mass spectrometry imaging for visualizing organic analytes ... · mass spectrometry imaging for...

MASS SPECTROMETRY IMAGING FOR VISUALIZINGORGANIC ANALYTES IN FOOD

Eric Handberg, Konstantin Chingin, NannanWang, XimoDai, andHuanwen Chen*Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation,Department of Applied Chemistry, East China Institute of Technology,Nanchang 330013, P.R. China

Received 15 November 2013; revised 18 February 2014; accepted 18 February 2014

Published online 28 March 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.21424

The demand for rapid chemical imaging of food productssteadily increases. Mass spectrometry (MS) is featured byexcellent molecular specificity of analysis and is, therefore, avery attractive method for chemical profiling. MS for foodimaging has increased significantly over the past decade, aidedby the emergence of various ambient ionization techniquesthat allow direct and rapid analysis in ambient environment. Inthis article, the current status of food imaging with MSI isreviewed. The described approaches include matrix-assistedlaser desorption/ionization (MALDI), but emphasize desorptionatmospheric pressure photoionization (DAPPI), electrospray-assisted laser desorption/ionization (ELDI), probe electrosprayionization (PESI), surface desorption atmospheric pressurechemical ionization (SDAPCI), and laser ablation flowingatmospheric pressure afterglow (LA-FAPA). The methods arecompared with regard to spatial resolution; analysis speed andtime; limit of detection; and technical aspects. The performanceof each method is illustrated with the description of a relatedapplication. Specific requirements in food imaging are discussed.# 2014 Wiley Periodicals, Inc. Mass Spec Rev 34:641–658,2015

Keywords: chemical imaging; food; ambient ionization; foodsafety; antioxidant; vitamin

I. INTRODUCTION

The global population constantly increases and has exceeded 7billion (World Health Organization, 2012). The global popula-tion challenges us to improve food science and technologyfor more qualified foods. Much of the food for Europe and NorthAmerica is grown in Latin America, the Caribbean, and sub-Sahara Africa. Although the majority of internationally tradedfood is safe, food contaminants are frequently reported andinclude adulterants (e.g., melamine; Tittlemier, 2010; Arnoldet al., 2011), plasticizers (e.g., phthalates; Sørensen, 2006),chain-transfer agents (e.g., styrenes; Bentayeb et al., 2011), and

many other chemicals accumulated during food processing,storage, and transportation. Besides the extent of chemicalcontamination, the two-dimensional (2D) (Zaima et al., 2010) orthree-dimensional (3D) (Eberlin et al., 2010, #2108) spatialdistribution of nutrients like lipids is another important factorthat determines food quality. Identifying the location ofchemicals in food is used for authentication purposes (Zaimaet al., 2011); evaluation of nutritional value (Goto-inoue, Setou,& Zaima, 2010; Franceschi et al., 2012; Yoshimura et al., 2012);differentiation between wild type and mutant crops or cropvariants (Korte et al., 2012); and visualization of allergens(Cavatorta et al., 2009).

Mass spectrometry (MS) possesses excellent molecularspecificity of analysis and is, therefore, a very attractive methodfor chemical identification (Cifuentes, 2013). For a long time,the modest capabilities of MS in surface sampling limitedthe range of its imaging application. Until recently, massspectrometry imaging (MSI) was largely performed withmatrix-assisted laser desorption/ionization (MALDI) andsecondary ion mass spectrometry (SIMS) on samples placed invacuum (Pacholski & Winograd, 1999). Offering submicronresolution of analysis, these methods found broad application ininorganic chemistry (Pacholski & Winograd, 1999), materialsscience (Pacholski & Winograd, 1999), biology (McDonnell &Heeren, 2007; Schwartz & Caprioli, 2010; Lee et al., 2012b),and pharmacokinetic studies (Chen et al., 2008; Romppet al., 2011; Shahidi-Latham et al., 2012). However, foodimaging with vacuum MALDI tested only blueberries(Yoshimura et al., 2012), beef (Zaima et al., 2011), GoldenDelicious apples (Franceschi et al., 2012), soybean cotyledon(Grassl, Taylor, & Millar, 2011), rice (Zaima et al., 2010),peaches (Pastorello Elide et al., 1999), wheat (Burrell,Earnshaw, & Clench, 2007), and potatoes (Ha et al., 2012),where food safety, adulteration, nutritional analysis, and cropdevelopment were selection criteria; food imaging with SIMSwere not found. Both MALDI and SIMS imaging instrumentsare dedicated and expensive; hence their use is justified whenhigh-resolution spatial resolution is necessary (e.g., for qualitycontrol of inorganic materials or for biological imaging ofcells and tissues) (Burrell, Earnshaw, & Clench, 2007).Conversely, the required spatial resolution in food imagingapplications is usually lower; therefore, cheaper approachesare commonly preferred (e.g., immunostaining methods)(Marzban et al., 2005; Borges et al., 2006). In addition, MALDIand SIMS are relatively slow imaging techniques due to therequirement of evacuation and multistep sample preparation(e.g., deposition of organic matrix). The slow speed of MSIanalysis is a serious problem for quality control. Finally, matrix

Contract grant sponsor: National key scientific instrument development

projects (2011YQ14015008); Contract grant sponsor: The Open Fund

of the State Key Laboratory of Analytical Chemistry for Life Science

of Nanjing University (No. SKLACLS1106); Contract grant sponsor:

Program for New Century Excellent Talents in University (No. NCET-

11-0999).�Correspondence to: Huanwen Chen, Jiangxi Key Laboratory for Mass

Spectrometry and Instrumentation, Department of Applied Chemistry,

East China Institute of Technology, Nanchang 330013, P.R. China.

E-mail: [email protected]

Mass Spectrometry Reviews 2015, 34, 641–658# 2014 by Wiley Periodicals, Inc.

deposition in MALDI-MS is a disadvantage in food imagingapplications because it involves analyte diffusion that affects theoriginal molecular distribution. Usually a suitable matrix must bedetermined for a particular analyte prior to analysis; that trial-and-error process is time-consuming (Dreisewerd et al., 2007).

Mass spectrometry (MS) for surface analysis has consider-ably increased over the past decade, aided by the emergence ofvarious ambient ionization techniques that include desorptionelectrospray ionization (DESI) (Takats et al., 2004), directanalysis in real time (DART) (Cody, Laramee, & Durst, 2005),surface desorption atmospheric pressure chemical ionization(SDAPCI) (Chen et al., 2007b), laser-assisted electrosprayionization (LAESI) (Nemes & Vertes, 2007), flowing atmo-spheric pressure afterglow (FAPA) (Andrade et al., 2008), andseveral other methods reviewed recently (McDonnell andHeeren, 2007; Gode & Volmer, 2013). Ambient ionizationsources for MSI of tissues was reviewed by both Nemes andVertes (2012) and Wu et al. (2013). Ambient MS methods allowdirect surface sampling in front of a mass spectrometer withsubmillimeter resolution and no sample preparation. Ambiention sources rapidly gained popularity from the simplicity ofdesign, operation convenience, and cost-efficiency (Cody,Laramee, & Durst, 2005; Takats, Wiseman, & Cooks, 2005;Cooks et al., 2006; Chen, Gamez, & Zenobi, 2009; Huanget al., 2010). MALDI analyses have also advanced recently withthe introduction of an interface that allows surface sampling atatmospheric pressure (AP-MALDI) (Laiko, Baldwin, &Burlingame, 2000; Doroshenko et al., 2002).

