Common MSI organic acid matrices and their applications.
\\n\\n
More than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\\n\\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\\n\\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\\n\\nAdditionally, each book published by IntechOpen contains original content and research findings.
\\n\\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\\n\\n\\n\\n
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'
Simba Information has released its Open Access Book Publishing 2020 - 2024 report and has again identified IntechOpen as the world’s largest Open Access book publisher by title count.
\n\nSimba Information is a leading provider for market intelligence and forecasts in the media and publishing industry. The report, published every year, provides an overview and financial outlook for the global professional e-book publishing market.
\n\nIntechOpen, De Gruyter, and Frontiers are the largest OA book publishers by title count, with IntechOpen coming in at first place with 5,101 OA books published, a good 1,782 titles ahead of the nearest competitor.
\n\nSince the first Open Access Book Publishing report published in 2016, IntechOpen has held the top stop each year.
\n\n\n\nMore than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\n\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\n\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\n\nAdditionally, each book published by IntechOpen contains original content and research findings.
\n\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\n\n\n\n
\n'}],latestNews:[{slug:"intechopen-authors-included-in-the-highly-cited-researchers-list-for-2020-20210121",title:"IntechOpen Authors Included in the Highly Cited Researchers List for 2020"},{slug:"intechopen-maintains-position-as-the-world-s-largest-oa-book-publisher-20201218",title:"IntechOpen Maintains Position as the World’s Largest OA Book Publisher"},{slug:"all-intechopen-books-available-on-perlego-20201215",title:"All IntechOpen Books Available on Perlego"},{slug:"oiv-awards-recognizes-intechopen-s-editors-20201127",title:"OIV Awards Recognizes IntechOpen's Editors"},{slug:"intechopen-joins-crossref-s-initiative-for-open-abstracts-i4oa-to-boost-the-discovery-of-research-20201005",title:"IntechOpen joins Crossref's Initiative for Open Abstracts (I4OA) to Boost the Discovery of Research"},{slug:"intechopen-hits-milestone-5-000-open-access-books-published-20200908",title:"IntechOpen hits milestone: 5,000 Open Access books published!"},{slug:"intechopen-books-hosted-on-the-mathworks-book-program-20200819",title:"IntechOpen Books Hosted on the MathWorks Book Program"},{slug:"intechopen-s-chapter-awarded-the-guenther-von-pannewitz-preis-2020-20200715",title:"IntechOpen's Chapter Awarded the Günther-von-Pannewitz-Preis 2020"}]},book:{item:{type:"book",id:"6537",leadTitle:null,fullTitle:"Evolving Strategies in Peritoneal Dialysis",title:"Evolving Strategies in Peritoneal Dialysis",subtitle:null,reviewType:"peer-reviewed",abstract:"Evolving Strategies in Peritoneal Dialysis is intended as a concise compilation of articles designed to understand the basics of the current practice of the most cost-effective form of life support for patients with end-stage renal disease who require dialysis. Current strategies are understood best with a review of the historical development of catheter materials, solution packaging, and simplified machinery, which allow safe and effective nocturnal treatments. Quantitation of the efficacy of peritoneal dialysis is also reviewed because such calculations were also developed by the pioneers of nephrology to ensure adequacy of dialysis and daily fluid balance, which are responsible for the best chance for long-term patient survival. Comparison of methods for catheter placement is presented as well as the role that a dialysis center plays in the health and success of this form of end-stage renal disease patient care. The novel concept of assisted peritoneal dialysis for the infirm or institutionalized patients is probably the next direction needed to make available this treatment to many more patients than are currently eligible to receive it. This concept is explored in a separate chapter. Finally, professional dialysis staff must monthly assess individuals' nutritional status, bone health, and infection prevention and treatment to ensure the greatest functional status for these patients. This book concludes with a review of each of these topics to expand the mandatory monthly surveillance performed by dialysis centers for each patient who receives home peritoneal dialysis therapy.",isbn:"978-1-78923-533-3",printIsbn:"978-1-78923-532-6",pdfIsbn:"978-1-83881-537-0",doi:"10.5772/intechopen.71213",price:119,priceEur:129,priceUsd:155,slug:"evolving-strategies-in-peritoneal-dialysis",numberOfPages:114,isOpenForSubmission:!1,isInWos:null,hash:"76bb5dc2ce63f0026f7993b54f062861",bookSignature:"Edward T. 