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Strict optimization of matrix application technique seems to be more critical when working with DHB.

The matrix application technique is critical to the success of a matrix-assisted laser desorption/ionization (MALDI) experiment.

The matrix application technique plays a crucial role in the quality of mass spectral images, especially when obtaining high spatial resolution images.

Sublimation is a solvent-free matrix application technique that is becoming more and more popular for mass spectral imaging of metabolites and small molecules.

Improving the matrix application technique for high spatial resolution imaging of small molecules holds promise for better mechanistic understanding of biological pathways and processes in M. truncatula, and the methodology developed for small molecule MSI can be transferred to many other important biological systems and applications.

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Herein, we present a detailed optimization process for three different matrix application techniques with a focus on studying endogenous small molecules.

All three matrix application techniques produce slightly different matrix ion peak patterns; the automatic sprayer and sublimation techniques could be complementary ways to detect low molecular weight metabolites that are masked by high intensity matrix peaks.

This work presents a comprehensive evaluation of three major MALDI matrix application techniques with the two most widely used matrices (DHB and CHCA) for MS imaging of small molecules.

This work presents a systematic study aiming to evaluate three different matrix application techniques for MALDI mass spectrometric imaging (MSI) of endogenous metabolites from legume plant, Medicago truncatula, root nodules.

The combined use of solvent-free (sublimation) and solvent-based (automatic sprayer) matrix application techniques can provide complementary matrix peak profile results, providing the possibility to detect metabolites that were masked by interfering matrix ion peaks in one matrix application method but not masked in the other.

In this work, we optimized and compared the utilization of three matrix application techniques, exploiting the two most widely used MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA), using the metabolome of Medicago truncatula root and nodule tissue as a model.

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