**3. Methodology of MALDI-IMS**

The important experimental steps for visualizing endogenous molecules or administered pharmaceutical agents in tissue using MALDI-IMS are sample preparation (such as fixation, sectioning, and washing), choice of matrix and matrix application, measurement, and data analysis. To obtain meaningful biological images, all steps need to be carefully controlled. In this section, the basic experimental MALDI-IMS procedures are described. The schema of MALDI-IMS is presented in Figure 2.

Fig. 2. Schema of MALDI-IMS.

438 Pharmacology

application of matrix results in the *in situ* extraction of molecules from biological tissues. The cocrystal of matrix and analyte molecules in tissue is irradiated with a pulsed laser of appropriate energy, leading to desorption and ionization of the matrix and analyte molecules. The fragmentation of analyte molecules is prevented by the incorporation of the analyte molecules into matrix crystals. The role of the optical absorption of the matrix in the transfer of energy from the laser beam to the analyte molecules is governed by Beer's law, as described previously (Karas et al., 1985). However, the mechanisms underlying the formation of charged matrix and analyte molecules in the MALDI process are not fully

The matrix molecules absorb the laser energy and facilitate desorption and ionization of analyte molecules in the tissue. The homogeneous matrix cover is important for MALDI-IMS, because a heterogeneous distribution of matrix results in different ionization

The important experimental steps for visualizing endogenous molecules or administered pharmaceutical agents in tissue using MALDI-IMS are sample preparation (such as fixation,

understood.

Fig. 1. Schema of MALDI-MS.

**3. Methodology of MALDI-IMS** 

efficiencies of analyte molecules based on their location.

After biological study (a), the tissue of interest should be appropriately isolated (b). A thin section of isolated tissue is mounted on a glass slide (c), coated with matrix (d), and measured by a mass spectrometer (e). The resultant mass spectra (f) can be used for a data mining approach (g). Molecules of interest can be visualized (f) and identified by MS/MS on tissue (f).

## **3.1 Biological sample preparation**

The samples for MALDI-IMS come from a variety of biological sources, including organs, whole animal body dosed with a pharmaceutical compound, or pathological tissues. Optimization of the sample preparation procedure according to the chemical and physical properties of analytes is important. Here, the basic sample preparation steps for MALDI-IMS are described.