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He trained at the University of\nCalifornia at Los Angeles (UCLA) from 1973 to 1978. His faculty\npositions include UCLA, University of Utah, Medical College of\nVirginia, and University of South Dakota. Other positions include professor and chairman emeritus, Department of Internal\nMedicine, University of South Dakota, Sanford School of Medicine; and Bush Foundation of Minnesota Sabbatical Fellowship in Critical Care, Department of Anesthesiology at the University of Iowa in 2009. Dr. Zawada Jr. is board certified by the\nAmerican Board of Internal Medicine in Internal Medicine, Nephrology, Geriatrics, and Critical Care Medicine. Other board certifications include Nutrition and\nClinical Pharmacology. He is a Master of the American College of Physicians and\nFellow of the American College of Critical Care Medicine, the American Society of\nNephrology, the American Society of Hypertension, the American College of Chest\nPhysicians, the American College of Clinical Pharmacology, the American College\nof Nutrition, and the American Heart Association.",institutionString:"University of South Dakota",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"8",totalChapterViews:"0",totalEditedBooks:"5",institution:{name:"University of South Dakota",institutionURL:null,country:{name:"United States of America"}}}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"1163",title:"Nephrology",slug:"nephrology"}],chapters:[{id:"60480",title:"Introductory Chapter: Peritoneal Dialysis, Overview and Current Concepts",doi:"10.5772/intechopen.75627",slug:"introductory-chapter-peritoneal-dialysis-overview-and-current-concepts",totalDownloads:324,totalCrossrefCites:0,totalDimensionsCites:0,signatures:"Edward T. 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\r\n\tThe textbook would target the recent most developments in the chromophores (dyes, pigments, organic compounds with extended conjugation and their metal-complexes) and bio-chromophores (natural biomolecules such as metalloenzymes and artificial biomolecules for instance those developed by integrating biomolecules with chromophores) based research.
\r\n\r\n\tThis book will entail design, development, and industrial applications of new and novel dyes and pigments from wide areas of organic and inorganic chemistry along with biological sciences. Besides, this book will comprise recent design and development on organic molecules and their metal-complexes towards the detection of biologically and environmentally concerned cations, anions, neutral molecules via chromogenic, fluorometric, and electrochemical signaling responses using UV/vis, emission, and electrochemical techniques. Further, the advancements in the bio-chromophores for detection of biologically vital as well as harmful ions and molecules using colorimetric changes via UV/vis technique, fluorimetric signaling through emission technique, and electrochemical changes by cyclic voltammetry (CV), LSV, etc. will also be included in this book.
",isbn:"978-1-83969-341-0",printIsbn:"978-1-83969-340-3",pdfIsbn:"978-1-83969-342-7",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,hash:"4aca0af0356d8d31fa8621859a68db8f",bookSignature:"Dr. Rampal Pandey",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/10572.jpg",keywords:"Organic Probes, Metal-Complex Probes, Nano-Probes, Fluorometric Readout, Electrochemical Response, Environmentally Concerned Analytes, Bio-Chromophores, Biomolecular Detection, Multichannel Signaling Response, Absorption, Emission, Electrochemical",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"November 17th 2020",dateEndSecondStepPublish:"December 15th 2020",dateEndThirdStepPublish:"February 13th 2021",dateEndFourthStepPublish:"May 4th 2021",dateEndFifthStepPublish:"July 3rd 2021",remainingDaysToSecondStep:"a month",secondStepPassed:!0,currentStepOfPublishingProcess:3,editedByType:null,kuFlag:!1,biosketch:"A leading young researcher in the chromophore, MOF, and soft material research, Appointed Associate Dean at NIT Uttarakhand, received Presidents Inspire Teacher Award, published quality international papers, registred patents, developed MOOC and e-PG Pathshala contents.