridine (9-AA)8 and 1,5-diaminonapthalene (DAN)7 are considered as being



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potentially cancerogenous. Considerable effort is currently on the way to identify new matrices with improved properties for MALDI-MS imaging of lipids. Furthermore, the lateral resolution of a MALDI-MSI analysis depends on the cross-section of the focal laser beam profile and the precision of the laser stage movement. Benchmark figures as provided to this end by most commercial MALDI imaging mass spectrometers range between approximately 10–50 µm. In particular abundant phospholipids can now routinely be analyzed by MALDI-MSI with these benchmark figures.7,9 MALDI-MSI with sub-10 µm resolution has also been reported and was, for instance, achieved by use of a high numerical aperture (N.A.) objective for laser focusing, mounted in front of the target such that the samples are irradiated at normal incidence.9,10,11 In contrast, in most standard MALDI instruments the samples are irradiated at an angle of incidence of ~45° and at a lower N.A. Zavalin et al. demonstrated that also under these conditions a sub-10 µm resolution is, however, possible if laser beams of improved mode purity are used.12 A lateral resolution surpassing the focal laser spot dimensions can be achieved by applying the so-called oversampling method. Here the sample stage is moved by an increment smaller than the laser beam cross-section, after essentially all material has been ablated within the previously irradiated, overlapping pixel.13 For example, using a Synapt mass spectrometer Snel and Fuller applied oversampling for the analysis of neutral GSLs in spleen tissue with an approximate resolution of 15 µm.14 However, at least if a common Gaussian-shaped MALDI laser beam is used,15 MSI oversampling is associated with a few significant drawbacks, in particular a reduced sensitivity, less defined material ejection area, and reduced data acquisition speed. Here we present the first example of performing MALDI-MS imaging with a Synapt G2-S HDMS mass spectrometer at a true lateral resolution of about 10 microns. Coronal mouse brain sections were investigated as constituting widely used “standard samples” for the ACS Paragon Plus Environment

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development and test of the MALDI MSI methodology. The small laser spot size was produced by laser beam shaping using a 4:1 beam expander, a circular aperture for spatial mode filtering, and by replacement of the default focusing lens. A high sensitivity for the analysis of acidic phospho- and glycolipids at this lateral resolution was achieved by using dithranol (1,8-dihydroxyanthracen-9(10H)-one) as MALDI matrix. Dithranol is an established matrix for MALDI-MS of polymers16 but was only recently described by Le et al. for MALDI-MSI of bioactive lipids in rat liver and bovine calf lens tissue.17 That work focused exclusively on analyzing the positive ion mode.

EXPERIMENTAL SECTION

Chemicals Dithranol (>99 %) was from Seratec (Courville-sur-Eure, France), red phosphorus (≥ 97 %), used as mass calibrant, and all other chemicals were from Sigma-Aldrich (Steinheim, Germany). Cardiolipin (72:8) was from bovine heart.

Mouse Brains Mouse brains were dissected from six week old female CBA/J mice according to standard protocols and approval by the local ethical commission. Whole organs were snap-frozen in liquid N2 and stored at –75 °C before further use. Single brains were embedded into 2hydroxyethylcellulose; 15–20 µm-thick tissue slices were prepared with a cryomicrotome



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(Jung Frigocut 2800-E, Leica, Wetzlar, Germany) at –20 °C and thaw-mounted on standard (non-coated) histological glass slides.

Matrix Coating Dithranol was dissolved to 8 mg/mL in chloroform/methanol (CHCl3/CH3OH ; 2:1, v/v). Matrix was sprayed onto the tissue by use of an airbrush, affixed to a pillar with a 150 µmnozzle (infinity solo, Harder&Steenbeck, Norderstedt, Germany). The distance between the airbrush outlet and the sample was set to 10 cm. Matrix was sprayed in 10 cycles of 3 s duration, followed by 15 s drying intervals. A back pressure of 3 bar of N2 was used and the gas flow maintained during drying cycles to support solvent evaporation.