The progress of ambient ionization methods revivedinterest in MSI. Particular successes have been achieved inmetabolite imaging of animal tissues at high resolution (Burrell,Earnshaw, & Clench, 2007) or whole-body sample (Burrell,Earnshaw, & Clench, 2007; Stoeckli, Staab, & Schweitzer,2007) Most recent biological applications of MSI are summa-rized in two review articles and book sections (Furuta, Sugiura,& Setou, 2010; Rubakhin & Sweedler, 2010; Lee et al., 2012b).Plant imaging (Grassl, Taylor, & Millar, 2011; Kasparet al., 2011; Matros & Mock, 2013) especially of Arabidopsis(Jun et al., 2010; Korte et al., 2012; Sarsby et al., 2012),lipidomics (Woods & Jackson, 2010; Goto-Inoue et al., 2011),and food science (Cifuentes, 2013) are other fields where MSIpublications are increasing. Reports have been published overrecent years about MSI analyses of nutrients in various foodproducts that use AP-MALDI (Li, Shrestha, & Vertes, 2006,2008), desorption atmospheric pressure photoionization (DAPPI)(Po�l et al., 2009), SDAPCI (Yang et al., 2009a), laser ablationflowing atmospheric pressure afterglow (LA-FAPA) (Shelley,Ray, & Hieftje, 2008), electrospray ionization-assisted laserdesorption (ELDI) (Jhang et al., 2012, #49), and probe electro-spray ionization (PESI) (Hiraoka et al., 2007) for strawberries,sage leaf, potato, potato, a cooked chicken egg, and turkey/celery, respectively. The analytes in most reported applicationsinclude both endogenous and exogenous organic molecules, suchas vitamins, antioxidants, lipids, contaminants, and adulterants.

In this article, the current status of food imaging by MSI isreviewed. Different introduced methods are compared withregard to spatial resolution; analysis time and speed; acquisitionrate; limit of detection; and technical aspects. The performanceof each method is illustrated with a description of an applicationin food imaging. Because the current review focuses ontraditional food samples, MALDI-MS analyses of related

samples are left beyond the scope (e.g., brains (Hsiehet al., 2006; Chen et al., 2009d; Nemes, Woods, & Vertes, 2010;Shrestha et al., 2010; Pirman & Yost, 2011), neuronal tissue ofcrabs (Ye et al., 2013), and tomato plant roots (Nihorimbereet al., 2009; Nihorimbere et al., 2012)). Likewise IR-MALDIand LAESI imaging results of mouse brain (Shresthaet al., 2010) and rat brain (Nemes, Woods, & Vertes, 2010) areexcluded. DART and DESI are not included in this reviewbecause we are not aware of imaging results for food with theseionization sources. The authors are aware of DART profilingresults for beverages (Cajka et al., 2008, 2010), cooking oils(Vaclavik et al., 2009; Hajslova, Cajka, & Vaclavik, 2011),wheat (Schurek et al., 2008; Vaclavik et al., 2010b), milk powder(Dane & Cody, 2010; Vaclavik et al., 2010a), yogurt (Morlock &Schwack, 2006), and garlic (Kubec et al., 2010) and of thediterpene profile from Stevia leaves with DESI (Jacksonet al., 2009). We are also aware of DESI imaging results forhuman samples (Wu et al., 2009; Dill et al., 2010, 2011; Elliset al., 2010), non-edible plant samples (Lane et al., 2009; Mulleret al., 2011), agar (Watrous et al., 2010), organs from smallmammals (Wiseman et al., 2006, 2008; Dill et al., 2009; Eberlinet al., 2010; Girod et al., 2010; Paglia et al., 2010; Wuet al., 2010), a mouse body (Kertesz et al., 2008) and medicinalplants (Van Berkel, Tomkins, & Kertesz, 2007; Kennedy &Wiseman, 2010; Ifa et al., 2011; Thunig, Hansen, & Janfelt,2011; Lee, Kim, & Jang, 2012). The narrow scope was necessaryfor a cogent, concise document for the foodomics readership.Authors have published peer-reviewed articles with DESI,LAESI, and others (Nemes & Vertes, 2012; Wu et al., 2013) forMSI of biological tissues, and we and others await mass spectralimaging reports with food samples. Food-only MSI applicationsare presented in Table 1. The review is written to be comprehen-sible for a broad scientific audience and to welcome newresearchers to this interesting and rapidly growing field. We hopethe combination of instrumentation and applications introducesresearchers and lab managers to affordable, new technology infood science; Table 2 is meant to aid this goal.

Abbreviations used throughout the text are explained in thedesignated section at the end of this article. For the generaloverview of ambient ionization methods and their application inMSI, readers are referred to recent review articles (Pacholski &Winograd, 1999; Cooks et al., 2006; Chen et al., 2009a; Harris,Galhena, & Fernandez, 2011;Wu et al., 2013;Monge et al., 2013).

II. INSTRUMENTATION

Traditional MSI workflow (Fig. 1) based on vacuum-MALDIionization requires six major steps: (1) sample preparation (e.g.,cryo-sectioning, drying, freezing, etc.); (2) matrix application;(3) sample loading into a mass spectrometer and evacuate themass analyzer; (4) 2D sample scanning; (5) collection of massspectra; and (6) image construction (Lee et al., 2012b). Signifi-cantly, MS imaging with AP-MALDI or ambient ionization isperformed in the ambient atmosphere without any need to loadthe sample and evacuate the instrument. Further, ambientionization allows one to bypass matrix deposition (Fig. 1). In thissection, instrumentation and principles of operation are describedfor the MSI techniques employed in imaging studies of food:vacuum-MALDI, AP-MALDI, DAPPI, ELDI, PESI, SDAPCI,and LA-FAPA. Note that, for each method, the describedexperimental configuration is specific to its application in food

642 Mass Spectrometry Reviews DOI 10.1002/mas

& HANDBERG ET AL.

TABLE 1. Recent applications of MS in food imaging

noitazinoI etylanA eussit/elpmaSsource

Matrix cryocutting reference

Wheat/grain Amino acids, sugar and sugar phosphates

Vacuum MALDI

THA Yes (Burrell, et al., 2007)

muucaV sdipiL niarg/yelraBMALDI

DHB Yes (Peukert et al., 2012a,b)

Rice/grain Lipids and γ-oryzanol phytic acid Vacuum MALDI

DHB Yes (Zaima, et al., 2010)

Eggplant/fruit γ-aminobutyric acid, amino acids and sugars

Vacuum MALDI

DHB Yes (Goto-inoue, et al., 2010)

Plant/medule and skin

Glycoalkaloids Vacuum MALDI

DHB Yes (Ha, et al., 2012)

Palm/leaf Neutral lipids Vacuum MALDI

Lithium DHB

No (Vrkoslav et al., 2009)

Strawberry, banana, and grapes/fruit

Sugar monomers, oligomers and citric acid

AP-MALDI None No (Li, et al., 2006)

Blueberry/fruit and skin

Anthocyanins Vacuum MALDI

DHB Yes (Yoshimura, et al., 2012)

Apple/fruit and skin

Flavonoids and dihydrochalcones Vacuum MALDI

α-CHCA

No (Franceschi, et al., 2012)

oN enoN ISED sdiolaklA tiurf/gemtuN (Ifa, et al., 2011)

oN enoN ISED senepretiD fael/aivetS (Jackson, et al., 2009)

Peach/fruit and skin

muucaV 3 p urPMALDI

SA Yes (Cavatorta, et al., 2009)

muucaV sdipiL taem/feeBMALDI

DHB Yes (Zaima, et al., 2011)

Soybean/cotyledon

muucaV sdipiLMALDI

DHB/CHCA

Yes (Grassl, et al., 2011)

Sage/leaf α-tocopherol DAPPI None No (Pól, et al., 2009)

Potato/medulla and skin

α-solanine and α No None ELDI -chaconine (Jhang, et al., 2012)

Potato/medulla and skin

α oN enoN ISEP eninalos- (Hiraoka, 2012)

Egg/yolk and egg white

oN enoN ICPADS enimaleM (Yang, et al., 2009)

Turkey luncheon meat/meat; celery/flesh

Lidocaine and caffeine LA-FAPA None No (Shelley, et al., 2008)

Mass Spectrometry Reviews DOI 10.1002/mas 643

MS IMAGING OF FOOD &

imaging and can differ for other types of application. Theircharacteristics (spatial resolution, data acquisition speed, andtime of analysis) and essential parts are compared in Table 2.