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"338234",title:"Dr.",name:"Rampal",middleName:null,surname:"Pandey",slug:"rampal-pandey",fullName:"Rampal Pandey",profilePictureURL:"https://mts.intechopen.com/storage/users/338234/images/system/338234.jpg",biography:"DR RAMPAL PANDEEY\nAssistant Professor (Chemistry) & Associate Dean (Faculty Welfare & International Affairs) \n\t \nACADEMIC and PROFESSIONAL DETAILS\n1. Assistant Professor (Gr-1), The President’s Inspired Teacher, NIT Uttarakhand, India July 2018-To date \n2. Assistant Professor (DST INSPIRE Faculty), Dr. H. S. Gour University, Sagar, 2013-2018. \n3. Research Associate, September 2012-June, 2013 BHU, Varanasi (U.P.),\n4. Senior Research Fellow (2010-2012) Junior Research Fellow: (2009-2010), BHU, Varanasi. \n5. Teaching Assistant: 2012-13 (BHU) and Teaching Assistant: 2005-06 (APS University, Rewa). \n6. Post PG Project: (06 Months) 2004, BARC, Mumbai.\n7. M.Sc. (Inorganic Chemistry) in 2004; B.Sc. in 2002 from A.P.S. University, Rewa, India. \nADDITIONAL QUALIFICATION\n1.\tNET Qualified\n2. GATE Qualified\nRESEARCH GUIDANCE \n1.\tPhD Awarded: 01 (one); On-going 04 (four). One JRF trained under my project.\nRESEARCH PROJECTS \n1. DST Project (Rs. 35,00,000/-) 01/07/2013 to 30/06/2018.\n2. Co-ordinating MOOC programme (Rs. 13,00,000/-), Status: On-going \nHONORS and AWARDS \n1.\tVDGOOD International Outstanding Scientist Award-2020\n2.\tExcellent work appreciation from NIT Uttarakhand (present employer)-2020\n3.\tThe President’s Inspired Teacher (Recognized in 2015) (https://en.wikipedia.org/wiki/Inspired_Teacher)\n4.\tAmerican Chemical Society Award (3 yr membership) for significant research-2015\n5.\tBest poster award in RSC-CRSI at NCL Pune, India-2015\n6.\tBest poster award (IYL) Conference at Dr. H. S. Gour University, Sagar, India-2015\n7.\tDST-INSPIRE Faculty Award-2012\n8.\tSenior Research Fellowship, CSIR, New Delhi, India-2010\n9.\tBest (1st) poster award at IISc, Bangalore, India-2010.\n10.\t1st ranker in university entrance examination for MBA-IR-2002\nMEMBERSHIP \n1.\tThree (03) year member of American Chemical Society (ACS), USA\n2.\tAnnual member of Royal Chemical Society (RSC), UK (No. 574194) \n3.\tLife member of Chemical Research Society of India (CRSI) (No. LM 1051).\n4. Life member of Society of Material Chemistry of India (SMC-INDIA) (No. LM-647).\n\nRESEARCH PUBLICATIONS (Cumulative Impact Factor 131; h-index 15, i10 23)\nInternational (41) in ACS, RSC, Elsevier, Wiley-Intersciences \nNational (03) \nPATENTS\nOne (01) GRANTED; one (01) under revision \n\n(a) Online Invited lectures delivered: 02\n(b) Invited lectures: 06 (02 International; 04 National), \n(c) Oral lectures: 03; Poster: 15 \n(d) Resource Person: 01\n(e) Conferences/meetings organized: 08 \n\nBook Chapters Written: 01 \n\ne-CONTENT DEVELOPMENT \n1. MHRD-UGC e-pathashala programme \n(a) Developed e-texts of 20 modules. \n(b) Delivered e-lectures of 09 modules available on YouTube by MHRD-UGC\nhttps://youtu.be/9nDUfojnjNk https://youtu.be/YKlQQodPXMo https://youtu.be/1WCpvT7wLso \n \n2. MOOC programme on-going (Role: Co-coordinator) \n(a) Developed 40 modules under my supervision\n(b) Seven (07) e-content developed as content writer.",institutionString:"National Institute of Technology Uttarakhand",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:null}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"8",title:"Chemistry",slug:"chemistry"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"252211",firstName:"Sara",lastName:"Debeuc",middleName:null,title:"Ms.",imageUrl:"https://mts.intechopen.com/storage/users/252211/images/7239_n.png",email:"sara.d@intechopen.com",biography:"As an Author Service Manager my responsibilities include monitoring and facilitating all publishing activities for authors and editors. 