Microscopy Scanning electron microscopy (SEM) images were acquired with a field-emission SEM (Hitachi S800; Tokyo, Japan). High-resolving optical microscopy images were produced with an Axiovert 40 C (Carl Zeiss, Jena, Germany).

Mass Spectrometer Mass spectra were recorded with a MALDI Synapt G2-S HDMS mass spectrometer (Waters/Micromass, Manchester, UK). To enable adjustment of the buffer gas pressure, the MALDI ion source was modified by adding a gas supply line that is directly connected to the region of ion generation (Figure S-1A in the Supporting Information); the default gas line connected to the hexapole ion guide compartment was shut off. In order to more precisely control the gas flux between the sample region and the hexapole ion guide compartment, a ACS Paragon Plus Environment

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custom-made shielding ring aperture (made of PEEK and Viton) was mounted onto the ion extraction cone (Figure S-1A). Upon mounting the (detachable) hexapole/cone assembly, the PEEK/Viton assembly snug-fits with the sparing of the chamber housing; gas flow, thereby, becomes constraint to the inner opening of the ion extraction cone. Pressure was monitored and continuously adjustable (within a range of 0.04–4 mbar) by custom-made soft- and hardware. The pressure control system is described in more detail in Figure S-2. In order to prevent sparking between the rods of the hexapole ion guide upon increasing the overall gas pressure, the RF voltage driving the hexapole was reduced from its default value of 450 V to 200 V. A change in ion signal intensities was not visible upon this operation. All MS measurements were performed with a cooling gas pressure (N2) of approximately 0.7 mbar at the MALDI target.

Lasers Experiments with a ~35 µm-wide laser spot were performed using the standard Nd:YAG laser (Flare PQ UV 1000-30, InnoLight Laser, Kaiserslautern, Germany) of the mass spectrometer, mounted in its default position (i.e., within the MALDI ion source housing). This laser generates pulses of 355 nm wavelength and 2 ns duration at a fixed pulse repetition rate of 1 kHz. The diameter of the circular laser beam at the exit aperture is ~2 mm (beam divergence, ≤0.5 mrad). Experiments with ~10 µm-wide laser spots were performed using a second identical Nd:YAG laser. In a few experiments, a N2 laser (MNL 100 LD; LTB Lasertechnik Berlin, Germany) was moreover used. This laser emits pulses of 3 ns duration at a wavelength of 337 nm and a pulse repetition rate of 30 Hz (beam divergence, ≤0.5 mrad). Due to the different resonator geometry the N2 laser has a rectangular beam shape of ~5 mm × 3 mm. The pulseto-pulse energy stabilities of the three lasers were better than 5% (standard deviation) and ACS Paragon Plus Environment


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pulse energies were found to be “constant” throughout the maximum of 12–60 h of data acquisition time realized in the course of the MSI experiments. The “external” lasers were mounted onto an optical bench for improved flexibility regarding beam shaping and laser pulse energy control. Details of the applied beam shaping procedure are described in the Results and Discussion section. A set of aluminum surface mirrors was used for beam steering and coupling of the modified laser beams to the MALDI ion source. A custom-made interface was installed to allow for mounting an aluminum surface mirror with appropriate orientation (to accept the “external” laser beam) and for using a focusing lens with a reduced focal length f of 125 mm (at 355 nm wavelength) in lieu of the default lens (f ~ 200 mm); a snapshot of this device is plotted in Figure S-3. The same laser port was used for coupling of “internal” and “external” lasers, such that all laser beams impinged on the surface at an angle of incidence of 45°. Pulse energies were adjusted with gradient density filters and measured with a laser pulse energy meter. Typical pulse energies applied to the samples were ~3 µJ for the 35 µm-spot and ~100 nJ for the 10 µm-spots.

