Article

Fiducial Markers for Combined 3-Dimensional Mass Spectrometric and Optical Tissue Imaging

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Abstract

Mass spectrometric imaging (MSI) has become widely used in the analysis of a variety of biological surfaces. Biological samples are spatially, morphologically, and metabolically complex. Multimodal molecular imaging is an emerging approach that is capable of dealing with this complexity. In a multimodal approach, different imaging modalities can provide precise information about the local molecular composition of the surfaces. Images obtained by MSI can be coregistered with images obtained by other molecular imaging techniques such as microscopic images of fluorescent protein expression or histologically stained sections. In order to properly coregister images from different modalities, each tissue section must contain points of reference, which are visible in all data sets. Here, we report a newly developed coregistration technique using fiducial markers such as cresyl violet, Ponceau S, and bromophenol blue that possess a combination of optical and molecular properties that result in a clear mass spectrometric signature. We describe these fiducial markers and demonstrate an application that allows accurate coregistration and 3-dimensional reconstruction of serial histological and fluorescent microscopic images with MSI images of thin tissue sections from a breast tumor model.

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... In contrast to their human counterpart, the murine optic nerve is very small (4 mm in length, 1 mm across the main body, and 300 µm for the diameter of the optic nerves, with a total of around 500 µm in depth at the chiasm). This poses significant challenges for proteomic discovery efforts, which is further amplified for studies in which mass spectrometry is combined with MALDI IMS to assess the spatial distributions of identified proteins in 2-D [4][5][6] and 3-D space [7][8][9][10][11]. ...
... The use of fiducials for image registration has been incorporated into a number of imaging modalities including MRI, CT, PET and MALDI IMS. Methods for incorporating the reference points within the sample vary from one technique to another, and a number of approaches have been previously reported for MALDI IMS [7,10,12]. As such, some investigators have utilized printed fiducials to register optical images to images generated by signals observed through MALDI IMS analysis [7], while others have used fiducial markers to help align breast cancer explants grown in mice [10]. ...
... Methods for incorporating the reference points within the sample vary from one technique to another, and a number of approaches have been previously reported for MALDI IMS [7,10,12]. As such, some investigators have utilized printed fiducials to register optical images to images generated by signals observed through MALDI IMS analysis [7], while others have used fiducial markers to help align breast cancer explants grown in mice [10]. These fiducials were produced by injecting dyes into gelatin surrounding the sample tissue. ...
Article
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Biological significance: The current work addresses a number of challenges in 3-D MALDI IMS, driven by the small size of the mouse optic nerve and the need to maintain consistency across multiple 2-D IMS experiments. The 3-D IMS data yield both molecular similarities and differences between glioma-bearing and wild-type (WT) tissues, including protein distributions localizing to different anatomical subregions, which could then be targeted for identification and related back to the biology observed in gliomas of the optic nerve from this model.
... MSI distinguishes itself from regular mass spectrometry (MS) by the ability to spatially localize compounds across surfaces in the case of 2D imaging [1,2]. In the case of 3D imaging volumes are analyzed by dividing the 3D volume into multiple 2D images [3]. With the introduction of ion mobility separation, even 4D datasets appear, with the ion drift time as the fourth dimension. ...
... The MSI datasets employed to evaluate and test the methods described in this paper were acquired from samples taken from a xenograft breast tumor model as described in [3]. A MDA-MB-231 breast cancer cell line was purchased from the American Type Culture Collection (ATCC) and genetically modified to express a red fluorescent protein (tdTomato) under control of hypoxia response elements as described in [11,12]. ...
... When the tumors reached a volume of approximately 500 mm 3 , the mice were sacrificed and tumors were removed. Each tumor was embedded into a gelatin block and cresyl violet fiducial markers [3] were injected at three different positions inside the block next to the tumor. The block was sectioned into serial 2-mm thick fresh tumor sections which were snap-frozen immediately. ...
Article
Mass spectrometry imaging (MSI) produces such large amounts of high-resolution data that fast visualization of full data sets in both high spatial and spectral resolution is often problematic. Instrument specific software tools are available, but often struggle with the size and the complexity of the MSI data sets. We describe new methods to improve the handling of these large MSI data sets by means of innovative data structures and visualization strategies developed specifically for MSI. Two new software instruments implement these new methods for rapid data exploration and visualization of both 2D and 3D data sets in full spatial and spectral resolution, and can handle existing MSI data formats, including imzML and BioMap.
... Corresponding three-dimensional CSI images of the unsuppressed water signal and lipids were acquired of the same volume with TE of 15 milliseconds and NA of 2, and all other parameters remaining the same as for the water-suppressed MRSI. After these MRI/MRSI studies, mice were killed, and each tumor was marked with a novel fiducial marker system [27,31] and cut into 2-mm-thick sections for hypoxia imaging as described in our previous report [27,31]. ...
... Corresponding three-dimensional CSI images of the unsuppressed water signal and lipids were acquired of the same volume with TE of 15 milliseconds and NA of 2, and all other parameters remaining the same as for the water-suppressed MRSI. After these MRI/MRSI studies, mice were killed, and each tumor was marked with a novel fiducial marker system [27,31] and cut into 2-mm-thick sections for hypoxia imaging as described in our previous report [27,31]. ...