A. Matrix-Assisted Laser Desorption Ionization

1. VacuumMatrix-Assisted Laser Desorption/Ionization(MALDI)

Because vacuum MALDI is the most popular imaging methodtoday, we anticipate that most readers know it and will compareit to the ambient ionization methods. Briefly, in vacuum-MALDIthin tissue slices are prepared firstly by mounting on a targetplate and secondly by applying a suitable matrix manually orautomatically. After the matrix has been dried, the target plate isloaded for MALDI analysis, which includes partial or totalventing of the vacuum chamber, fixation of the target plate, andevacuating the chamber with a turbomolecular vacuum pump(10�4–10�7 Pa). The spatial distribution of molecular species inthe sample slice (e.g., peptides, proteins, or small molecules) isrecorded by scanning the UV laser beam across the target plate.Suitable imaging software is used to import the generated arrayof mass spectra from the mass spectrometer and to create themolecular image, which can be directly compared with theoptical image of the sample.

The following brief literature survey guides newcomers tohighlights in MSI for food science only. Vacuum MALDIimaging is described extensively in books (Rubakhin &Sweedler, 2010; Setou, 2010) and reviews (Pacholski et al.,1999; McDonnell and Heeren, 2007; Schwartz et al., 2010), andfood is a subset of biological samples suitable for vacuumMALDI-MS. Briefly, researchers described tissue extraction

TABLE 2. Characteristics of ionization sources in MS food imaging

Source Essential parts MSI Analyte

Spatial Resolution

(μm)

Data Acquisition Rate (Hz)

Time of Analysis

(min) vacuum- MALDI

nitrogen or Nd:YAG laser, matrix sprayer Various 10–100 10 180-600

AP-MALDI

IR laser and 3rd-party software

Fruits and vegetables 40 10 45

DAPPI N2 tank, gas lines, microchip,

UV lamp, power supply, extension and heater

Sage leaf 1000 60c N/A

ELDI nitrogen laser and electrospray Potato 500b 2 13

PESI

Power supply, current monitoring circuit, needle, actuator, tee joint, liquid

supply and capillary

Potato 60 2–5 120

SDAPCI N2 tank, gas lines, needle,

microsampler, and 3rd-party software

Cooked chicken

egg 250a 0.05 125

LA-FAPA

N2 tank, gas lines, laser ablation cell, FAPA, and 1-m

tube

Turkey, Celery 20-200 20 30

N/A, not available.aChen et al. reported a resolution of 0.06mm2, the product of 0.25mm� 0.25mm, where the step size was 0.25mm.bSee text.cSee text.

FIGURE 1. Workflow for mass spectrometry imaging experiments.Adapted from Lee et al. (2012b), which is a US Government work and is inthe public domain in the USA.


& HANDBERG ET AL.

(Yao & Setou, 2010), biological sample preparation (Sugiura &Setou, 2010), matrix selection (Sugiura et al., 2010a), methodsof matrix application (Sugiura et al., 2010b), and a mechanismfor MALDI (Lambert et al., 1998; Hoffmann & Stroobant,2001). To explain commercial products, other researchers wrotedocumentation: ImagePrep (Schuerenberg & Deininger, 2010),AXIMA-quadrupole ion trap (QIT) by Shimadzu (Furuta,Sugiura, & Setou, 2010; Hayasaka, 2010), Applied Biosystems(Kokaji, 2010a), Bruker Daltonics (Kokaji, 2010b), and Waters(Oshikata et al., 2010).

2. Atmospheric-Pressure Matrix-Assisted LaserDesorption Ionization (AP-MALDI)

Atmospheric-pressure matrix-assisted laser desorption ioniza-tion (AP-MALDI) is a relatively recent MALDI outgrowth that

allows analyte ionization at atmospheric pressure and tempera-ture (Laiko, Baldwin, & Burlingame, 2000). It might beconsidered as an intermediate between vacuum methods forsurface analysis (e.g., vacuum-MALDI or SIMS) and ambientionization sources, which allow direct analyte sampling fromnative environment. An alternative to the traditional vacuum-MALDI, AP-MALDI usually employs infrared (IR) irradiationfor analyte desorption/ionization (Lambert et al., 1998). Benefi-cially, water can be used as a matrix in AP-MALDI. Excitationis achieved with pulsed irradiation (l� 3–4mm, n� 10Hz,t� 4 nsec). The distance and the voltage between the MS inletand the target plate are normally ca. 2mm and 1–5 kV,respectively. Laser spot size is ca. 50mm. Irradiation incidenceangle is ca. 45˚ (Li, Shrestha, & Vertes, 2006). The essentialparts of AP-MALDI are shown in Figure 2A. The schematicdrawing shows the infrared laser, stage, and mass spectrometer.

FIGURE 2. Schematic drawings for the MS methods in food imaging: (A) AP-MALDI-MS, (B) DAPPI-MS,(C) ELDI-MS, (D) PESI-MS, (E) SDAPCI-MS, and (F) LA-FAPA-MS. (A) Adapted with permission from‘Atmospheric Pressure Infrared MALDI Imaging Mass Spectrometry for Plant Metabolomics,’ by Li, Shrestha,Vertes in 55th Annual Conference of the American Society for Mass Spectrometry and Allied Topics.# 2007, Dr.Akos Vertes. (B) Adapted with permission from ‘Desorption Atmospheric Pressure Photoionization,’ by Haapalaet al., in Analytical Chemistry.# 2007, American Chemical Society. (C) Adapted with permission from ‘AmbientMolecular Imaging of Toxins within a Sprouted Potato Slice by Electrospray-assisted Laser Desorption IonizationMass Sepctrometry (ELDI/MS),’ by Jhang et al. in 60th ASMS Conference on Mass Spectrometry and AlliedTopic. # 2012, Jhang, Huang, Shiea. (D) Adapted with permission from ‘Ambient Imaging Mass Spectrometryby Electrospray Ionization Using Solid Needle as Sampling Probe,’ by Lee et al. in J Mass Spetrom. # 2009,John Wiley & Sons, Ltd. (E) Adapted with permission from ‘Imaging Melamine in Egg Samples by SurfaceDesorption Atmospheric Pressure Chemical Ionization Mass Spectrometry,’ by Yang et al. in Chin J Anal Chem.# 2009, Chinese Chemical Society. (F) Adapted with permission from ‘Laser Ablation Coupled to a FlowingAtmospheric Pressure Afterglow for Ambient Mass Spectral Imaging,’ by Shelly, Ray, Hieftje in AnalyticalChemistry.# 2008, American Chemical Society.



The stage might be at ambient temperature or cooled with eithera Peltier element (ca. �10˚C) (Von Seggern, Gardner, & Cotter,2004) or a combination of a liquid nitrogen and Peltier element(ca.�120˚C) (Pirkl et al., 2012).

B. Ambient-ionization Techniques

1. Desorption Atmospheric Pressure Photoionization(DAPPI)

A DAPPI source is a thermal desorption and photoionizationsource, which is operated at atmospheric pressure (Haapalaet al., 2007) and can ionize both polar and nonpolar molecules.Figure 2B is a schematic diagram of the source, includinggas line, solvent line, microchip, UV lamp, sample, and inlet.The sample is simultaneously exposed to a heated vapor jetand UV irradiation. The jet is created with a heated microchip(up to 500˚C) directed toward the sample and the inlet at a45˚ angle. The jet is used to extract and evaporate analytemolecules from the surface. The sample support is also heated(ca. 350˚C) (Wu et al., 2013). Normal to the sample supportsurface, a 10-eV vacuum discharge lamp shines UV light toionize desorbed molecules. The choice of solvent is largelydictated by the target compound. For analysis of nonpolarcompounds such as anthracene and tetracyclone, toluene is anefficient solvent, but for more polar analyte compounds, such astestosterone and verapamil, acetone is more efficient. Thesurface material is important because it determines the rate ofthermal diffusion during desorption. Analyte ions enter themass spectrometer through an extension that is heatedexternally.