Data Acquisition Unless noted, all mass spectra were recorded in the “sensitivity mode” providing a mass resolution of ~14,000 (FWHM). MALDI-MSI data were acquired by irradiating each pixel with 250 (Nd:YAG laser) or 60 pulses (N2 laser). Only for recording data with the 35 µmwide laser spot initially 500 instead of 250 Nd:YAG laser pulses were applied. Generally, this did, however, not further increase the ion signals, presumably due to sample depletion during the first 250 pulses. Mass calibration was performed using red phosphorus cluster ions.

Processing of Mass Spectra If high-resolving MS images are recorded from larger areas (e.g., whole brain cross-sections) large data file sizes of several to a few ten Gigabyte can be generated. Evaluation of such ACS Paragon Plus Environment

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large files is not straightforward with current software solutions. Here a bin size of 0.02 Da was used for processing all mass spectra recorded in the sensitivity mode. HDImaging software (Waters) was applied for initial file conversion to enable use of BioMAP (v3.8.0.4, Novartis, Basel, Switzerland) for further data evaluation. In order to extract mass spectra of a given region of interest, the data processed with HDImaging were processed with a recently modified version of DatacubeExplorer (v.2.1).18 All MALDI-MS images and mass spectra presented are displaying raw data without smoothing or interpolation.

Safety Hazard Note Dithranol can cause irritation of the respiratory system, eyes, and skin. Sample preparations should be handled in a hood and protective goggles worn. The employed lasers are of safety class 3B. Safety precautions should be taken when working with free beams of such lasers (e.g., by wearing protective goggles).

RESULTS AND DISCUSSION

Physicochemical Properties of the Dithranol Matrix Dithranol exhibits a high optical absorption over a wide wavelength range (Figure S-4). At the classical MALDI wavelengths of 337 and 355 nm decadic molar extinction coefficients in solution (acetonitrile) of 21.500 and 28.000 l mol-1 cm-1, respectively, are determined. These high values facilitate a sensitive MALDI-MS analysis.19 Due to the photo-reactivity of dithranol20 it is advisable to store matrix-coated slices in a protective environment.



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Another advantageous feature, which was already noted in reference 17, is that dithranol is well soluble in CHCl3:CH3OH (2:1, v/v), one of the most widely used solvent systems for lipid extraction. It can be assumed that using this solvent mixture also enables an efficient micro-extraction of lipids from tissue slices upon application of matrix droplets. Because of the high volatility of the solvent system, only small, rapidly evaporating droplets are presumably produced on the tissue surface when the matrix is sprayed as an aerosol. Probably, this forms one reason why a particular uniform coating of tissue with the dithranol matrix is obtained, as is illustrated by the SEM images shown in Figure 1A. The formation of small crystals exhibiting average dimensions between ~0.5–3 µm (Figure 1B) facilitates MALDI-MSI analyses with high lateral resolutions. The random generation of larger crystals on top of the uniform micro-crystalline layer, as visible in Figure 1A, did not affect the lateral resolution of the analysis, presumably because these are not containing sizable concentrations of analyte. Analyte diffusion as a result of the matrix coating was not notable. This is, for example, demonstrated by the MS image in Figure S-5, in which analyte and matrix ion signals were recorded across sharp tears in a tissue slice. As illustrated in Figure 1C, the uniform matrix coating also enables a straightforward determination of the effective laser spot dimensions by measuring the sizes of the “ablation craters”. Naturally, a precise knowledge of the laser spot dimensions is an important parameter in MS imaging applications. Although only a thin dithranol film was covering the tissue slices, stable analyte and matrix ion signals were obtained over acquisition times of up to 48 h. Acquisition times in excess of 2½–3 d were accompanied by precipitously declining matrix and analyte ion signals, caused by complete evaporation of matrix material (data not shown).


