**3. Alternative matrix materials**

As an alternative to the traditional organic acid matrices, contemporary studies have returned to the inorganic materials that were originally proposed for MALDI-MS by Tanaka *et al*. [45]. Nanoparticles (NPs) made of gold [46–49], silver [50–53], carbon based substrates [54–57], and silicon surfaces [58, 59] have been demonstrated on the target plate as materials that facilitate ionization of biomolecules. In particular, gold NPs (AuNPs) have the potential to be a more universal material to help facilitate ionization of small molecules and seem to have fewer reproducibility issues across multiple instrument platforms, locations, and organisms/biofluids [46, 60, 61]. Specific advantages for ionization of small molecules using AuNPs on the target plate include: (i) less chemical noise in the range where small molecules are found (below *m/z* 300), (ii) flexible analyte solution preparation conditions, including tolerances for salts, surfactants, and pH, and (iii) broad applicability across chemical classes [46, 48, 57, 60, 62].

The general success, though not broad usage, of these materials on the target plate have led to several different approaches for using alternative inorganic

**103**

embryos.

*Mass Spectrometry Imaging of Neurotransmitters DOI: http://dx.doi.org/10.5772/intechopen.93835*

which are described in the paragraphs below.

the molecules detected is shown in **Figure 2**.

acids, nucleotide precursors, and more in mouse colon [71].

materials for MSI, such as sputtering of metals or the use of metallic NPs, both of

Sputtering of metals over tissue sections has produced a number of quality articles that have utilized silver [50], platinum [63], and gold [64]. Sputtering deposits highly pure and homogeneous metal or metal oxide nanolayers onto biological tissue sections. Magnetron sputtering systems utilize a plasma gun under high- or ultra-high vacuum and deposits layers of metal onto the substrate of interest. Deposition times range from under a minute to several minutes, with total sample preparation time at least several minutes long because of the need for a vacuum-based system. Sputtered layers of silver or gold are typically reported in the 20-50 nm range [49, 50, 65], which is a narrower size distribution than solutionbased NPs. The biggest disadvantages of sputtering are the need for expert users, the time involved for sample preparation, and the equipment cost (e.g., sputtering

apparatuses are up to tenfold more expensive than pneumatic sprayers).

Molecules that have been successfully detected using Ag or Au sputtering experiments are largely neutral lipids, with cholesterol being of high interest [49, 50, 52, 66]. Pt sputtering has been demonstrated on lipids in tissue [67] and in leaves where metabolites of interest were detected, including many with molecular features similar to NTs, such as acetamiprid [63]. Rafols *et al.* showed an Au sputtering MSI experiment that resulted in the potential detection of 25 different compounds, but only 1 of which could be called a small molecule metabolite (i.e., citrulline) [64]. A significant advantage that sputtering demonstrates, compared to organic acid matrices [22], is the lack of analyte delocalization [64]. The largest survey of sputtering materials was done by Hansen *et al.,* where Ag, Au, Cu, Ni, Pt, and Ti were sputtered for varying times on plant tissues [68]. Noble metals (e.g., Ag, Au, Pt) were found to be more effective than transition metals (e.g., Cu, Ni, Ti) for overall ionization in positive- and negative-ion modes. While lipids were the most prevalent biomolecule class examined, this is a rare demonstration of the detection of amine-based structures, including choline, asparagine, glutamic acid, and leucine. DHB was used for comparison in positive-ion mode and DAN in negative-ion mode, with primarily insoluble lipids being effectively ionized with organic matrices. A summary of

Nanomaterials in suspension form, such as colloidal NPs, could potentially be deposited or sprayed onto tissue sections for analysis, yet there are only select demonstrations of this application for MSI, which are described herein. This area of research has again been applied primarily to lipidomics, with successful detection of fatty acids and their derivatives, sterols, phospholipids (e.g., phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, etc.), sphingomyelins, and ceramides. Silver NPs (AgNPs), including those that have been surface-derivatized are most popular. Polyvinylpyrrolidone-capped AgNPs have been utilized for analysis of brain [69], and AuNPs with alkylamine surface modifications have been used for imaging of glycosphingolipids in the brain [70]. Fluorinated AuNPs have been shown to detect carbohydrates, lipids, bile acids, sulfur metabolites, amino

