**1. Introduction**

Insect molecular biology studies the molecular basis of biological processes in insects, including molecular synthesis, modification, mechanisms, and interactions [1]. Metabolites play key roles among all these aspects of insect molecular biology. Therefore, understanding the distribution of metabolites contributes to revealing the mechanisms of insect biology, including ontogeny, metabolism, and physiology. Research methodologies such as liquid chromatography-mass spectrometry (LC-MS) and immunofluorescence are generally used in visualizing the distribution of metabolites. However, all of them have their shortcomings. LC-MS or gas chromatography-mass spectrometry (GC**-**MS) usually uses the homogenate of a certain weight of specific tissue(s) or organ(s), resulting in losing *in situ* spatiotemporal information. Insect body sizes are mostly small, let alone certain tissues; so tissue-specific researches, in most case, consume a large number of insect individuals [2–4]. On the other hand, ordinary *in situ* characterization technologies such as immunofluorescence assay and fluorescence *in situ* hybridization (FISH) require labeling at specific biomolecules [5–7]. Hence, operating processes such as synthesizing probes and antibodies are usually time-consuming, inefficient, and limited to only one molecule.

As a new molecular visualization technology, mass spectrometry imaging (MSI) has drawn more and more attention in recent years. MSI can visualize the spatial distribution of molecules in specific samples without any labeling and enable simultaneous evaluation and identification of hundreds of molecules *in situ*. In comparison with LC-MS and GC-MS, MSI requires only one sample for biomolecular localization, which makes it a powerful tool to visualize the changes in organism physiology and biochemistry. The basic principle of MSI is to scan target samples such as tissue slice for desorption and ionization of molecules or ions on the surface of samples by a laser or a high-energy ion beam [8]. Mass analyzer obtains mass-to-charge ratio (m/z) and ion intensity of the molecules or ions from pixels. Mass peaks are obtained from the database of imaging software such as FlexImaging and used to visualize one-dimensional linear profiling, two-dimensional spatial distribution of molecules, or threedimensional anatomic structure [8]. MSI has been widely applied in life sciences, such as histology [9, 10]; pathology [11, 12]; pharmacology [13, 14]; food science [15]; botany [16–19]; and microbiology [20, 21].

In this chapter, we introduce the major types of *in situ* measurement by MSI and present an example of matrix-assisted laser desorption ionization (MALDI) to elucidate the operating processes. We also discuss the advances of MSI in insect physiology and biochemistry to better promote the research in entomology.

#### **2. Mass spectrometry imaging method**

Among all the MSI technologies, we can divide them into two major groups, vacuum ionization mass spectrometry imaging system and ambient ionization mass spectrometry imaging system, based on whether the environment of the instruments is a vacuum [8]. Based on desorption or ionization ion, vacuum ionization mass spectrometry imaging system can be further divided into different categories, namely MALDI [22], secondary ion mass spectrometry (SIMS) [23], surface-assisted laser desorption ionization (SALDI) [24], and laser desorption ionization (LDI) [25]; ambient ionization mass spectrometry imaging system can be further divided into different categories, namely desorption electrospray ionization (DESI) [26], laser ablation electrospray ionization (LAESI) [27], laser electrospray mass spectrometry (LEMS) [28], electrospray laser desorption ionization (ELDI) [28], atmospheric pressure matrix-assisted laser desorption ionization (AP-SMALDI) [29], and air flow-assisted ionization (AFAI) [30]. Among all these above-mentioned technologies, MALDI-MSI is the most popular technology in life science research because it not only can be applied to a wide range from inorganic ion, small molecules to proteins but also has the characteristics of high accuracy and sensitivity [31]. Here, we provide a further explanation of the basic principle of MADLI-MSI and elucidate the workflow for MALDI.

The basic working principle of MALDI is that target analytes on the surface of tissue are crystallized with matrix (e.g., α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxyacetophenone) to form a complex. When the complex is exposed by infrared laser at 2.94 or 10.6 μm and/or ultraviolet laser at 337, 355, or 266 nm, it absorbs the laser energy and converts these analytes into a phase of gas, which causes molecule ionization. The ionized molecules automatically enter a mass spectrometer where the molecules are detected and mapped [19].

