The tissue images and MS data presented in this document were provided courtesy of Dr. Alain Brunelle of the French National Centre for Scientific Research (CNRS), Paris, France.

Application & Background

Mass spectrometry imaging (MSI) is the only analytical method that allows researchers to locate and identify various chemical compounds without selection a priori a chemical class or compound (1,2). Since this method does not require targeting compounds prior to the analysis, it makes it possible to draw anatomical images of any ion detected in the mass spectra in one single experiment. It is known that lipids are important cell signaling molecules, acting as neurotransmitters and precursors in the regulation of various cellular functions (3). Here, we demonstrate lipid imaging in rat brain sections using two different matrices. α-cyano-4-hydroxycinamic acid (CHCA) is an excellent matrix for in situ analysis of lipids on tissue sections in the positive ion mode (4), while in negative ion mode 9-aminoacridine (9-AA) leads to very good signal-to-noise ratio and contrast of the ion images, as well as enhanced capabilities of direct MS/MS measurements (5).

Experimental

Sample Preparation

For imaging experiments, rat brains were immediately frozen on dry ice upon necropsy. The samples were then stored at -80°C until sectioning at 16 μm. Samples were thaw-mounted onto indium-tin oxide (ITO) microscope slides.

Matrix Deposition

Both CHCA and 9-AA were applied to tissue sections using the HTX TM-Sprayer. The unique capability of the HTX TM-Sprayer to spray matrices at different temperatures allowed for optimized protocols for each individual matrix to be used to obtain the best results. Specifically, as the use of 9- AA as an imaging matrix intrinsically leads to a better signal-to- noise ratio, a higher spray temperature, which results in a drier spray, could be used. Additionally, only one pass was needed for the coating of 9-AA to obtain sufficient signal, while two was needed for CHCA. The ability to easily change these parameters between spraying protocols not only helps optimize MSI results, but also saves researchers time in the lab. The spraying parameters used for each matrix are displayed in Table 1.

Table 1. Spray parameters for MALDI MSI in both positive and negative mode. — **Table 1.** Spray parameters for MALDI MSI in both positive and negative mode.

Figure 4. High resolution image of DHB matrix crystal size and coverage on a glass slide when applied with the HTX TM-Sprayer compared to application via sieve. — **Figure 4.** High resolution image of DHB matrix crystal size and coverage on a glass slide when applied with the HTX TM-Sprayer compared to application via sieve.

MALDI Mass Spectrometry Imaging

Spectra were collected across the entire tissue area using the UltrafleXtreme (Bruker Daltoniks, Bremen, Germany) with a tripled-frequency Nd/YAG pulsed laser (355 nm) operating at 2 kHz in reflector mode. The emitted ions were accelerated with a voltage of 20 kV and a delayed extraction time of 120 ns. Analog offset was 1.8 mV. Ten μm laser spot size was set for cerebellum imaging while 50 μm laser spot size was used for the whole sagittal brain section, with 500 laser shots per pixel for both positive and negative ion modes. Mass spectra were acquired over a mass range of m/z 140 to 1200 with a resolution of about 20,000 at m/z 885.6.

DATA PROCESSING

Data acquisition and preprocessing were performed using FlexControl 3.4 and FlexImaging 4.0 (Bruker Daltoniks, Bremen, Germany), respectively. All the spectra were normalized by total ion counts.

Figure 2. Representative mass spectra recorded on rat brain sections in (A) positive ion mode, CHCA matrix, cerebral cortex, (B) positive ion mode, CHCA matrix, corpus callosum (white matter), (C) negative ion mode, 9-AA matrix, corpus callosum (whi… — **Figure 2.** Representative mass spectra recorded on rat brain sections in (A) positive ion mode, CHCA matrix, cerebral cortex, (B) positive ion mode, CHCA matrix, corpus callosum (white matter), (C) negative ion mode, 9-AA matrix, corpus callosum (white matter), and (D) negative ion mode, 9-AA matrix, cortex. Many lipid peaks are visible in spectra.

Results & cONCLUSIONS

MALDI MS images of compounds detected in the agar and the bacterial colony when matrix was applied using the method above were compared to images acquired when the matrix was applied via dry coating with a sieve.

Figure 3. MALDI MSI image of rat brain recorded in negative ion mode (9-AA matrix). Two color overlay, Red m/z 885.55 ([PI38:4-H]-) Green m/z 888.63 ([ST42:2-H]-). Pixel size: 50 µM, acquisition time ~15 h. Scale bar represents 2 mm. — **Figure 3.** MALDI MSI image of rat brain recorded in negative ion mode (9-AA matrix). Two color overlay, Red *m/z* 885.55 ([PI38:4-H]-) Green *m/z* 888.63 ([ST42:2-H]-). Pixel size: 50 µM, acquisition time ~15 h. Scale bar represents 2 mm.

Figure 4. MALDI MSI image of rat brain recorded in positive ion mode (CHCA matrix). Two color overlay, Red m/z 772.7 ([PC32:0+K]+); Green m/z 826.8 ([PE40:0+Na]+). Pixel size: 50 µM, acquisition time ~15 h. Scale bar represents 2 mm. — **Figure 4.** MALDI MSI image of rat brain recorded in positive ion mode (CHCA matrix). Two color overlay, Red *m/z* 772.7 ([PC32:0+K]+); Green *m/z* 826.8 ([PE40:0+Na]+). Pixel size: 50 µM, acquisition time ~15 h. Scale bar represents 2 mm.

Figure 5. MALDI MSI image of rat cerebellum recorded in negative ion mode (9-AA matrix), Three color overlay, Red m/z 885.55 ([PI38:4-H]-); Green m/z 888.63 )[ST42:2-H]-); Blue m/z 806.55 ([ST36:1-H]-). Pixel size 10 µm, acquisition time ~5 h. Scal… — **Figure 5.** MALDI MSI image of rat cerebellum recorded in negative ion mode (9-AA matrix), Three color overlay, Red m/z 885.55 ([PI38:4-H]-); Green *m/z* 888.63 )[ST42:2-H]-); Blue *m/z* 806.55 ([ST36:1-H]-). Pixel size 10 µm, acquisition time ~5 h. Scale bar represents 500 µm.

Figure 6. MALDI MSI image of rat cerebellum recorded in positive ion mode (CHCA matrix). Two color overlay, Red m/z 772.7 ([PC32:0+K]+); Green m/z 826.8 ([PE40:0+Na]+). Pixel size 10 µm, acquisition time ~5.5 h. Scale bar represents 500 µm. — **Figure 6.** MALDI MSI image of rat cerebellum recorded in positive ion mode (CHCA matrix). Two color overlay, Red m/z 772.7 ([PC32:0+K]+); Green *m/z* 826.8 ([PE40:0+Na]+). Pixel size 10 µm, acquisition time ~5.5 h. Scale bar represents 500 µm.

references

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