Small molecule examples, specifically NTs, are notably lacking in the MSI literature. We have been developing a method for the detection of endogenous NTs from biological samples using citrate-capped AuNPs that are pneumatically sprayed on tissue sections [47]. Successful detection of acetylcholine, dopamine/octopamine, epinephrine, glutamine, GABA, norepinephrine, and serotonin was achieved in rabbit brain tissue sections, zebrafish embryos, and neuroblastoma cells [61, 72]. See **Figure 3** for an image of seven different NTs in 5 day-post fertilization zebrafish

#### *Mass Spectrometry Imaging of Neurotransmitters DOI: http://dx.doi.org/10.5772/intechopen.93835*

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

**Matrix and structure Common analytes** Sinapinic acid (SA) Proteins [12–15]

2,5-dihydroxybenzoic acid (DHB) Peptides [16–18], lipids [19, 20],

**α**-cyano-4-hydroxycinnamic acid (CHCA) Neuropeptides [23], peptides [24],

N-(1-napthyl)ethylenediamine dihydrochloride (NEDC)• 2HCl Small molecule metabolites [29],

1,5-diaminonapthalene (DAN) Lipids [31–33], small molecule

some small molecules [4, 21, 22]

proteins [25], lipids [26, 27],

drugs [28]

lipids [30]

metabolites [34]

**102**

**Table 1.**

**3. Alternative matrix materials**

*Common MSI organic acid matrices and their applications.*

applicability across chemical classes [46, 48, 57, 60, 62].

As an alternative to the traditional organic acid matrices, contemporary studies have returned to the inorganic materials that were originally proposed for MALDI-MS by Tanaka *et al*. [45]. Nanoparticles (NPs) made of gold [46–49], silver [50–53], carbon based substrates [54–57], and silicon surfaces [58, 59] have been demonstrated on the target plate as materials that facilitate ionization of biomolecules. In particular, gold NPs (AuNPs) have the potential to be a more universal material to help facilitate ionization of small molecules and seem to have fewer reproducibility issues across multiple instrument platforms, locations, and organisms/biofluids [46, 60, 61]. Specific advantages for ionization of small molecules using AuNPs on the target plate include: (i) less chemical noise in the range where small molecules are found (below *m/z* 300), (ii) flexible analyte solution preparation conditions, including tolerances for salts, surfactants, and pH, and (iii) broad

The general success, though not broad usage, of these materials on the target plate have led to several different approaches for using alternative inorganic

materials for MSI, such as sputtering of metals or the use of metallic NPs, both of which are described in the paragraphs below.

Sputtering of metals over tissue sections has produced a number of quality articles that have utilized silver [50], platinum [63], and gold [64]. Sputtering deposits highly pure and homogeneous metal or metal oxide nanolayers onto biological tissue sections. Magnetron sputtering systems utilize a plasma gun under high- or ultra-high vacuum and deposits layers of metal onto the substrate of interest. Deposition times range from under a minute to several minutes, with total sample preparation time at least several minutes long because of the need for a vacuum-based system. Sputtered layers of silver or gold are typically reported in the 20-50 nm range [49, 50, 65], which is a narrower size distribution than solutionbased NPs. The biggest disadvantages of sputtering are the need for expert users, the time involved for sample preparation, and the equipment cost (e.g., sputtering apparatuses are up to tenfold more expensive than pneumatic sprayers).