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Víctor García",authors:[{id:"99504",title:"Prof.",name:"Maurizio",middleName:null,surname:"Ceccarelli",fullName:"Maurizio Ceccarelli",slug:"maurizio-ceccarelli"}]},{id:"19143",title:"Complications of Liposuction",slug:"complications-of-liposuction",signatures:"Francisco J. Agullo, Humberto Palladino and Sadri O. Sozer",authors:[{id:"49318",title:"Dr.",name:"Sadri",middleName:"Ozan",surname:"Sozer",fullName:"Sadri Sozer",slug:"sadri-sozer"},{id:"49319",title:"Dr.",name:"Frank",middleName:null,surname:"Agullo",fullName:"Frank Agullo",slug:"frank-agullo"},{id:"128585",title:"Dr.",name:"Humberto",middleName:null,surname:"Palladino",fullName:"Humberto Palladino",slug:"humberto-palladino"},{id:"57330",title:"Dr.",name:"Humberto",middleName:null,surname:"Palladino",fullName:"Humberto Palladino",slug:"humberto-palladino"}]}]}]},onlineFirst:{chapter:{type:"chapter",id:"73803",title:"Mass Spectrometry Imaging of Neurotransmitters",doi:"10.5772/intechopen.93835",slug:"mass-spectrometry-imaging-of-neurotransmitters",body:'Mass Spectrometry Imaging (MSI) is an incredibly powerful label-free technique that can determine qualitative and quantitative information of hundreds of compounds in a tissue section in one experiment [1, 2]. Small molecule detection, especially of neurotransmitters (NTs), currently relies heavily on histochemical, immunohistochemical, and ligand-based assays. Antibody-based methods suffer from limitations in cost and availability of antibodies, lack of specificity for target molecules of interest, and low throughput [3, 4]. The development of MSI has overcome many of these challenges and will be discussed throughout. The basic methodology of MSI is to section tissue using a cryostat to approximately 10-20 μm thickness; tissues may or may not be embedded in a cryomatrix such as Shandon™ M-1 (ThermoFisher Scientific). Next, matrix must be deposited on the tissue section, which is most often done by spray-coating the tissue using a pneumatic sprayer. Variations on typical organic matrices, such as inorganic nanoparticles (NPs), have been explored by numerous researchers and will be commented on here. Mass spectral data is collected at discrete locations on the sample surface via a raster pattern, which can then be assembled into a heat-map image of molecule location. Figure 1 depicts the typical MSI scheme [5]. A number of overall reviews of MSI have appeared in recent years [6] that address broad topics like ionization of small molecules [7], clinical applications [2], and high-resolution analyses [5].
General scheme of the mass spectrometry imaging process. (a) The tissue section is covered with matrix and irradiated by a pulsed laser beam. (b) Mass spectrum acquired from one spatial location on the tissue section. (c) MS images of different m/z peaks compiled from all spatial locations. Reprinted with permission from Ref. [5]. Copyright 2013 Springer.
The broader scientific community is not yet fully utilizing MSI as there are still challenges to be overcome [7, 8], including: (i) low ionization efficiency for small molecules, (ii) chemical noise interferents/overlapping signals of small molecules with traditional matrices [9], (iii) reproducibility issues across laboratories which limits universal procedures for MSI in pre-/clinical research, (iv) limits to lateral spatial resolution inherent to the matrix crystallization process which affects the ability to clearly define tissue margins, and (v) delocalization of analyte molecules during sample preparation. This chapter will focus on the analysis of small molecules, specifically neurotransmitters (NTs), due to the complex biological processes that occur in the brain and have broad implications in disease states and overall health. This chapter is broken down in two main categories, as strategies to improve ionization must either focus on (i) the chemical nature of the analyte and changing its properties to better facilitate ionization, or (ii) on utilizing a different mechanism of ionization to favor small molecules of interest.
Small molecule NTs are the chemical messengers of the central nervous system. Having a complete picture of NT location and abundance will aid in understanding of many different disease states and developmental processes. NTs are difficult to detect in situ via mass spectrometry due to their low physiological abundance (e.g., nM to pM concentration) within a complex biological tissue with many different classes of biomolecules, and overlapping low molecular mass range with most traditional matrices. Prior to the analyses discussed here, NTs were localized based on their protein-receptors or transporters, which does not always give an accurate accounting of present location.