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Increasing the MALDI Signal Intensities by Elevating the Buffer Gas Pressure in the MALDI Ion Source Numerous studies have demonstrated that collisional cooling mediated by a buffer gas (typically N2) in the MALDI ion source has a significant impact on the stability of the generated MALDI ions.21–24 These studies suggested that for an electrical field E of a few 10– 100 V/mm, as typically used in MALDI ion sources that are coupled to hybrid orthogonal extracting mass analyzers, the N2 buffer pressure p should generally exceed 0.5–1 mbar to achieve an optimal efficiency in the collisional cooling process (the exact values depend on the detailed ion extraction conditions).22 Because the energy densities that are generated by a tightly focused MALDI laser beam increase with decreasing focal beam diameter,25 it could be speculated that efficient collisional cooling is especially relevant for highly-resolved MALDI-MSI measurements. We have previously developed a system with which the pressure in oMALDI2 ion sources of AB Sciex QStar instruments can be varied continuously between 0.05–4 mbar. As sketched in Figures S-1A and S-2 this technical solution was adopted in this work for the Synapt G2-S mass spectrometer. The increase of p from its default value (about 0.2 mbar) and reversal of the gas flow between the sample and hexapole compartments improved the detection of several lipids, not least that of labile sialic acid-bearing gangliosides.29 For example, if analyzed from brain extract (to increase the reproducibility), for the monosialoganglioside GM1 (II3Neu5Ac-Gg4Cer) a signal increase by a factor of 2–3 was obtained by increasing the buffer gas pressure to values above about 0.7 mbar, and the signal increase moreover continues until the maximum pressure so far tested (2.5 mbar) was reached (Figure S-6). For the more stable phospholipids different signal intensity-cooling gas pressure relations seem to be obtained. For example, for cardiolipin 72:8, having a similar molecular weight (1451.95 g/mol) as GM1, the maximum signal intensity peaked around p ~ 0.7 mbar and slightly drops



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for pressures above 1 mbar (Figure S-6). This may be attributed to the general pressure dependence of the ion transmission – for the labile gangliosides the beneficial effect of enhanced collisional cooling may compensate the reduced ion transmission . Because also the matrix cluster ion background was found to increase with p, we chose a pressure of 0.7 mbar for all reported MSI experiments.

Decreasing the Focal Spot Size by Laser Beam Shaping In its default configuration the Synapt G2-S HDMS mass spectrometer enables MALDI-MS imaging with a lateral resolution of about 50 µm (without oversampling). To change the spot size the focal lens (f ~ 200 mm) can moreover be moved along the beam axis until minimal and maximal stop positions are reached. We noted that in our instrument the minimal position was, however, not fully matching the focal length of the lens. Removal of a mounting unit enabled shifting the lens further toward the vacuum window and placing the lens at its “correct” position. This decreased the effective laser spot size dimensions (area of visible material ejection) to values of about 35 µm × 30 µm (hor. × ver.) at typical MALDI fluences, a factor of 1.6–2 above the ion detection threshold (note that the lateral dimensions are fluence-sensitive). The minimum diameter d = 2ω0 to which a free laser beam can be focused depends on several parameters:

ω0 =

2

π

λ

f (λ ) 2 M D

(1)

where f is the focal length of the lens, D the beam diameter or effective diameter of the decisive aperture (whatever is smaller), and M2 the beam parameter product or beam quality factor. For an ideal TEM00 Gaussian laser beam M2 is equal to 1. However, compact lasers as ACS Paragon Plus Environment

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commonly used for MALDI have larger M2 factors. For instance, for the Nd:YAG and N2 lasers used in this study M2 is specified to ≤2. Inspection of Equation (1) suggests three straightforward means of reducing the focal laser spot size: (i) reduction of the focal length f, (ii) increase of the effective diameter D to make use of a larger N.A., and (iii) improvement of the beam quality factor M2. The diagram in Figure S-1B illustrates the measures that were specifically taken for improving the laser focusing for the Synapt instrument (with regard to the beam shaping, a similar approach was previously adopted by Zavalin et al.12): (i) The default focusing lens was replaced by a plano-convex CaF2 lens with a shorter focal length of 125 mm (at λ = 355 nm). (ii) To increase the beam diameter D a 4:1 telescope was assembled. This “beam expander” consists of two fused silica plano-convex lenses (f1 = 50 mm, f2 = 200 mm; φ1,2 = 25 mm); note that use of lenses with smaller focal lengths would presumably allow one to mount the expander inside the MALDI source housing. After beam expansion, the now approximately 8 mm-wide laser beam illuminated the focusing lens by about 30% (by crosssection). (iii) To further improve the beam quality by spatial mode filtering, a circular 30 µmwide aperture made of tungsten was placed in the intermediate image plane. As is illustrated in Figure 1C these measures resulted in minimal effective (elliptical) laser spot sizes of ~12 µm × 8 µm in diameter (at a fluence a factor of 1.6–2 above the ion detection threshold).