A typical experimental workflow for MALDI is as follows (**Figure 1**):

a.Insect tissues are flash-frozen (with or without fixation) in an embedding media with gelatin, carboxymethylcellulose, or water;

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**Figure 1.**

*MALDI-MSI imaging workflow.*

*Current Advances in Mass Spectrometry Imaging for Insect Physiology and Metabolism*

b.Each sample is cryo-sectioned at 10–20 μm thickness and mounted onto glass slides coated with indium tin oxide, which is then lyophilized for tissue

c.The lyophilized slide is subject to three irregular fiducial markings on the

*DOI: http://dx.doi.org/10.5772/intechopen.92584*

surface of each sample for localization;

imaging;

*Current Advances in Mass Spectrometry Imaging for Insect Physiology and Metabolism DOI: http://dx.doi.org/10.5772/intechopen.92584*


**Figure 1.** *MALDI-MSI imaging workflow.*

*Pests, Weeds and Diseases in Agricultural Crop and Animal Husbandry Production*

[15]; botany [16–19]; and microbiology [20, 21].

**2. Mass spectrometry imaging method**

MADLI-MSI and elucidate the workflow for MALDI.

spectrometer where the molecules are detected and mapped [19].

media with gelatin, carboxymethylcellulose, or water;

A typical experimental workflow for MALDI is as follows (**Figure 1**):

a.Insect tissues are flash-frozen (with or without fixation) in an embedding

As a new molecular visualization technology, mass spectrometry imaging (MSI) has drawn more and more attention in recent years. MSI can visualize the spatial distribution of molecules in specific samples without any labeling and enable simultaneous evaluation and identification of hundreds of molecules *in situ*. In comparison with LC-MS and GC-MS, MSI requires only one sample for biomolecular localization, which makes it a powerful tool to visualize the changes in organism physiology and biochemistry. The basic principle of MSI is to scan target samples such as tissue slice for desorption and ionization of molecules or ions on the surface of samples by a laser or a high-energy ion beam [8]. Mass analyzer obtains mass-to-charge ratio (m/z) and ion intensity of the molecules or ions from pixels. Mass peaks are obtained from the database of imaging software such as FlexImaging and used to visualize one-dimensional linear profiling, two-dimensional spatial distribution of molecules, or threedimensional anatomic structure [8]. MSI has been widely applied in life sciences, such as histology [9, 10]; pathology [11, 12]; pharmacology [13, 14]; food science

In this chapter, we introduce the major types of *in situ* measurement by MSI and present an example of matrix-assisted laser desorption ionization (MALDI) to elucidate the operating processes. We also discuss the advances of MSI in insect physiology and biochemistry to better promote the research in entomology.

Among all the MSI technologies, we can divide them into two major groups, vacuum ionization mass spectrometry imaging system and ambient ionization mass spectrometry imaging system, based on whether the environment of the instruments is a vacuum [8]. Based on desorption or ionization ion, vacuum ionization mass spectrometry imaging system can be further divided into different categories, namely MALDI [22], secondary ion mass spectrometry (SIMS) [23], surface-assisted laser desorption ionization (SALDI) [24], and laser desorption ionization (LDI) [25]; ambient ionization mass spectrometry imaging system can be further divided into different categories, namely desorption electrospray ionization (DESI) [26], laser ablation electrospray ionization (LAESI) [27], laser electrospray mass spectrometry (LEMS) [28], electrospray laser desorption ionization (ELDI) [28], atmospheric pressure matrix-assisted laser desorption ionization (AP-SMALDI) [29], and air flow-assisted ionization (AFAI) [30]. Among all these above-mentioned technologies, MALDI-MSI is the most popular technology in life science research because it not only can be applied to a wide range from inorganic ion, small molecules to proteins but also has the characteristics of high accuracy and sensitivity [31]. Here, we provide a further explanation of the basic principle of

The basic working principle of MALDI is that target analytes on the surface of tissue are crystallized with matrix (e.g., α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxyacetophenone) to form a complex. When the complex is exposed by infrared laser at 2.94 or 10.6 μm and/or ultraviolet laser at 337, 355, or 266 nm, it absorbs the laser energy and converts these analytes into a phase of gas, which causes molecule ionization. The ionized molecules automatically enter a mass

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h.Hematoxylin-eosin staining is optional for displaying tissue localization.