Instrumentation used for MSI can vary widely, but most laser-based work is performed by time-of-flight (TOF) instruments. The other common setup is using desorption electrospray ionization (DESI) as an ion source, which is not the focus here, but is another option gaining in popularity [10, 11]. Other hardware configurations can favor small molecules (e.g., ion mobility, triple quadrupole instruments, Fourier transform – ion cyclotron resonance) and so there is no one-size-fits-all set-up for small molecule MSI experiments. In contrast to instrument choice, sample preparation/derivatization and ionization conditions are areas that can be standardized in order to achieve similar results across different platforms. The focus here is not on the many instrument combinations as other reviews have adequately explored this topic [6, 7].
Sample handling and preparation of tissue sections are integral to maintaining sample integrity; after cryo-sectioning, tissue is typically thaw mounted onto a solid surface. The surface must be conductive in order to apply a potential to the sample and accelerate ions out of the instrument source. Common materials include coated metal targets (expensive, cannot be archived, and not histology compatible) or indium-tin oxide (ITO) coated glass slides. Matrix application to the tissue section is ideally a homogenous coating of small crystals that provide optimum extraction conditions of analyte. After application, the key process is the co-crystallization that must occur between matrix and analyte. Spraying parameters affect the “wetness” of the surface of the tissue and are a balance between molecular diffusion and effective extraction. Crystal size is one of the more critical factors for a successful MSI experiment and multiple studies determined the parameters important for optimum crystallization.
Commonly used organic acid matrices for MSI are shown in Table 1 and include sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DHB), and α-cyano-4-hydroxycinnamic acid (CHCA). These matrices work for a broad variety of biomolecules including peptides and lipids, but do not always translate well to small molecule detection. The introduction of N-(1-napthyl)ethylenediamine dihydrochloride (NEDC) and 1,5-diaminonapthalene (DAN) have improved the detection of small molecules, though these matrices have different preparation needs (e.g., sublimation and recrystallization) which increases the time required for sample processing [12, 19]. DAN and NEDC matrices have not been fully explored yet in the literature. Moreover, the propensity for organic matrices to self-ionize and create chemical noise in the low mass range prevents effective analysis of most metabolites. Recently, this has led toward the rational design [35] or selection [36] of matrices that can address this, but the lack of consistency in performance can still be an issue. Matrix applications are notorious for behaving differently across laboratories, and significant research in the past 25 years has been devoted to identifying preparation methods that result in the most consistent data [37, 38, 39].
Common MSI organic acid matrices and their applications.
Sublimation procedures, mentioned vida supra, require that matrix and sample are placed in a vacuum chamber which is evacuated [32]. The sample is cooled while the matrix is heated, resulting in sublimation of the matrix which will condense on the cool sample surface. Recrystallization of the matrix is often coupled with this technique. There are advantages and disadvantages to all of the aforementioned matrix application techniques, which have been discussed thoroughly in the literature [40, 41, 42]. Automated sprayers have become increasingly popular and help with consistency of matrix application, though the size and spacing of the matrix droplets will ultimately affect spatial resolution of the experiment. This topic has been frequently discussed and reviewed in the literature [2, 6], so only a basic introduction is given here.
Despite these challenges, there are a few examples of successful metabolomics imaging experiments, though they have utilized purposefully designed matrices that do not generate interfering signals [35] or have used high-resolution instruments that have the high mass accuracy to distinguish between isobaric signals [43, 44]. These approaches are not an all-encompassing solution, and the next sections explore other strategies to achieve broader success with MSI of small molecules, specifically NTs.
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, 47, 48, 49], silver [50, 51, 52, 53], carbon based substrates [54, 55, 56, 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 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 solution-based 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, 67]. Pt sputtering has been demonstrated on lipids in tissue [68] 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 [66]. 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.
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. [66]. Copyright 2018 American Society for Mass Spectrometry.
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.
MSI of a sagittal zebrafish tissue section at 5 μm 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.
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 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.
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, 80, 81, 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-coating 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.
(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.
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 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.
(A) LATT setup and (B) schematic diagram of the system. Reprinted with permission from Ref. [89]. Copyright 2020 American Chemical Society.