Molecules that have been successfully detected using Ag or Au sputtering experiments are largely neutral lipids, with cholesterol being of high interest [49, 50, 52, 66]. Pt sputtering has been demonstrated on lipids in tissue [67] and in leaves where metabolites of interest were detected, including many with molecular features similar to NTs, such as acetamiprid [63]. Rafols *et al.* showed an Au sputtering MSI experiment that resulted in the potential detection of 25 different compounds, but only 1 of which could be called a small molecule metabolite (i.e., citrulline) [64]. A significant advantage that sputtering demonstrates, compared to organic acid matrices [22], is the lack of analyte delocalization [64]. The largest survey of sputtering materials was done by Hansen *et al.,* where Ag, Au, Cu, Ni, Pt, and Ti were sputtered for varying times on plant tissues [68]. Noble metals (e.g., Ag, Au, Pt) were found to be more effective than transition metals (e.g., Cu, Ni, Ti) for overall ionization in positive- and negative-ion modes. While lipids were the most prevalent biomolecule class examined, this is a rare demonstration of the detection of amine-based structures, including choline, asparagine, glutamic acid, and leucine. DHB was used for comparison in positive-ion mode and DAN in negative-ion mode, with primarily insoluble lipids being effectively ionized with organic matrices. A summary of the molecules detected is shown in **Figure 2**.

Nanomaterials in suspension form, such as colloidal NPs, could potentially be deposited or sprayed onto tissue sections for analysis, yet there are only select demonstrations of this application for MSI, which are described herein. This area of research has again been applied primarily to lipidomics, with successful detection of fatty acids and their derivatives, sterols, phospholipids (e.g., phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, etc.), sphingomyelins, and ceramides. Silver NPs (AgNPs), including those that have been surface-derivatized are most popular. Polyvinylpyrrolidone-capped AgNPs have been utilized for analysis of brain [69], and AuNPs with alkylamine surface modifications have been used for imaging of glycosphingolipids in the brain [70]. Fluorinated AuNPs have been shown to detect carbohydrates, lipids, bile acids, sulfur metabolites, amino acids, nucleotide precursors, and more in mouse colon [71].

Small molecule examples, specifically NTs, are notably lacking in the MSI literature. We have been developing a method for the detection of endogenous NTs from biological samples using citrate-capped AuNPs that are pneumatically sprayed on tissue sections [47]. Successful detection of acetylcholine, dopamine/octopamine, epinephrine, glutamine, GABA, norepinephrine, and serotonin was achieved in rabbit brain tissue sections, zebrafish embryos, and neuroblastoma cells [61, 72]. See **Figure 3** for an image of seven different NTs in 5 day-post fertilization zebrafish embryos.

#### **Figure 2.**

*Summary of sputter-coated metal screening for small metabolite analysis in (a) positive and (b) negative ion mode. Asterisks indicate a fragment ion was detected. Reprinted with permission from Ref. [68]. Copyright 2018 American Society for Mass Spectrometry.*

With the intention of demonstrating the flexibility of AuNPs in terms of sample preparation, spraying parameters have been explored, including matrix concentration, solvent composition, spray temperature, and linear flow rate (which determines overall spray density of material). Early data and statistical analysis by principal component analysis (PCA) suggests that: (i) AuNP concentration can be varied over 4 orders of magnitude, (ii) a variety of organic:aqueous solvent mixes are possible, and (iii) temperatures from 30 to 60°C can be utilized [73, 74]. The flexibility in spray parameters means that less-experienced users with varying spraying capabilities can still get optimum data from their tissue sections, opening the application of MSI to more areas of study.

As previously mentioned, delocalization is an issue with organic acid matrices, often because of a "wet" matrix spray that results in true molecular diffusion instead of analyte extraction from the tissue. A standard literature method for CHCA has been compared with AuNPs, where CHCA gives only extreme delocalization outside of the tissue margins, and the AuNPs result in distinct anatomical visualization, as well as the ability to see subtle differences in analyte concentration [61]. Another advantage of AuNPs that we have discovered is flexibility in sample storage. For example, AuNP-sprayed tissue sections on slides were stored overnight at −20°C and imaging runs were repeated after 24 hours. Nearly identical data resulted and up to 8 imaging runs were completed on the same tissue section without loss of signal or the need to reapply AuNPs. The ability to archive slides for later

**105**

**Figure 3.**

**4. Derivatization strategies**

*Mass Spectrometry Imaging of Neurotransmitters DOI: http://dx.doi.org/10.5772/intechopen.93835*

examination could allow for follow-up data to be collected as well as the potential to

