Sample preparation is a crucial step in producing reproducible and reliable mass spectral images. The quality of the images greatly depends upon factors such as tissue fixing methods, embedding medium, and slice thickness. Microwave irradiation and heat denaturation with the Denator Stabilizer T1 (Gothenburg, Sweden) have been reported to prevent postmortem protein degradation by deactivating proteolytic enzymes [11]. For imaging applications, ideal section thickness should be the width of one cell; 8–20 μm thickness is appropriate. Thicker sections tend to have better tissue integrity (i.e., less tearing or folding) but thinner sections typically result in better sensitivity.

MALDI requires deposition of an organic, crystalline compound, typically a weak acid, on the tissue of interest to assist analyte ablation and ionization [12]. Choosing a MALDI matrix and its application method is essential for quality mass spectrometric imaging experiments. Conventional matrices include CHCA (α-cyano-4-hydroxycinnamic acid) and DHB. Less traditional matrices such as DAN (1,5-diaminonaphthalene), DMAN (1,8-bis(dimethylamino)naphthalene), DHPT (2,3,4,5-tetra(3′,4′-dihydroxylphenyl)thiophene), TiO₂ nanoparticles, and ionic matrices are being used and are reported to improve spectral quality, crystallization, and vacuum stability [13–17]. Different matrices provide different amounts of coverage, signal intensity, matrix interference, and ionization efficiency. It is important to choose a matrix that gives the best results for the particular analytes of interest. The matrix application technique also plays a role in the quality of mass spectral images. Three matrix application methods are presented here: airbrush, automatic sprayer, and sublimation. Airbrush matrix application has been widely used in MALDI imaging and is relatively fast and easy, but is less reproducible and sometimes causes diffusion of analytes [18]. Automatic sprayer systems, like the TM-Sprayer, have been developed which remove the variability seen with manual airbrush application, making the spray more reproducible, but is more time consuming. Sublimation is a dry matrix application technique that is becoming more and more popular for mass spectral imaging of metabolites and small molecules [19]. Sublimation reduces analyte diffusion, but lacks the solvent necessary to observe higher mass compounds.