2. Electrospray Laser Desorption Ionization (ELDI)

Electrospray laser desorption ionization (ELDI) combines laserdesorption of solid materials and post-ionization with electro-spray under ambient conditions. Analyte molecules are desorbedfrom either conductive or insulating substrate with a pulsednitrogen laser and without matrix. Post-ionization with ESIallows generation of multiply charged analyte ions to makeELDI suitable for protein analysis (Shiea et al., 2008). Figure 2Cis a schematic drawing of an ELDI experiment to show a massspectrometer, sample, laser beam, and electrospray source,sample plate, and stage (Huang et al., 2006). The irradiationwavelength is normally 337 nm; repetition rate 10Hz; incidentangle ca. 45˚; focal length 15 cm; and spot size ca. 100mm� 150mm. The needle and the target plate are grounded, and thesampling cone is at�4.5 kV (Huang et al., 2006).

3. Probe Electrospray Ionization (PESI)

Probe electrospray ionization (PESI) is another ESI-basedtechnique, in which the capillary for sample solution transfer-ence is replaced by a solid needle with a sharp tip (Chenet al., 2008). Compared with conventional electrospray ioniza-tion, PESI features high-salt tolerance, direct sampling, and lowsample consumption. In contrast to ESI, PESI employs adiscontinuous spray and has two different modes of operationfor dry and wet samples. Figure 2D is a schematic drawing ofPESI for wet samples and shows the power supply, currentmonitoring circuit, solid needle, inlet, sample, and stage. The

electrospray voltage is 2–2.4 kV, and volume of secondarydroplets is 0.3–0.4 pL (Chen et al., 2009b). The needle movesbetween the up- and down-positions, and the analysis occurs inthree steps. First, the de-energized needle moves from thestarting position to the invasion depth. Second, the needlereturns from the invasion depth to the starting position. Third,the needle is energized. At the up-position, a Taylor coneforms as sample sprays from the needle. The spatial resolutionof PESI analysis is ca. 60mm and dwell time is 10 sec (Chenet al., 2009b).

4. Surface Desorption Atmospheric Pressure ChemicalIonization (SDAPCI)

Surface desorption atmospheric pressure chemical ionization(SDAPCI) is a combination of corona discharge and neutraldesorption. Figure 2E is a schematic drawing of the samplingprocess in SDAPCI: desorption of molecules with the nitrogenjet followed by ionization with charged water clusters (Chenet al., 2007a; Yang et al., 2009a; Huang et al., 2011). SDAPCIis distinct from corona discharge due to the use of annular flowof humidified nitrogen around a polarized needle (Chen et al.,2007a). Figure 2E shows the major parts of a SDAPCI source,including a discharge needle, T-unit for gas introduction, sampleholder, and MS inlet. Nitrogen is delivered at a pressure of ca.1MPa (10 bar) at ambient temperature, and the outer diameterof gas tubing is 1.6mm (Chen et al., 2007b; Schwartz et al.,2010). The nitrogen gas can be humidified with an Erlenmeyerflask-stopper-tubing assembly (not shown). Nikolaev et al.reported that nitrogen humidification and inlet capillary temper-ature are important to achieve a stable corona discharge. Theyreported Hþ(H2O)n varied from n¼ 55 to n¼ 4 with ambienthumidified air at an inlet capillary temperatures of 50˚C and130˚C; the high number of water clusters at 50˚C interfereswith interpretation and, therefore, a steady MS signal inSDAPCI experiments (Nikolaev et al., 2004). The formation ofthe water clusters has been discussed (Brubaker, 1968; Pavlik &Skalny, 1997). The corona discharge occurs between the needleand the inlet. Figure 2E shows blue circles, green circles, andbrown circles to represent the ionization water clusters, neutralnitrogen, and sample. The voltage applied to the needle inSDAPCI is ca. 4 kV, the spatial resolution ca. 250mm, and dwelltime 10 sec.

5. Laser Ablation Flowing Atmospheric PressureAfterglow (LA-FAPA)

Laser ablation flowing atmospheric pressure afterglow(LA-FAPA) combines laser ablation of analyte molecules withpost-ionization with atmospheric pressure plasma. The ionsource is a combination of an ablation chamber and ionizationchamber, which are connected to each other with a Teflon tube.Figure 2F is a schematic drawing of the LA-FAPA experiment(Shelley, Ray, & Hieftje, 2008). Analyte molecules are desorbedwith pulsed a UV laser (226 nm, 20Hz). The area exposed to asingle laser pulse is between 10 and 300mm in diameter. Sampleaerosol is transferred out of the ablation chamber into theionization volume with the flow of room-temperature nitrogengas (0.8 Lmin�1). The FAPA chamber contains an atmosphericpressure glow discharge, which produces a background ofprimarily Hþ(H2O)2 at m/z 37 and of NOþ, H2O

þ, and O2þ to a


& HANDBERG ET AL.

lesser extent (Andrade et al., 2008). The cathode voltage inFAPA experiments is ca. 500V, and discharge current is 25mA(Andrade et al., 2008). The pin-to-plate distance is ca. 5mm andthe cell-to-inlet distance is ca. 10 mm, respectively. The spatialresolution of LA-FAPA is ca. 20mm; the dwell time is 4 sec.

C. Image Construction

Although ionization mechanism and principles of operationfor the ion sources described above differ significantly, verysimilar methods and instrumentation are employed for imageconstruction. The sample surface is scanned with an automated-or manually- controlled stage that moves in the x� y domain,while the ion probe remains stationary during the experiment.Automated stages are driven by a stepper motor that iscomputer-controlled and can be equipped with piezoelectricactuators and displacement encoders. The sample surface isdivided into small areas (pixels) that are scanned individuallyand sequentially in time (Lee et al., 2012b). The size of a pixelis limited by the spatial resolution of desorption, usually from30 to 500mm. Scanning the surface can be achieved eitherincrementally (i.e., in discrete steps) or continuously. In the firstcase, a single mass spectrum is normally collected for eachpixel. In the second case, the signal for each pixel is averagedfrom collected mass spectra while the pixel passes the focus ofionization probe. The number of spectra per pixel in thecontinuous mode depends on the speed of scanning, size of thepixel, and ablated area. The eventual spatial resolution of MSimage is determined by the probe diameter (Nemes & Vertes,2012), the sample area instantaneously exposed to desorptionprocess. In addition, it also depends on many experimentalparameters such as translational speed, step size, analytecarryover during the scanning process, etc. Because foodimaging is less demanding on resolution compared to biological,forensic, or materials imaging, the ion sources are operated at asuboptimal resolution to allow much shorter total analysis time.A good rule is to use the minimum resolution necessary toaddress the problem being investigated (Lee et al., 2012b). Tovisualize an MS image, the operator records mass spectra into a2D image file. The file size can range from a few megabytes upto gigabytes because the size depends on the number of pixels,resolution of MS detection, and mass range. The generateddata file is processed with dedicated commercial software(e.g., FlexImaging or TissueView) or freeware programs (e.g.,Biomap and MMSITE; http://www.maldi-msi.org) to produce2D images for selected ions. Ion intensity is usually color-codedand is displayed with a false-color scale with the relative ionintensity reflected by the intensity of the color. More detail onhowMS image is constructed can be found in a recent review byWu et al. (2013).