 *image, (c) is the epinephrine* 

*MSI of a sagittal zebrafish tissue section at 5* μ*m lateral spatial resolution with eye (E), forebrain (FB),* 

*image, (d) is the histidine image, (e) is the acetylcholine image, (f) is the glutamine image, (g) is the dopamine/octopamine image, (h) is the norepinephrine image, and (i) is the serotonin image. Reprinted with* 

The improvements in delocalization, reproducibility, and long-term stability from pneumatically-sprayed AuNPs warrant further investigation of this method. Finally, the quick and low-cost preparation may enable a broad range of new appli-

Chemical derivatization of functional groups is a common strategy to improve detection in MS techniques for a variety of molecular classes. On-tissue derivatization has been explored for many different purposes, including tryptic digestion [75, 76], phospholipid digestion [77], N-terminal peptide derivatization [78], and

reduce the number of organisms needed in a given research project.

*midbrain (MB), and hindbrain (HB) indicated in (a), (b) is the [GABA + Na]+*

*permission from Ref. [61]. Copyright 2020 American Chemical Society.*

cations in neuroscience, pharmacology, drug discovery, and pathology.

*Mass Spectrometry Imaging of Neurotransmitters DOI: http://dx.doi.org/10.5772/intechopen.93835*

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

With the intention of demonstrating the flexibility of AuNPs in terms of sample preparation, spraying parameters have been explored, including matrix concentration, solvent composition, spray temperature, and linear flow rate (which determines overall spray density of material). Early data and statistical analysis by principal component analysis (PCA) suggests that: (i) AuNP concentration can be varied over 4 orders of magnitude, (ii) a variety of organic:aqueous solvent mixes are possible, and (iii) temperatures from 30 to 60°C can be utilized [73, 74]. The flexibility in spray parameters means that less-experienced users with varying spraying capabilities can still get optimum data from their tissue sections, opening

*Summary of sputter-coated metal screening for small metabolite analysis in (a) positive and (b) negative ion mode. Asterisks indicate a fragment ion was detected. Reprinted with permission from Ref. [68]. Copyright* 

As previously mentioned, delocalization is an issue with organic acid matrices,

often because of a "wet" matrix spray that results in true molecular diffusion instead of analyte extraction from the tissue. A standard literature method for CHCA has been compared with AuNPs, where CHCA gives only extreme delocalization outside of the tissue margins, and the AuNPs result in distinct anatomical visualization, as well as the ability to see subtle differences in analyte concentration [61]. Another advantage of AuNPs that we have discovered is flexibility in sample storage. For example, AuNP-sprayed tissue sections on slides were stored overnight at −20°C and imaging runs were repeated after 24 hours. Nearly identical data resulted and up to 8 imaging runs were completed on the same tissue section without loss of signal or the need to reapply AuNPs. The ability to archive slides for later

the application of MSI to more areas of study.

*2018 American Society for Mass Spectrometry.*

**104**

**Figure 2.**

#### **Figure 3.**

*MSI of a sagittal zebrafish tissue section at 5* μ*m lateral spatial resolution with eye (E), forebrain (FB), midbrain (MB), and hindbrain (HB) indicated in (a), (b) is the [GABA + Na]+ image, (c) is the epinephrine image, (d) is the histidine image, (e) is the acetylcholine image, (f) is the glutamine image, (g) is the dopamine/octopamine image, (h) is the norepinephrine image, and (i) is the serotonin image. Reprinted with permission from Ref. [61]. Copyright 2020 American Chemical Society.*

examination could allow for follow-up data to be collected as well as the potential to reduce the number of organisms needed in a given research project.

The improvements in delocalization, reproducibility, and long-term stability from pneumatically-sprayed AuNPs warrant further investigation of this method. Finally, the quick and low-cost preparation may enable a broad range of new applications in neuroscience, pharmacology, drug discovery, and pathology.