MALDI-MS Images and Mass Spectra Recorded from Mouse Brain with Effective Laser Spots of Approximately 35 µm and 10 µm in Diameter Three MALDI-MS images displaying the distribution of phosphatidylinositol (PI, 38:4), sulfatide (ST, d18:1/24:1), and GM1 (d18:1/18:0) in a coronal mouse brain slice at ~50 µm lateral resolution (effective laser spot cross sections, 35 µm × 30 µm; step size, 50 µm × 50



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µm) are plotted in Figure 2A–C. A H&E stain of the same tissue slice prepared after washing off the matrix with CHCl3/CH3OH (2:1, v/v) is shown in Figure 2D for comparison. All compounds were detected in the negative ion mode as deprotonated molecular species [M–H]. The identity of the three species was confirmed by tandem mass spectrometry. MALDI-MS images that were recorded from the cerebellum at the smaller laser spot size of 12 µm × 8 µm are shown in Figure 2E,F and the corresponding H&E stain in Figure 2H; note that these data were recorded from a different intraaural brain slice located approximately 1 mm caudal from the one shown in Figure 2A–D. The high-resolving images confirm the widely complementary distribution of PI (38:4) and ST (d18:1/24:1) in the cerebellum, as was already notable in the overview spectra of Figure 2A,B, and was also discussed in previous studies.8 With regard to GM1 an enrichment is notable in the cortex at both lateral resolutions (Figure 2C,G). As another example, the distribution of ST (d18:1/24:1) in the olfactory bulb system, recorded at ~10 µm resolution, is shown in Figure S-7. Tentatively, the subtle tissue structures seen in both the MSI and the H&E stain could be attributed to the intrabulbar part of anterior commission (white matter) inside the clearly visible neuronal structure. The sponge-like distribution around the neuronal structure might belong to the piriform cortex. In principle, the lateral resolution may be further improved by combining the 10 micron spot with oversampling. However, due to the current limitation on the minimal sample stage step of 3.75 µm this approach was – at present – not further evaluated Summed mass spectra (m/z range 500–1600), that were recorded from the tissue slices used to generate Figure 2A–C and 2G, respectively, are displayed in Figure 3A,C. The mass spectra were acquired from 3600 and 22500 irradiated pixels, respectively, corresponding to scanned areas of 9.0 mm2 and 3.4 mm2 (the total areas of actual material ablation may be


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estimated to 3.0 mm2 and 1.7 mm2); the tissue areas from which the mass spectra were recorded are marked with red and yellow boxes in Figure 2A-C and 2G, respectively. A lower signal intensity (by about a factor of 200) is obtained upon reducing the laser spot diameter. This “over-proportional” reduction in signal intensity (compared to the area ratio of ~10) can be attributed to the well-known spot size effect in MALDI.25,26 Despite of the reduction in ion yield all major lipids (if present) are well detected from a single 10 µm-pixel (Figure 3D). Overall, about 45 distinct endogenous lipid ion species, that produced sufficient ion signal intensities for MALDI-MSI in the negative ion mode, could (tentatively) be identified in the m/z range above 600, based on exact mass and comparison with literature data;27,28 these species are marked by an arrow in Figure 3 and their experimental and theoretical m/z values are summarized in Table S-1A in the Supporting Information. With regard to brain gangliosides, next to GM1 (d18:1/18:0) and GM1 (d18:1/20:0), two disialogangliosides, GD1 (d18:1/18:0) and GD1 (d18:1/20:0) were furthermore detected (Figure 3A,B), that displayed the same spatial distribution in all so far analyzed tissue slices (data not shown); note that the GD1 species are not displayed in Figure 3C,D because the m/z range had in this particular experiment been limited to 1600 to reduce the size of the recorded data files. The intensity of three times sialylated GT1 did generally not suffice to generate useful MSI data, even with the 35 µm-laser spot. Compared to dithranol tests with a few other matrices that were successfully applied in other studies for the MALDI-MS analysis gangliosides (in particular DHB and 2,4-dihydroxyacetophenone29,30) have in our hands so far produced inferior results.