III. APPLICATIONS

The use of vacuum-MALDI in food imaging is well-documentedin the literature (Zaima et al., 2010; Yoshimura et al., 2012).Applications with atmospheric pressure ionization (API) sour-ces are fairly recent and, yet, have not been reviewed withemphasis on food (Wu et al., 2013). In this section, we focusdiscussion to MSI food analyses with API sources, includingAP-MALDI (strawberry imaging), DAPPI (sage leaf imaging),ELDI (potato imaging), PESI (potato imaging), SDAPCI

(cooked chicken egg), and LA-FAPA (turkey and celery). Targetcompounds include metabolites, vitamins, adulterants, andcontaminants.

A. Vacuum MALDI

Matrix-assisted laser desorption/ionization (MALDI) is theoldest MSI method in food imaging. Lipids in rice were studiedwith the Kawamoto method and vacuum-MALDI (Kawamoto,2003; Zaima et al., 2010). Pru p 3 is a protein allergen at 9,1 kDain the peach skin of three peach varieties (Cavatorta et al., 2009),and the allergen was imaged. Lipids in beef were imaged andwere used to distinguish beef from three locations (Zaimaet al., 2011). Metabolites in barley (Peukert et al., 2012a,b),soybean cotyledon (Grassl, Taylor, & Millar, 2011), and wheat(Burrell, Earnshaw, & Clench, 2007) were imaged during thedevelopment of the seeds for selecting food crop varieties; wheatstems (Robinson et al., 2007) were also imaged for the samereason. The spatial distribution of gabba-alphabutyric acid ineggplant was reported by Goto-inoue, Setou, and Zaima (2010)for identifying the location of functional food factors. Thespatial distributions of sugars in both blueberries and GoldenDelicious apples were reported by Yoshimura et al. (2012) andFranceschi et al. (2012). Vacuum-MALDI for high-resolutionimaging is well developed (Guenther et al., 2010; Lin et al.,2011).

Although many food imaging examples were reported withvacuum-MALDI-MS, we selected the spatial distribution of anallergen Pru p 3 to emphasize protein imaging in food (Cavatortaet al., 2009). The complete amino acid sequence of Pru p 1was reported and was renamed to Pru p 3 (Pastorello Elideet al., 1999). To complement the previous work, Pru p 3 wascharacterized with exact mass, complete amino acid sequenceand imaging (Cavatorta et al., 2009). The spatial distribution ofPru p 3 provides valuable information for the producers andregulators of peach products without Pru p 3. Figure 3 comparesthe photograph of a peach slice with three molecular imagesobtained with vacuum-MALDI-MS. Pru p 3 is in the skin ofeach molecular image, but the 6,996; 7,311; and 7,745 Th ionsare exclusively found in the pulp of the peach. The red spots onthe perimeter correspond to Pru p 3 signals from the peach, andthe red arrow highlights the location of Pru p 3. The spatialdistribution of Pru p 3 was observed in three peach varieties:white flesh “Italia K2,” yellow flesh “Toscana” and nectarineyellow flesh “Rita star.” Although only three varieties weretested in the study, that spatial distribution might apply to a largenumber of peach varieties.

B. AP-MALDI

Li, Shrestha, and Vertes (2007) used AP-MALDI for chemicalimaging of a strawberry. The stainless steel MALDI target platewas cooled with either liquid nitrogen or dry ice for 2–3minbefore analysis. Because strawberries largely consist of water(91%), no additional water matrix was needed to assist ioniza-tion. Scanning was achieved with a computer-controlled, steppermotor-driven stage. The stage had piezoelectric actuators anddisplacement encoders. The travel range in the x- and y-directionwas 4mm, the spatial resolution was 250mm, and the totalanalysis time was 45min (Li, Shrestha, & Vertes, 2007). Massspectra produced with up to 80 laser shots were averaged for



http://www.maldi-msi.org/

each spot on the sample surface and stored as a function of time.A LabVIEW program that rendered times to the correspondingx� y coordinates was used to convert data sets into two-dimensional distributions. A graphics program was used toproduce false-color images of the analyte distribution (Li,Shrestha, & Vertes, 2007). Figure 4 shows the strawberry opticalimage and AP-MALDI-MS images for sucrose, citric acid, andfructose or glucose; the tentative molecular assignments forsucrose, citric acid and fructose or glucose are from thepotassium-sucrose adduct ion (m/z 381.1), potassium-citric acidadduct ion (m/z 231.0), and potassium–fructose or potassium–glucose adduct ion (m/z 219) (Li, Shrestha, & Vertes, 2007). Allthree metabolites were observed at high intensity; sucrose andfructose or glucose had the most-abundant potassium adductsignal in the corresponding spectra. However, some compounds,such as anthocyanins, ascorbic acid, flavanols, ellegic acid, andphenolics could not be revealed with AP-MALDI-MSI despitethe known high abundance of these compounds in strawberries.The most probable reason for the absences of these chemicalsignals is the lower desorption/ionization efficiency of thesecompounds compared to sugar molecules.

C. DAPPI

Distribution of the antioxidant a-tocopherol in a sage leaf wasimaged with DAPPI-MS (Po�l et al., 2009). A grid array wasprogrammed with a custom software program. The spatialresolution of imaging was 1mm. MS images were created form/z 430.3810, 310.2166, and 315.0863 were tentatively assigned

to a-tocopherol radical cation [Mþ], methyl carnosic acid[Mþ], and an unknown leaf phytocompound; the accurate masserrors were 0.9, �0.09 and �0.3 ppm, respectively (Po�l et al.,2009). Despite the high resolution of the Fourier transformion cyclotron resonance (FTICR) mass spectrometer, it isimpossible to characterize the actual structures without compar-ing tandem mass spectra of standards; the tentative assignmentswere made from the existing literature for tocopherol (Abreuet al., 2008) and methyl carnosic acid (Schwarz & Ternes, 1992)in sage. Tocopherol was observed in the region between m/z 300and 600, where most of the esters and terpenoids are found.Figure 5A is the tocopherol radical cation [Mþ•] image, whichclearly reveals the highest concentration of tocopherol in themiddle of the leaf (Po�l et al., 2009). Figure 5A is an overlay ofthe tocopherol MS image (m/z 430.3810) with the optical image,which is in Figure 5C. Figure 5B is also an overlay, but two ionsare represented for methyl carnosic acid and the unknown peak,not one ion. The unknown peak is interesting because itsmaximum intensity occurs in areas where no carnosic deriva-tives were detected (see Fig. 5B).

D. ELDI

The distribution of a-solanine and a-chaconine in a potato slicewas imaged with ELDI MSI (Jhang, Huang, & Shiea, 2012).The identity of a-chaconine was confirmed with tandem massspectrometry of m/z 852, which produced fragments at m/z 706[M� (b-L-rhamnose)]þ, m/z 560 [M� 2(b-L-rhamnose)]þ, andm/z 398 [M� 2(b-L-rhamnose)� (b-D-glucose)]þ. The identity

FIGURE 3. VacuumMALDI-MS imaging of a peach for Pru p 3, an allergen in peaches: (A) Optical image of thepeach slice. (B,C) and (D) are molecular images of the signals from the slice and include Pru p 3 (in red, indicatedby the red arrow in B) and three ions detected exclusively in the pulp. (E) The averaged spectrum for theslice. Reprinted with permission from ‘Unambiguous Characterization and Tissue Localization of Pru P 3Peach Allergen by ElectrosprayMass Spectrometry andMALDI Imaging,’ by Cavatorta et al. in J Mass Spectrom.# 2009, JohnWiley & Sons, Ltd.