### **4. Derivatization strategies**

Chemical derivatization of functional groups is a common strategy to improve detection in MS techniques for a variety of molecular classes. On-tissue derivatization has been explored for many different purposes, including tryptic digestion [75, 76], phospholipid digestion [77], N-terminal peptide derivatization [78], and

derivatization of various metabolites/drugs of interest [79–82]. One particularly attractive advantage of derivatization is that small mass species typically have a change in molecular weight which avoids interferences from low mass matrix peaks.

Some of the aforementioned studies were on small molecular weight species, but derivatization was typically for only one analyte of interest. Examples of NT derivatization have met with varied success in terms of how many different molecular classes are accessible. Coniferyl aldehyde has been used to derivatize primary amines in pig adrenal glands and rat brains [83]. Methods included pre-coating target plates and then incubating after tissue was affixed for several minutes. Spraycoating with an organic matrix followed.

Specific reactions focused on NTs have utilized pyrylium salts (e.g., 2,4-diphenyl pyranylium (DPP)) that are reactive toward primary amines. The reaction scheme with a common NT, dopamine, is shown in **Figure 4a**. The reaction can proceed at room temperature but requires 30-80 spray passes of the derivatizing agent, followed by drying time, and then application of an organic matrix [84]. Additionally, preparation of the derivatizing agent is required and can be a multi-day process. While smaller crystals than typical organic matrix preparations have been reported, there are still limitations on overall spatial resolution. **Figure 4b** shows dopamine derivatized with 3 different pyrylium salts and the resulting images that are generated; without derivatization no dopamine was observed.

Derivatization with DPP has been demonstrated in multiple instances, with the generation a 3D mouse brain atlas of dopamine, norepinephrine and serotonin [85] as well as detection of up to 23 amino metabolites [86].

Additional derivatization methods have been developed since the initial report on primary amines only. For example, fluoromethylpyridinium-based materials

#### **Figure 4.**

*(a) Reaction of dopamine with pyrylium salts. MALDI-MSI images of dopamine derivatized with DPP (b, c), PBDPP (d, e) or TMP (f, g). Signal intensity is indicated using a rainbow scale. Reprinted with permission from Ref. [84]. Copyright 2015 American Society for Mass Spectrometry.*

**107**

*Mass Spectrometry Imaging of Neurotransmitters DOI: http://dx.doi.org/10.5772/intechopen.93835*

species from other potential species [88].

*Copyright 2020 American Chemical Society.*

**5. Quantitation**

**Figure 5.**

in LATT as compared to other derivatization techniques.

which feature spiking of tissue homogenates [96].

are reactive with phenolic hydroxyl and/or primary or secondary amines, which expand the potential range of NTs that can be detected [87]. Charge-tagging using 2-(4-bromophenyl)-4,6- diphenylpyranylium (Br-TPP) results in distinctive isotopic distributions in the mass spectrum, making it easier to identify derivatized

*(A) LATT setup and (B) schematic diagram of the system. Reprinted with permission from Ref. [89].* 

The last example here is a laser-induced tissue transfer (LATT) system that enhances on-tissue derivatization of small molecules [89]. An electrosprayer applies the derivatization reagent and matrix solution on tissue and is then irradiated with a 450 nm laser beam in transmission mode, which results in transfer of a thin film of tissue to a second slide. **Figure 5** shows the setup and diagram of the LATT system. Chemicals used for derivatization include coniferyl acetate or Girard's T reagent. Preparation time requires multiple hours (overnight) and additional matrix application. Multiple classes of biomolecules were analyzed, including amino acids, NTs, polyamines, dipeptides, and others. The issue of analyte delocalization is improved

MSI has been applied to quantitative analysis of drugs [90, 91], metabolites [92], and biomarkers in tissue [93] using pneumatic sprayers and sublimation techniques described in this chapter. Nearly all of these demonstrations have utilized organic acid matrices such as DHB, CHCA, and trihydroxyacetophenone (THAP), with one research group utilizing TiO2 NPs [94, 95]. Methods of quantitation are still being investigated [96], as many of the consistency issues with MALDI-MSI that have been discussed in this chapter are even more relevant with quantitative MSI (qMSI). **Figure 6** shows a summary of two of the more common methods used for generation of a calibration curve for qMSI, on-tissue spotting and tissue mimetic models