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 which feature spiking of tissue homogenates [96].
Description of qMSI experiments where on-tissue spotting and homogenate spiking are two popular methods. Reprinted with permission from Ref. [94]. Copyright 2019 Elsevier Ltd.
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 better representation of the ionization process for analyte embedded within tissue. However, it is more time consuming, labor intensive, and requires more tissue for the calibration curve. Each method has been correlated with LC-MS data, the current primary method used for quantification [97].
Specific examples with clinical relevancy are briefly described here. First, epertinib and lapatinib were quantified in a metastatic brain cancer mouse model using stable isotope labeling, and with liquid chromatography (LC)-MS validation [91]. The topical drugs roflumilast, tofacitinib, ruxolitinib, and LEO 29102 were examined in human skin explants to determine drug penetration and evaluate lipid markers [90]. qMSI data had a much lower quantitation range than LC-MS data of individual skin layers. Rifampicin in mouse liver tissue was quantified via a fragment ion of the intact molecule. The method used an in-house synthesized stable isotope and correlated the results with LC-MS/MS [98]. Lastly, there is one example that specifically focused on comprehensive mapping of NTs in Parkinson’s disease lesioned mouse brain and demonstrated quantitation of dopamine using a stable isotope [87]. All of the drug molecules listed in this paragraph are above the general size range that NTs and metabolites fall within, ranging from 400 to 800 Da, but present possible future avenues of research for the NT-focused methods discussed in this chapter.
The tissue mimetic model first gained popularity with examination of lapatinib and nevirapine in mouse liver by Groseclose and Castellino [99]. In addition to demonstrating high spatial resolution, they examined reproducibility and drug distribution within the homogenate. Fewer applications of the tissue mimetic model have been done, especially with small molecules as opposed to lipids [100]. A notable example includes the determination of the spatial distribution of gemcitabine, a chemotherapeutic agent, and its metabolites in mouse model pancreatic tumors using AuNPs and a traditional matrix as comparison [101]. Further experiments also work on the computational side of MSI and determining the best ways to normalize spectra [102, 103].
This chapter has introduced the utility of mass spectrometry imaging (MSI) for small molecules, with a specific focus on neurotransmitters (NTs). Methods that have resulted in enhanced signals of NTs were highlighted, with alternative matrix materials and chemical derivatization of analytes the two main points of discussion. Future research is needed in both of these areas to determine optimum conditions and applications, as well as establishing standard procedures so that broad application of MSI can continue. Finally, an area not discussed here that is relevant to these techniques and that will likely be explored in the future is the quantitative determination of small molecules.
KS thanks the University of Scranton and the Chemistry Department for providing facilities, equipment, and financial support for any referenced experiments. KS thanks the Johns Hopkins Applied Imaging Mass Spectrometry (AIMS) Core Facility at the Johns Hopkins University School of Medicine for undertaking the imaging experiments referenced in this chapter. Lastly, KS thanks Nolan McLaughlin and Tyler Bielinski for tagging along for the science journey.
The author declares no conflict of interest.
DAG | diacylglycerol |
DAN | 1,5-diaminonapthalene |
DESI | desorption electrospray ionization |
DHB | 2,5-dihydroxybenzoic acid |
DPP | 2,4-diphenyl pyranylium |
CHCA | α-cyano-4-hydroxy cinnamic acid |
Glucose 6-P | glαucose 6-phosphate |
HTP | high throughput |
ITO | indium tin oxide |
LATT | laser-assisted tissue transfer |
LC-MS | liquid chromatography mass spectrometry |
MALDI | matrix-assisted laser desorption ionization |
MSI | |
qMSI | quantitative mass spectrometry imaging |
NEDC | N-(1-napthyl)ethylenediamine dihydrochloride |
NT | neurotransmitter |
NP | nanoparticle |
PA | phosphatidic acid |
PCA | principal component analysis |
PChol | phosphocholine |
PE | phospatidylethanolamine |
PEP | phosphoenolpyruvic acid |
PG | phosphatidylglycerol |
PI | phosphatidylinositol |
PC | phosphatidylcholine |
SA | sinapic (or sinapinic) acid |
TAG | triacylglycerol |
THAP | trihydroxyacetophenone |
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