& HANDBERG ET AL.

of a-solanine was confirmed with tandem mass spectrometry ofm/z 868, which produced fragments atm/z 850 [M�H2O]

þ, m/z722 [M� (b-L-glucose)]þ, andm/z 706 [M� (b-L-rhamnose)]þ.A potato with sprouts was sliced (35mm� 15mm� 2mm), andit was attached to a stainless steel MALDI sample plateand analyzed with ELDI. A control MSI analysis was done withUV-MALDI-MS assisted with 2,5-dihydroxybenzoic acid(DHB) matrix. Also, bulk extract of another potato slice wastested with MALDI. Figures 6A–C show three mass spectra forELDI (Fig. 6A), MALDI (Fig. 6B), and bulk MALDI analysis ofpotato extract (Fig. 6C) (Jhang, Huang, & Shiea, 2012). Muchhigher signal abundance is indeed observed for the extractedsample compared to image spectra. Jhang, Huang, and Shiea(2012) attributed the low abundance in the MSI experiments ofpotato to charge buildup on the analyzed slice. The electricfield between the potato and MS inlet is attenuated by water in athick potato sample. Figures 6D–F show an optical image ofthe potato sprout and the molecular images for a-chaconine(m/z 852) and a-solanine (m/z 868) with different spatialdistributions. ELDI imaging was done with the following

settings: repetition rate 2Hz; x, y stage moving speed 0.05 cmsec�1; and spot size 300 nm in diameter. The results agree withvacuumMALDI imaging of Ha et al. (2012).

E. PESI

The distribution of a-solanine in a potato was analyzed withPESI-MS (Hiraoka, 2012). The tentative identification ofa-solanine was based on the isotopic envelope of the molecularion at m/z 868. A green, dry potato was cut in half, and thesheath-flow PESI stethoscope was pressed to the potato slice inthe middle of the medulla region (spot A in Fig. 7A). Spectrawere acquired in the following manner as paraphrased fromChen et al. (2009b). First, the needle tip was moved to thelowest (sampling) position, and the sampling stage was movedslowly upward (along z-axis) until the sample surface was incontact with the needle tip. The stage was further elevateduntil the needle tip penetrated the sample to a preset samplingdepth (e.g., 100mm). The loaded needle was actuated forMS acquisition over 10 sec. The accumulated signal that

FIGURE 4. Imaging of a strawberry surface with AP-MALDI: (A) optical image, (B) sucrose image, (C) citricacid image and (D) fructose or glucose image. Reprinted with permission from ‘Atmospheric Pressure MolecularImaging by Infrared MALDI Mass Spectrometry,’ by Li, Shrestha, Vertes in Analytical Chemistry. # 2007,American Chemical Society.



corresponds to the molecular ion of a-solanine (m/z 868)was saved as a separate file according to the pixel coordinate.The imaging stage was moved to the next sampling location ata step of 60mm in x- or y-direction. The above steps were

repeated until a predefined area was analyzed. The sameworkflow was applied to analyze other spots on the potato slice(spots B and C in Fig. 7A). The total analysis time was�10min.Figure 7B shows the mass spectra for spots A, B, and C.

FIGURE 5. Imaging of a sage leaf with DAPPI: (A) distribution of m/z 430.3810 on sage leaf; (B) combineddistributions of peaks atm/z 301.2166 (green) andm/z 315.0863 (red); (C) optical image. Adapted with permissionfrom ‘Automated Ambient Desorption–Ionization Platform for Surface Imaging Integrated with a CommercialFourier Transform Ion Cyclotron Resonance Mass Spectrometer,’ by Po�l et al. in Analytical Chemistry. # 2009,American Chemical Society.

FIGURE 6. (A) ELDI-MS from a potato slice; (B) UV-MALDI-MS from a potato slice; (C) UV-MALDI-MSfrom extract solution of a sprouted potato. The matrix was 2,5-dihydroxybenzoic acid. (D) Photograph of a potatosprout slice. (E, F) Molecular images from m/z 852 and 868 with ELDI-MS. Adapted with permission from‘Ambient Molecular Imaging of Toxins within a Sprouted Potato Slice by Electrospray-assisted Laser DesorptionIonization Mass Sepctrometry (ELDI/MS),’ by Jhang et al. in 60th ASMS Conference on Mass Spectrometry andAllied Topics.# 2012, Jhang, Huang, Shiea.


& HANDBERG ET AL.

The amount of a-solanine in the potato skin was greater than theamount of a-solanine in the medulla (see Fig. 7A). Like theELDI result, the results agree with vacuum MALDI imaging ofHa et al. (2012).

F. SDAPCI

Distribution of endogenous melamine in a boiled chicken eggfrom mainland China was imaged with SDAPCI-MS (Yanget al., 2009a). Egg slices were placed on a motor-driven 3Dmoving stage so that the samples were 1–2 mm away from thetip of the desorption sprayer. The position of the stage waschanged incrementally by 0.25mm in x- or y-direction with anelectronic control unit. Spectra were acquired in MS/MSmode on the precursor ion at m/z 127, protonated melamine[MþHþ]. The three product ions of protonated melamine are[M�NH3

þ], [M�NH2CN], and [M�C2HN3] at m/z 110, 85at 60 (Yang et al., 2009a). Although m/z 85 is smaller than thebase peak at m/z 99 in the tandem mass spectrum, the signalintensity of characteristic fragment at m/z 85 was used in theimaging experiments. For each pixel, data were recorded for10 sec, and the averaged intensity of the m/z 85 fragment pairedwith the corresponding XY-coordinates were saved into aspreadsheet.

Figure 8 shows an optical image of an egg and thedistribution of melamine. The relative intensity of the m/z 85 ionwas binned in 20 equal intervals, from zero to the maximumintensity, and each interval was displayed with a different color.The chemical image revealed that melamine was mostlyconcentrated in the egg white rather than yolk. The authorsattributed the observation to the strong hydrogen bondingbetween melamine amino groups and proteins in the egg white.

At the same time, melamine is much weaker bound by theconstituents of the egg yolk, such as cholesterol or blastodisc,than melamine is bound weak much by the constituents of theegg white.

G. LA-FAPA

We describe two LA-FAPA imaging experiments of foodproducts by Shelley, Ray, and Hieftje (2008). In the firstexperiment, a slice of turkey luncheon meat was spotted withlidocaine (50 ng). Lidocaine solution contained a blue dyefor optical visualization. Lidocaine was clearly identified in theLA-FAPA mass spectrum, and the chemical image (Fig. 9A)matched well with the photographic layout (Fig. 9B). In anotherexperiment, a piece of celery was soaked overnight in caffeinesolution (Shelley, Ray, & Hieftje, 2008). A blue dye was addedto the caffeine solution for optical visualization. The celery wassliced perpendicular to the direction of the stock and imageddirectly. The protonated caffeine molecular ion mass [MþHþ]was tentatively identified at m/z 195 (Fig. 9C). The caffeinechemical image shows two high concentration spots thatcorresponded to the two vascular bundle locations in the sample.Spatial resolution of analysis was ca. 200mm, scanning speedwas 80mmsec�1, and the total time of imaging experiment,including data processing, was�by 30min.

IV. COMPARISON FOR ION SOURCES

This section compares the ion sources employed in foodimaging with regard to spatial resolution, speed, total analysistime, limits of detection, acquisition speed, and technicalaspects.

FIGURE 7. (A) Optical image of the green, dry potato and (B) PESI-MS of the potato slice sampled fromdifferent spots (A–C). Adapted with permission from ‘Development of Probe Electrospray Using a Solid Needle,’by Hiraoka et al. in Rapid CommunMass Spectrom.# 2007, JohnWiley& Sons, Ltd.