On-tissue spotting uses either a standard molecule that is chemically similar to the analyte or a stable isotope of the analyte for making the calibration curve. Ion intensities between the analyte and standard are used to estimate the drug concentration in dosed tissue. Disadvantages include difficulty in maintaining uniform application of standards and differences in ionization for sprayed on standards vs. analyte molecules embedded within tissue. Advantages are that this method is fast and straightforward. The tissue mimetic model uses a surrogate tissue that is homogenized and spiked with the analyte of interest, frozen, sectioned, then prepared with matrix. The advantage of this method is that there is

*Mass Spectrometry Imaging of Neurotransmitters DOI: http://dx.doi.org/10.5772/intechopen.93835*

**Figure 5.**

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

coating with an organic matrix followed.

ated; without derivatization no dopamine was observed.

as well as detection of up to 23 amino metabolites [86].

derivatization of various metabolites/drugs of interest [79–82]. One particularly attractive advantage of derivatization is that small mass species typically have a change in molecular weight which avoids interferences from low mass matrix peaks. Some of the aforementioned studies were on small molecular weight species, but derivatization was typically for only one analyte of interest. Examples of NT derivatization have met with varied success in terms of how many different molecular classes are accessible. Coniferyl aldehyde has been used to derivatize primary amines in pig adrenal glands and rat brains [83]. Methods included pre-coating target plates and then incubating after tissue was affixed for several minutes. Spray-

Specific reactions focused on NTs have utilized pyrylium salts (e.g., 2,4-diphenyl pyranylium (DPP)) that are reactive toward primary amines. The reaction scheme with a common NT, dopamine, is shown in **Figure 4a**. The reaction can proceed at room temperature but requires 30-80 spray passes of the derivatizing agent, followed by drying time, and then application of an organic matrix [84]. Additionally, preparation of the derivatizing agent is required and can be a multi-day process. While smaller crystals than typical organic matrix preparations have been reported, there are still limitations on overall spatial resolution. **Figure 4b** shows dopamine derivatized with 3 different pyrylium salts and the resulting images that are gener-

Derivatization with DPP has been demonstrated in multiple instances, with the generation a 3D mouse brain atlas of dopamine, norepinephrine and serotonin [85]

Additional derivatization methods have been developed since the initial report on primary amines only. For example, fluoromethylpyridinium-based materials

**106**

**Figure 4.**

*(a) Reaction of dopamine with pyrylium salts. MALDI-MSI images of dopamine derivatized with DPP (b, c), PBDPP (d, e) or TMP (f, g). Signal intensity is indicated using a rainbow scale. Reprinted with permission* 

*from Ref. [84]. Copyright 2015 American Society for Mass Spectrometry.*

*(A) LATT setup and (B) schematic diagram of the system. Reprinted with permission from Ref. [89]. Copyright 2020 American Chemical Society.*

are reactive with phenolic hydroxyl and/or primary or secondary amines, which expand the potential range of NTs that can be detected [87]. Charge-tagging using 2-(4-bromophenyl)-4,6- diphenylpyranylium (Br-TPP) results in distinctive isotopic distributions in the mass spectrum, making it easier to identify derivatized species from other potential species [88].

The last example here is a laser-induced tissue transfer (LATT) system that enhances on-tissue derivatization of small molecules [89]. An electrosprayer applies the derivatization reagent and matrix solution on tissue and is then irradiated with a 450 nm laser beam in transmission mode, which results in transfer of a thin film of tissue to a second slide. **Figure 5** shows the setup and diagram of the LATT system. Chemicals used for derivatization include coniferyl acetate or Girard's T reagent. Preparation time requires multiple hours (overnight) and additional matrix application. Multiple classes of biomolecules were analyzed, including amino acids, NTs, polyamines, dipeptides, and others. The issue of analyte delocalization is improved in LATT as compared to other derivatization techniques.