MALDI-MSI with a N2 Laser



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Adopting the same beam shaping approach for the available N2 laser (see Experimental Section) a similar laser spot diameter of ~10 µm was obtained. Qualitatively, also similar MALDI-MS images were obtained. An example, showing the distribution of ST (d18:1/24:1) in the corpus callosum, anterior commission, cerebral cortex, and basal ganglia (zoomed region) of a coronal brain slice, is displayed in Figure 4. Sixty laser pulses (at 30 Hz) were in this case applied per pixel to record the mass spectra.

MALDI-MSI with the Dithranol Matrix in the Positive Ion Mode In line with the work of Le et al.17 dithranol also produced high signal intensities for numerous more basic phospholipids if the positive ion mode was used. Two examples showing

the

distribution

of

(potassiated)

phosphatidylcholine

(PC)

36:1

and

phosphatidylglycerol (PG) 36:2 in the cerebellum are shown in Figure S-8. A list of ~60 positive ions of which the signal intensity was sufficient for generating meaningful MS images and their proposed identity can be found in Table S-1B.

MS Imaging in Tandem MS Mode MALDI-MS images that were recorded in tandem MS mode are presented in Figure 5, a representative MS/MS spectrum is shown in Figure S-9. The mass selecting quadrupole was set to a width of approximately ±3 Da. The motivation behind this was to simulate realistic MSI conditions – generally, the limited amount of material available will not allow numerous tandem MS scans to be performed from the same tissue – and to enable the simultaneous transmission of both ST (d18:1/24:1; m/z 888.65; Figure 5A) and PI (38:4; m/z 885.57; Figure 5B) precursor ions.


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On the basis of the tandem MS data, the sulfatide is readily distinguished by the occurrence of the characteristic SO4- fragment at m/z 96.96. The MS image produced with this ion (Figure 5C) reflects exactly the distribution of its precursor in the tissue. The signal at m/z 283.27 (Figure 5D) corresponds to stearic acid (18:0), released from PI (18:0/20:4). In addition, signals of 18:1 and 18:2 fatty acids are detected with m/z values of 281.25 and 279.23; both show exactly the same lateral distribution in the tissue as the precursor (see Figure 5E for the 18:1 species). It must, therefore, be concluded that PI (38:4) is expressed as a mixture of three isomeric species, namely PI (18:0/20:4,), PI (18:1/20:3), and PI (18:2/20:2). This assumption is corroborated by the presence of the 20:3 and 20:4 fatty acyl fragments, which again show the same lateral distribution (data not shown); the low-abundant 20:2 fragment became only detectable above noise level when a large number of scans were accumulated. Interestingly, the detection of three PI 38:3 isomers is contrasting other work that reported the presence of exclusively PI (18:0/20:4) in rodent brain.29,27 Using our enhanced MALDI tandem MSI option thus offers the intriguing possibility to distinguish between isobaric species which produce different fragmentation patterns.

