**2. MALDI-MS**

MALDI-MS was developed from laser desorption/ionization mass spectrometry (LDI-MS). The first LDI-MS experiment for high-mass molecules was reported in 1987 (Tanaka et al., 1987). In this experiment, a powder of cobalt metal in glycerol was used for the observation of ions with a mass to charge (*m/z*) ratio of 34,000. Soon afterward, MALDI-MS results of serum albumin (67,000 Da) were reported using nicotinic acid as the matrix (Karas & Hillenkamp, 1988). It was reported that MALDI-MS can detect a wide range of molecules ranging from small (*m/z* <1000) to large molecules (*m/z* >1,000,000) (Yates, 1998). The schema of MALDI-MS is shown in Figure 1.

In routine MALDI-MS analysis (i.e., non-imaging analysis), the analyte can be mixed with an excess of matrix. On the other hand, molecular imaging of tissue sections using MALDI-IMS requires the tissue surface to be homogeneously covered by a matrix. On-tissue

Application of Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry 439

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

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).

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-

Collection and treatment procedures need to be sufficiently fast to prevent rapid tissue degradation, because the sample degradation process starts immediately after the cessation of blood flow. The most preferred sample for MALDI-IMS is a chemically unmodified freshfrozen one. Fresh-frozen samples can be prepared using dry ice, liquid nitrogen, or liquid nitrogen-chilled isopentane, and can be preserved in a deep freezer until required. The samples should be well sealed to prevent drying during storage, and it is important to

ensure that the tissue section morphology is well preserved before MALDI-IMS.

MALDI-IMS is presented in Figure 2.

Fig. 2. Schema of MALDI-IMS.

**3.1 Biological sample preparation** 

**3.1.1 Sample condition for MALDI-IMS** 

IMS are described.

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 understood.

Fig. 1. Schema of MALDI-MS.

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 efficiencies of analyte molecules based on their location.
