Correlative mass spectrometry imaging, applying time‐of‐flight secondary ion mass spectrometry and atmospheric pressure matrix‐assisted laser desorption/ionization to a single tissue section

Rationale Mass spectrometry imaging (MSI) is a powerful tool for mapping the surface of a sample. Time‐of‐flight secondary ion mass spectrometry (TOF‐SIMS) and atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI) offer complementary capabilities. Here, we present a workflow to apply both techniques to a single tissue section and combine the resulting data for the example of human colon cancer tissue. Methods Following cryo‐sectioning, images were acquired using the high spatial resolution (1 μm pixel size) provided by TOF‐SIMS. The same section was then coated with a para‐nitroaniline matrix and images were acquired using AP‐MALDI coupled to an Orbitrap mass spectrometer, offering high mass resolution, high mass accuracy and tandem mass spectrometry (MS/MS) capabilities. Datasets provided by both mass spectrometers were converted into the open and vendor‐independent imzML file format and processed with the open‐source software MSiReader. Results The TOF‐SIMS and AP‐MALDI mass spectra show strong signals of fatty acids, cholesterol, phosphatidylcholine and sphingomyelin. We showed a high correlation between the fatty acid ions detected with TOF‐SIMS in negative ion mode and the phosphatidylcholine ions detected with AP‐MALDI in positive ion mode using a similar setting for visualization. Histological staining on the same section allowed the identification of the anatomical structures and their correlation with the ion images. Conclusions This multimodal approach using two MSI platforms shows an excellent complementarity for the localization and identification of lipids. The spatial resolution of both systems is at or close to cellular dimensions, and thus spatial correlation can only be obtained if the same tissue section is analyzed sequentially. Data processing based on imzML allows a real correlation of the imaging datasets provided by these two technologies and opens the way for a more complete molecular view of the anatomical structures of biological tissues.

analyzer. These two techniques allow access to the distribution of several classes of biomolecules from the surface of a tissue section. [2][3][4][5] Time-of-flight (TOF)-SIMS involves the bombardment of a sample by a focused beam of mono-or polyatomic ions, which induces desorption/ionization of secondary ions from the surface of the sample. [6][7][8] It also offers the possibility of localizing various ions produced from molecules, mainly lipids, drugs, xenobiotics and metabolites, with m/ z up to 1000-1500, good mass resolution (M/ΔM = 8000 (full width at half maximum) at m/z > 500) and a high lateral resolution from 400 nm to 1-2 μm. This makes TOF-SIMS a method of choice for the micrometric-scale analysis of lipids or other kinds of small molecules in biological samples. 5 Moreover, no matrix coating is required, i.e. no surface modification is made. One of the main important breakthroughs in TOF-SIMS during the last fifteen years concerns polyatomic ion sources. The introduction of such polyatomic ion sources and, in particular, ion guns providing metal clusters (e.g. bismuth and gold clusters) has improved the desorption/ionization of intact ions from molecules, significantly expanding the application of TOF-SIMS from a mapping tool of elements or small mass fragments to a powerful molecular microscope used in various fields, ranging from materials characterization to biological tissue imaging. 6,[8][9][10] Despite this improvement, two main limitations still exist: the high fragmentation rate induced by the high collision energy from the primary ion beam and the lack of tandem mass spectrometry capabilities. [11][12][13] After the first attempts a few years ago, 14,15 the latter issue is going to be addressed in the near future, with the recent advent of SIMS instruments with TOF/TOF and/or high-resolution Orbitrap mass analyzers. 16,17 MALDI imaging was described initially by Spengler et al 18,19 and tissue imaging was first shown by Caprioli et al. 20 Until recently, the main limitation of the MALDI method for MSI was its spatial resolution, which was typically in the range 50-200 μm. The Spengler group developed an efficient atmospheric pressure scanning microprobe MALDI method with a focused laser beam providing a high spatial resolution of 1.4 μm. 21 Moreover, coupling this with an orbital trapping mass spectrometer offers high mass resolution, mass accuracy and MS/MS capabilities. 21 In addition, an atmospheric pressure (AP)-MALDI ion source is perfectly suited for investigating biological samples and allows the detection of a wide range of biomolecule classes, including metabolites, 22,23 lipids 24 and peptides/proteins. 25 MSI is now widely used in many applications, mainly in biological sciences and medical research, [26][27][28][29] but also in cultural heritage research. 30 Correlated imaging has become an emerging strategy to combine complementary information from different analytical techniques. 31 The Cooks group combined desorption electrospray ionization (DESI) and MALDI imaging using a single tissue section, 32 achieving lipid and protein imaging by DESI-MS and MALDI-MS, respectively. 32 Despite the improvement concerning polyatomic ion sources, Brunelle et al showed the need to combine molecular information from TOF-SIMS and MALDI-MS imaging, and the possibility of performing a MALDI imaging experiment on the same sample after TOF-SIMS imaging. 33 Eijkel et al combined MALDI and SIMS imaging datasets applied to human cerebellum tissue. 34 Touboul et al also combined these two imaging techniques to study skin and kidney biopsies of patients suffering from Fabry disease by mapping globotriaosylceramides and digalactosylceramides, 35 showing good complementarity between the two techniques based on the identification and localization of biomolecules. In addition, Chughtai et al combined the elemental and small-molecule distribution provided by high lateral resolution SIMS with the specific distribution of lipids and peptides/proteins provided by MALDI for the study of musculoskeletal tissue. 36 Imaging dataset processing is a great challenge. The main difficulty for biologists or clinicians is to analyze, merge, compare and correlate data provided by different instruments on the same platform. Moreover, MSI data comprise a complex and huge dataset containing all relevant properties correlated to the mass spectral data. Vendors of MS instruments and many bio-informatics groups have come up with several pieces of software to analyze MSI datasets. Consequently, a common data format known as imzML 37 has been developed over the past few years. 38 The vendor-neutral data format imzML facilitates the flexible sharing of MSI data and their visualization into various software tools available without restriction to a proprietary vendor. 38 Additional details are provided in Roempp et al. 39 One of the most relevant examples is the data processing of a multicenter study. 40 The authors analyzed adjacent sections of mouse brain in five laboratories situated mainly in Europe and the USA. Five different instruments were used: MALDI-TOF/TOF, Orbitrap, QTOF, FT-ICR and TOF-SIMS. The imaging dataset was converted into imzML format using the appropriate converter tools 37 and displayed in a common open-source software to facilitate exchange and the comparison. 40 In the study reported here, we defined a workflow based on the investigation of lipids combining TOF-SIMS and AP-MALDI-Orbitrap.
In addition, this multimodal approach using these two imaging methods offers a strong complementarity, due, on the one hand, to the precise localization of biomolecules by the high spatial resolution provided by