Speed of Data Acquisition, Mass Resolution, and Ion Mobility Applying 250 laser pulses per pixel at a laser repetition rate of 1 kHz and using a sample stage step size of ~10 µm × 15 µm, the mean data acquisition time per pixel (including the stage movement) was about 0.4 s. For an area of 1 mm2, consisting of 6700 pixels, this corresponds to a total acquisition time of about 45 min. If the same area is scanned with a step size of 50 µm × 50 µm (400 pixels), the time reduces to 200 s (0.5 s/pixel). Using this laser step size is thus a convenient way to obtain overview images over larger tissue sections while still allowing to identify also smaller tissue structures. Overall, these acquisition times per pixel should be comparable to those obtained with current MALDI axial-TOF mass spectrometers ACS Paragon Plus Environment


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and below those consumed if Fourier transform ion cyclotron resonance (FTICR) mass spectrometers are used for high-resolving MSI.31 The Synapt G2-S mass spectrometer can be operated in three different “resolution” modes. Here we chose the “sensitivity mode” as providing the highest sensitivity at a typical mass resolution (FWHM) of ~14,000 (cf. Figure S-10). The initial mass accuracy after calibration was ≤1 ppm in all modes. If a sufficient number of scans is accumulated to achieve a sufficient ion statistics (smooth ion signals) an accuracy in the low ppm range is generally obtained. However, a gradual drift of the calibration with time (typically about 1-4 ppm/h) is notable. Although this is within the normally applied bin width for MSI data acquisition (0.01-0.02 Da), and this operation per se reduces the accuracy in the processed data to 10-20 ppm for the lipid mass range, if a higher accuracy is needed, the original data have to be used and spectra (from a lower scan number) eventually re-calibrated. Using an external lock mass was in our experience not really improving the general performance. Future software solutions might advantageously rather use “internal” lock masses (e.g., matrix-derived ions) and correct only the gradual drift with time, instead of applying a step-wise calibration correction each time an external lock mass is analyzed. Using the “resolution” and “high resolution” (HR) modes, the mass resolution increased to values of ~25,000 and 40,000, respectively (Figure S-10), while the sensitivity dropped concomitantly by factors of about 2 and 10. The MS images and mass spectra shown in Figure S-10 demonstrate that even in the HR mode the sensitivity is, however, high enough to image most lipids, in particular, if the reduced sensitivity was compensated by a slight increase in laser pulse energy. A distinct feature of the Synapt G2-S HDMS instrument is the incorporated travelling wave ion mobility cell. As has been shown by others, using ion mobility can potentially reduce the matrix-derived cluster ion background or separate “isobaric” compounds of ACS Paragon Plus Environment

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sufficiently different geometry.14,32,33 However, because the main focus of the present study was on improving the lateral resolution and to avoid convolution of several different factors, for the time-being we here used the mass spectrometer without ion mobility, thus in principle as a high-resolving QTOF instrument. That MSI at 10 µm-resolution can in principle be combined with ion mobility is, however, demonstrated in Figure S-11, that is again showing the complementary distributions of ST (d18:1/24:1) and PI (38:4) in the cerebellum.

CONCLUSIONS

By use of fundamental laser beam shaping the lateral resolution of MALDI-MS imaging with a MALDI Synapt G2-S instrument can be improved to a true 10 µm-level. Different to most other front-end MALDI mass spectrometers, the QTOF-type instrument is operated with an intermediate pressure ion source. Here we showed that increasing the cooling gas pressure above the default value further improves the collisional cooling conditions, a factor that may be particularly relevant for MALDI-MSI with small laser spot sizes. Dithranol was utilized as an efficient matrix for the analysis of numerous endogenous lipids in both the negative and positive ion mode. The particular fine, homogenous sample coating that is readily obtained with this matrix would lend itself even to measurements with sub 10 µm-wide laser spot diameters. Indeed, we expect that the lateral resolution of the instrument can in the near future be further improved to the 5 µm level, e.g., by using excitation lasers with a lower M2 factor or by mounting the final focusing lens inside the ion source. This would constitute another important step toward achieving a true cellular resolution in MALDI-MS imaging.