A. Spatial Resolution

The resolution of MS image is primarily dependent on thespatial resolution of a particular desorption/ionization method,but experimental factors, such as translational speed, step size,analyte carry-over during the scanning process, etc., can furtheraffect image resolution. The reported resolution in food MSIspans the range from 10 to 1,000mm (Table 2). MSI spatialresolutions for UV-MALDI, AP-MALDI, DAPPI, ELDI, PESI,SDAPCI, and LA-FAPA are 10–100, 40–50, 1,000, 500, 60, 250,and 20–200mm, respectively. As can be seen, the highestresolution can be achieved with laser-based sources (MALDIand LA-FAPA), which can be focused to�10–20mm. However,the operation of MALDI and LA-FAPA is commonly preferredat ca. 10 times lower resolution compared to the maximumbecause applications of food imaging usually do not requirespatial resolution <100mm. Therefore, larger spot size canallow much faster analysis without loss in information capacitythan the small spot (Cavatorta et al., 2009). The most commonspatial resolution in the reported analyses of food imaging spansthe range of �100–500mm, which is sufficient to identify theanalyte distribution in most food products.

B. Analysis Speed and Time

The enormous number of food products necessitates rapid andhigh-throughput analytical methods. The diversity renders thehigh speed of imaging in food applications more important thanhigh spatial resolution. Lee et al. (2012b) estimated that imaginga 4mm� 3mm area at 50-mm resolution can be accomplishedwithin 30min and that the analysis of the same area at 12-mmresolution would require 7–10 hr. Finally, high-resolution MSI

FIGURE 9. Imaging of food with LA-FAPA: (A) The chemical image ofprotonated lidocaine at m/z 235 and (B) optical image of lidocaine solutionon turkey luncheon meat. (C) The chemical image of protonated caffeine atm/z 195 (top) and optical image (bottom) of celery veins doped with caffeine.Celery was sliced perpendicular to the direction of the stalk. Lidocaine andcaffeine solutions both contained a blue dye for visalization. Adapted withpermission from ‘Laser Ablation Coupled to a Flowing AtmosphericPressure Afterglow for Ambient Mass Spectral Imaging,’ by Shelley, Ray,Hieftje in Analytical Chemistry.# 2008, American Chemical Society.

FIGURE 8. Optical image (left) and SDAPCI-MS/MS image (right) of m/z 85, fragment ion of protonatedmelamine at m/z 127, in a chicken egg that contains melamine. The color scale represents the binned intensitydata. Reprinted with permission from ‘Imaging Melamine in Egg Samples by Surface Desorption AtmosphericPressure Chemical IonizationMass Spectrometry,’ by Yang et al. inChin J Anal Chem.# 2009, Chinese.


& HANDBERG ET AL.

(e.g., with FTICR or Orbitrap) at 10-mm spatial resolutionrequires >40 hr. In most food applications, the analyte willsignificantly degrade during a 40 hr data acquisition process,and analysis might, therefore, result in wrong results. Efforts toimprove data acquisition rate are significant when massspectrometry is used to image biological and food samples. Thekey is to use the lowest possible spatial resolution and dwelltime. The total analysis time (i.e., time from the first to last pixelin an imaging experiment plus data processing and imageconstruction) reported in MSI experiments of foods are 45minwith AP-IR-MALDI (strawberry), 13min with ELDI (potato),120min with PESI (potato), 125min with SDAPCI (egg), and30min with LA-FAPA (celery) (Table 2). These values are eitherexplicitly specified in corresponding publications or derivedfrom relevant data. The data on the speed of DAPPI analysis wasnot reported and could not be derived from other reportedvalues. The shortest total analysis time is reported for analysis ofpotato with ELDI (13min), and the longest analysis time is toimage a chicken egg with SDAPCI (125min). The 10-timesfaster analysis by ELDI is primarily due to the lower spatialresolution of imaging (500mm in ELDI vs. 250mm in SDAPCI)and shorter dwell time (2 sec in ELDI vs. 10 sec in SDAPCI).

C. Limits of Detection

Because many endogenous chemicals and additives are presentin food in trace amounts, the limits of detection (LOD) areuseful. Vacuum-MALDI is the most-sensitive method, becausedesorption and ionization of analytes occur in vacuum. Typicallycombined with time-of-flight mass spectrometry (TOF-MS)detection, vacuum-MALDI can achieve sensitivity down tofemtomoles (fmol) and attomoles (amol) of analyte per pixel(Muddiman et al., 1997). API interfaces are usually from 1 to 2orders less sensitive than vacuum-MALDI (�1–100 fmol perpixel) from the ion losses at the MS inlet. The following LODsestimates can be derived based on the reported data for analysisof chemicals relevant to food imaging: 0.0001, 0.04, 0.06mganalyte per kg solution for sucrose, reserpine, and bradykininwith AP-MALDI; 0.024, 0.005, 0.002, and 0.005mg kg�1 foranthracene, testosterone, MDMA, and verapamil with DAPPI;0.5mg kg�1 for FD&C Red with ELDI; 0.02 and 0.05mg kg�1

for morphine and MDMA with PESI; 0.045mg kg�1 for mela-mine on an egg with SDAPCI; 0.2mg kg�1 for lidocaine withFA-LAPA. Of course, these values only give a general idea aboutMSI sensitivity and cannot be used for direct comparison of themethods because the LOD in each case is also influenced by thesensitivity of MS detection. For instance, the reported sensitivityof melamine in egg, 0.045mg kg�1, is less than the reportedsensitivity of melamine in milk powder, 0.5mg kg�1, withvacuum MALDI by Arnold et al. (2011). It is worthwhile tonote, however, that all derived LODs are better than the safetylimit for melamine in milk, 0.15mg kg�1 (World HealthOrganization Food and Agriculture Organization of the UnitedNations, 2011), and carbaryl on chili peppers, 0.5mg kg�1

(World Health Organization, 2011).Although the LODs are important for quantitative applica-

tions of MSI in food, reproducibility is also an important, butneglected requirement. Gurdak et al. (2013) reported therepeatability of absolute intensity of 49%, adhesive taperepeatability of the relative intensity of 10%, and averageconstancy of the relative intensity of 31% for DESI. The 49%

repeatability of DESI and the 39% repeatability of laserdesorption ionization sources (Barnes et al., 2004) are similardespite the difference in ionization mechanism. The coefficientof variation (CV) for vacuum MALDI on human adrenal glandswas reported to be less than 10% (Bucknall, Fung, &Duncan, 2002). The RSD for SDAPCI on boiled egg was 1.3%with six replicates (Yang et al., 2009a), and the RSD forLA-FAPA was reported to be 3.1% for caffeine with anacetaminophen internal standard on 10 replicates (Shelley, Ray,& Hieftje, 2008). Reproducibility values like CV, RSD, orrecoveries were not reported for AP MALDI (Li, Shrestha, &Vertes, 2007), DAPPI (Kauppila et al., 2007), ELDI (Linet al., 2007), or PESI (Hiraoka, 2012) in these food imagingpublications.