| TOF-SIMS imaging
The experiments were performed using a commercial TOF-SIMS IV mass spectrometer (ION-TOF GmbH, Münster, Germany). This mass spectrometer, described in detail elsewhere, 8 is fitted with a bismuth liquid metal ion gun delivering Bi n q+ bismuth cluster ions (Bi 3 + ions were selected). A low-energy electron flood gun was activated between two primary ions pulses to neutralize the sample surface, causing only minimum damage. 41 Only one mode of operation of the primary ion column was used during the experiments, which is called a "high-current bunched mode", 34 Consequently, in this mode the pixel stepsize was smaller than the beam diameter (2 μm), leading to oversampling. Another mode of operation could be used, which combines a higher spatial resolution of ca 400 nm and a mass resolution of M/ΔM = 8 × 10 3 , thanks to a delayed extraction of the secondary ions. 43 However in the present case the "high-current bunched mode" was preferred because it ensures the fastest acquisition time. Under these conditions, the fluence (also called the primary ion dose density) was maintained at 5.0 × 10 11 ions cm −2 , which is below the so-called static SIMS limit. 44 Because of the very low initial kinetic energy distribution of the secondary ions, the relationship between the TOF and the square root accuracy. 45,46 The data acquisition software used was SurfaceLab 6.2 (ION-TOF GmbH).

| AP-MALDI-MS imaging
After the static SIMS imaging experiments, a uniform matrix layer (pNA, 10 mg mL −1 in 1:1 acetone/water, 0.1% trifluoroacetic acid) was applied to the section using a pneumatic sprayer. 47 The MALDI-MS imaging analyses were performed using a high lateral resolution atmospheric pressure imaging ion source (AP-MALDI10, TransMIT GmbH, Giessen, Germany) coupled to an orbital trapping mass spectrometer (Q Exactive, Thermo Fisher Scientific GmbH, Bremen, Germany). 21 The mass spectrometer was operated in positive ion mode at a mass resolution of 140,000 at m/z 200 over a mass range of m/z 700 to 900. The ion source was equipped with a nitrogen laser (λ = 337 nm), operating at a repetition rate of 60 Hz, for desorption/ ionization. A useful spatial resolution from biological tissue down to a pixel size of 5 μm has been reported using this ion source. 2  Representative mass spectra for each mode are shown in Figure 2.
The mass spectra were acquired in positive and negative ion modes with TOF-SIMS (Figures 2A and 2B) and in positive ion mode only with AP-MALDI-MS ( Figure 2C) in the infiltrated submucosa.
The phosphatidylcholine head group, cholesterol and vitamin E were  Table S1 (supporting information).
Colon cancer spreads through the mucosa layer to the submucosa layer. Cancer cells infiltrate the submucosa and modify the cellular and extracellular composition. 53 Figure 3A shows This demonstrates that the described workflow results in a high reproducibility and can be applied to other tissue types.