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ACKNOWLEGEMENTS

We are thankful to Waters/Micromass (in particular Jeffery Brown, Paul Murray, Andrew Whatley, and Sasha Robbert) for providing a spare MALDI ion source and for sharing technical details, Frauke Spieker for dissection of mouse organs, Ann-Christin Niehoff and Sigrid Große Brinkhaus for helpful discussions on the embedding medium, Shane Ellis for helpful discussions on MALDI matrices, Katja Neukirch for help with the H&E staining, Ivo Klinkert for help with DatacubeExplorer software, Christine Rasch and Jürgen Klingauf for SEM images, Tanja Kuhlmann, Stefanie Albrecht, and Nicholas Schwab for expert support regarding identification of mouse brain areas, and Gottfried Pohlentz for assistance with lipid identification. Financial support by the German Science Foundation (DFG, grants DR 416/8-2 and DR 416/9-1) and the Interdisciplinary Center for Clinical Research (IZKF) Münster (grant Z03) is gratefully acknowledged.

ASSOCIATED CONTENT Additional information as denoted in the text: This material is available free of charge via the Internet at http://pubs.acs.org.


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FIGURE CAPTIONS Figure 1. (A,B) SEM images showing a dithranol-coated mouse brain slice at two different magnifications. (C) Optical microscope images of the ablation craters generated by the laser beam in the dithranol film (step size of the sample stage movement: 15 µm × 10 µm); removed material is differentiated by the brighter color.

Figure 2. MALDI-MS images acquired from coronal mouse brain slices. (A–C) Images obtained from a whole cross-section with a laser spot size of ~35 µm × 30 µm (step size: 50 µm × 50 µm). (E,F and G) Images obtained from two brain slices with an effective laser spot size of 12 µm × 8 µm (step size, 15 µm × 10 µm). The images show the distribution of PI (38:4, m/z 885.57), ST (d18:1/24:1, m/z 888.65) and GM1 (d18:1/18:0, m/z 1544.87). All lipids were detected as [M–H]- species. The red and yellow boxes in (A-C) and (G), respectively, denote the regions of interest used to acquire the mass spectra presented in Figure 3. (D,H): H&E stains obtained from the tissue slices evaluated in A-C and E,F, respectively.

Figure 3. Summed negative ion mode mass spectra, acquired at laser spot sizes of (A) ~35 µm × 30 µm and (C) ~12 µm × 8 µm, and (B,D) representative spectra acquired from two single pixels. Tentatively identified ion signals (cf. Table S-1A) are annotated by an arrow; a dash above an arrow indicates that two or more ion species with different masses are detected at that position. Asterisks denote matrix-derived ions. The two areas from which the mass spectra were recorded are depicted in Figure 2A-C and 2G by red and yellow boxes, respectively.



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Figure 4. MALDI-MS images of a coronal mouse brain slice recorded in the negative ion mode using the N2 laser at an effective laser spot size of about 12 µm × 8 µm (step size 15 µm × 10 µm). Displayed is the distribution of ST (d18:1/24:1, m/z 888.65). Visible structures: Corpus callosum (cc), anterior commission (ac), cerebral cortex (ctx) and basal ganglia (bg). Sixty laser pulses at 30 Hz were applied at each pixel.

Figure 5. MALDI tandem MSI data recorded from the cerebellum in the negative ion mode. A mass selection window of 888.6 ± 3 Da and a collision energy Elab of 70 eV were applied; Ar was used as collision gas. Images are showing the (residual) molecular ions [M–H]- of (A) ST (d18:1/24:1; m/z 888.64) and (B) of PI (38:4; m/z 885.55) and those of three characteristic fragments: (C) SO4- (m/z 96.96), (D) stearic acid (18:0; m/z 283.27), and (E) 18:1 (m/z 281.25, presumably linoleic acid). Data were recorded with a laser spot size of about 18 µm × 12 µm and a step size raster of 20 µm × 20 µm.


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Figure 1 676x255mm (96 x 96 DPI)



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MALDI Mass Spectrometry Imaging of Bioactive Lipids in Mouse Brain

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