D. Technical Aspects

A brief description is given about the technical aspects of ionsources. Readers are referred to the essential parts column ofTable 2. The ion sources in food imaging can be grouped intotwo categories. The first category comprises one-step ionsources: vacuum-MALDI, AP-MALDI, and SDAPCI. In thesemethods, the same probe is used for analyte desorption andionization. The second category comprises two-step techniques:LA-FAPA, ELDI, DAPPI, and PESI. In the two-step techniques,desorption and ionization are decoupled in space and time andrequire a hybrid interface. The one-step sources are generallyless complicated to learn and operate and have fewer functionalparts compared to two-step sources. Vacuum-MALDI is inte-grated into commercial TOF-MS instruments to make itparticularly easy to operate. The application of a UV matrix isusually achieved with automated spray systems. UV irradiationis generated with a nitrogen or Nd:YAG laser. These lasertypes are rather reliable, have long lifetime, and require fairlysimple maintenance, such as regular cartridge replacement.AP-MALDI is a commercially available unit that can beinstalled in front of an API mass spectrometer. AP-MALDIsource can also be built from commercially available parts,including IR laser, delay generator, HV switch, moving stageand LabVIEWequipment. IR lasers require user training and areless popular, rugged, or reliable than UV lasers. The rest of thedescribed sources do not have a commercial version and must bebuilt in-house. The SDAPCI equipment list includes gas-handling equipment, needle, humidifier assembly, stage, andspreadsheet. The equipment necessary for SDAPCI is low-costand is relatively simple to assemble in an analytical laboratory.The PESI equipment includes high-voltage power supply,custom current-monitoring circuit, needle, stage, actuator,fittings (e.g., tee, liquid supply, capillary), piezoelectric pump,and custom Cþþ program. The supply, needle, stage, actuator,fittings, and pump are commercially available. DAPPI uses gas-handling equipment (e.g., gas cylinder, gas lines), custommicrochip, commercial UV lamp, power supply, heater, andcapillary extension. Although the DAPPI microchip is notcommercially available, its fabrication is well-documented inthe literature (Haapala et al., 2007). The DAPPI program is alsodescribed in the literature (Haapala et al., 2007). For the DAPPI,the gas equipment, lamp, power supply, heater, and capillaryextension are commercially available, but the decoder, console,system platform, and software are custom made. The DAPPI’sdecoder, console, and system platform are significant obstacles



for most research laboratories. The ELDI equipment listincludes syringe pump, bracket, XYZ-micrometers, nitrogenlaser, and electrospray. The equipment for ELDI is reliable, low-cost, or easily fabricated, therefore, ELDI is a good choice forgeneral research laboratories. The LA-FAPA instrumentationlist includes parts related to laser desorption such as gashandling equipment, laser ablation cell, Nd:YAG laser, in-housestage, FAPA ion source, and tubing. The implementation of aLA-FAPA source in a research laboratory requires a high levelof engineering expertise.

V. REQUIREMENTS IN FOOD IMAGING

When food products are exposed to ambient environment, theyare prone to rapid spoilage, which can become evident on thetime scale of hours and even minutes. Therefore the high speedof imaging is of particular importance in food analysis. Scanningcan be accelerated by the intentional decrease in the spatialresolution of a method (e.g., defocus the laser beam, use largerscanning steps or shorter dwell times). As a rule of thumb, foodimaging should be completed within �1 hr at a resolution<500mm.

Chemical exposure of a food sample during analysis shouldbe minimal to preserve the sample in its original state. Chemicalexposure can include charged droplets of organic solvent (e.g.,in DESI) or matrix deposition (e.g., in MALDI). The formationof a solvent film on the sample surface can result in moleculartransformation as well as chemical extraction and significantredistribution of analyte molecules. Contamination can bealleviated with ambient ion sources that employ “dry” chemistry(e.g., LA-FAPA, PESI, SDAPCI).

Signal stability during analysis is crucial to obtain accuratechemical images. Frozen samples are widely used, but they areassociated with complications to the experimental procedure.A cold finger and a Peltier-cooled sample plate in MALDIexperiments were reported to prolong the analyte lifetime anddurability of ion signals for liquid samples (Li, Shrestha, &Vertes, 2006). However, more work is yet to be done toinvestigate the benefits of a cold sample plate in MSI. Enhancedsignal stability was observed in DAPPI and SDAPCI experi-ments when the gas flow to assist analyte desorption/ionizationwas heated (Yang et al., 2009b). However, only moderateheating should be used to prevent thermal degradation of a foodsample (Wu et al., 2013). Also, signal stability is generallyimproved when an ion source enclosure is applied to reduceambient factors (e.g., air convection, daily fluctuations intemperature/humidity) (Robichaud et al., 2013).

Finally, surface sampling inevitably results in materiallosses. To repeatedly image the same sample or study it withanother method, the penetration depth of desorption should bereasonably low (e.g., with laser ablation). Low analyte con-sumption also minimizes possible carryover effects due to thesputtering of desorbed material.

VI. CONCLUSION AND OUTLOOK

Mass spectrometry imaging (MSI) is a powerful method tocharacterize nutrients and contaminants in food products with ahigh chemical and spatial sensitivity. With the recent advancesin surface sampling, MSI is more accessible to researchers andend users. Several ambient ion sources have already been

developed into commercial products. Moreover, partially orfully automated imaging platforms were implemented to allowfaster imaging of food products without pretreatment. As aresult, the number of MSI applications for food analysissignificantly increased over recent years. Today, MSI is clearlycapable to simultaneously meet the required spatial resolution,sensitivity, and speed of analysis generally demanded in foodchemical imaging.

Food applications are particularly suited for ambient MSI,because scanning speed is usually more important than spatialresolution. It can be expected that the current trend towardambient sampling will expand in near future. In contrast,biological and materials science applications such as cellimaging or nanoelectronics are very demanding with regard tospatial resolution of analysis and still rely on vacuum sampling.

Currently, the reports of MSI of food are dominated bymetabolites which have a small molecular weight. Metaboliteimaging in food will continue to answer basic biologicalquestions, so we expect the publication rate of metabolite MSIto remain at a high level. In developing countries, pesticides,anti-microbial chemicals, inks, plasticizers, and adulterants willremain analytes of interest in MSI of food, so we expect thepublication rate of exogenous chemical MSI to grow marginally.In developed countries, protein and peptide allergens areanalytes of interest in MSI of food. Allergen imaging is arelatively new field and gives food-processing companies aneasy, visual method to justify “allergen-free” labeling on theirhigh-end products.

Because sampling can be easily performed in front of amass spectrometer, the standing challenge for MSI is to be ableto resolve isobaric interference and identify isomeric com-pounds. Until recently, chromatographic separation (e.g., withHPLC) was essentially the only approach to tackle the problemof isobaric interference. However, the steady development ofMS and hyphenated methods over recent years has broughta number of alternative methods. These methods includestructure-sensitive tandem MS techniques, such as electroncapture dissociation (ECD) and electron transfer dissociation(ETD) (Turecek & Julian, 2013), ion mobility spectroscopy,such as field asymmetric ion mobility spectroscopy (FAIMS)(Shvartsburg, Clemmer, & Smith, 2010) or differential mobilityspectrometry (DMS) (Blagojevic et al., 2011), and high-resolutiontandem mass spectrometry detection (e.g., Orbitrap or FTICR)(Yathavakilla et al., 2008). We expect that the above techniqueswill play a more important role in MS imaging of food in future.

ACKNOWLEDGMENTS

This work was supported by the National key scientificinstrument development projects (2011YQ14015008), the OpenFund of the State Key Laboratory of Analytical Chemistry forLife Science of Nanjing University (No. SKLACLS1106), andProgram for New Century Excellent Talents in University (No.NCET-11-0999).

VII. Abbreviations

API atmospheric pressure ionizationAP-MALDI atmospheric pressure matrix-assisted laser

desorption/ionizationDAPPI desorption atmospheric pressure photoionization


& HANDBERG ET AL.

DART direct analysis in real timeDESI desorption electrospray ionizationDMS differential mobility spectrometryECD electron capture dissociationELDI electrospray-assisted laser desorption ionizationESI electrospray ionizationETD electron transfer dissociationFAIMS field asymmetric ion mobility spectroscopyFAPA flowing atmospheric pressure afterglowFTICR Fourier transform ion cyclotron resonanceHV high voltageLAESI laser ablation electrospray ionizationLA-FAPA laser ablation flowing atmospheric pressure

afterglowHPLC high performance liquid chromatographyLOD limit-of-detectionMALDI matrix-assisted laser desorption/ionizationMS mass spectrometryMS/MS tandem mass spectrometryMSI mass spectrometry imagingNd:YAG neodymium-doped yttrium aluminum garnetPESI probe electrospray ionizationSIMS secondary ion mass spectrometrySDAPCI surface desorption atmospheric pressure chemi-

cal ionization

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