... For microscopic measurements, a given slide holding an embedded, fiducially marked [31] tumor slice was taken out of the icebox and placed under the microscope for imaging. In between microscopic measurements , the fresh tumor slices were returned to the icebox to keep the tissue at 4°C, and they were periodically moistened with saline to avoid dehydration of the tissue. ...
Article
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Tumor hypoxia triggers signaling cascades that significantly affect biologic outcomes such as resistance to radiotherapy and chemotherapy in breast cancer. Hypoxic regions in solid tumor are spatially heterogeneous. Therefore, delineating the origin and extent of hypoxia in tumors is critical. In this study, we have investigated the effect of hypoxia on different metabolic pathways, such as lipid and choline metabolism, in a human breast cancer model. Human MDA-MB-231 breast cancer cells and tumors, which were genetically engineered to express red fluorescent tdTomato protein under hypoxic conditions, were used to investigate hypoxia. Our data were obtained with a novel three-dimensional multimodal molecular imaging platform that combines magnetic resonance (MR) imaging, MR spectroscopic imaging (MRSI), and optical imaging of hypoxia and necrosis. A higher concentration of noninvasively detected total choline-containing metabolites (tCho) and lipid CH3 localized in the tdTomato-fluorescing hypoxic regions indicated that hypoxia can upregulate tCho and lipid CH3 levels in this breast tumor model. The increase in tCho under hypoxia was primarily due to elevated phosphocholine levels as shown by in vitro MR spectroscopy. Elevated lipid CH3 levels detected under hypoxia were caused by an increase in mobile MR-detectable lipid droplets, as demonstrated by Nile Red staining. Our findings demonstrate that noninvasive MRSI can help delineate hypoxic regions in solid tumors by means of detecting the metabolic outcome of tumor hypoxia, which is characterized by elevated tCho and lipid CH3.
... Fiducial markers are placed in the embedding block and may help to determine the position, orientation, and distortion of each section. They are also very suitable for use in the co-registration of MSI data with images from different imaging modalities [60][61][62]. ...
... It is of interest in many studies to align and stack the individual MSI images of a completed 3D-MSI dataset to construct a 3D volume, which can be used for an advanced interpretation of the spatial context of the molecular images. Embedded fiducial markers can be used as reference points to automatically co-register and stack the single sections (Fig. 4a) [60]. However, the 3D volume reconstruction can be completed without fiducial markers. ...
... a Alignment based on embedded fiducial markers requires a previous embedding of the sample into a medium but delivers good results. Adapted with permission from [60] (copyright American Chemical Society). b An alignment based on optical images requires the optical images to be linked to the MSI data before data analysis, e.g., during the experiment. ...
Article
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Mass spectrometry imaging (MSI) enables the visualization of molecular distributions on complex surfaces. It has been extensively used in the field of biomedical research to investigate healthy and diseased tissues. Most of the MSI studies are conducted in a 2D fashion where only a single slice of the full sample volume is investigated. However, biological processes occur within a tissue volume and would ideally be investigated as a whole to gain a more comprehensive understanding of the spatial and molecular complexity of biological samples such as tissues and cells. Mass spectrometry imaging has therefore been expanded to the 3D realm whereby molecular distributions within a 3D sample can be visualized. The benefit of investigating volumetric data has led to a quick rise in the application of single-sample 3D-MSI investigations. Several experimental and data analysis aspects need to be considered to perform successful 3D-MSI studies. In this review, we discuss these aspects as well as ongoing developments that enable 3D-MSI to be routinely applied to multi-sample studies.
... Copyright 2016 Elsevier B.V. (C) To avoid the "banana-problem" during volumetric reconstruction, i.e., the straightening of curved volumes, three-dimensional fiducial markers can be introduced next to the embedded tissue sample which serve as reference for a correct volumetric reconstruction. Adapted with permission from [44]. Copyright 2012 American Chemical Society. ...
... In order to address this challenge, additional information can be added in the form of fiducials. Chughtai et al. were the first to propose the insertion of fiducial marker compounds into the embedding matrix of tissues [44]. The authors injected various liquid compounds into gelatine blocks in proximity to the embedded breast cancer xenograft tissue. ...
... Current reference-free approaches suffer from the potential to create different errors, such as the banana-problem [49] or the propagation and accumulation of registration errors through the 3D stack [64]. While there have been attempts in the MSI community to address these by the introduction of spatial reference objects [44,50], the latter error occurs because the registrations are performed sequentially in pairs and with a directionality. Global optimisation approaches exist that optimise the alignment of more than two images either based on parallel algorithms, which couple all local neighbourhood transformations into a system of equations [65], or by modelling stack misalignment by using heat diffusion equations [64]. ...
Article
Full-text available
Mass spectrometry imaging (MSI) is used in many aspects of clinical research, including pharmacokinetics, toxicology, personalised medicine, and surgical decision-making. Maximising its potential requires the spatial integration of MSI images with imaging data from existing clinical imaging modalities, such as histology and MRI. To ensure that the information is properly integrated, all contributing images must be accurately aligned. This process is called image registration and is the focus of this review. In the light of the ever-increasing spatial resolution of MSI instrumentation and a diversification of multi-modal MSI studies (e.g., spatial omics, 3D-MSI), the accuracy, versatility, and precision of image registration must increase accordingly. We review application of image registration to align MSI data with different clinically relevant ex vivo and in vivo imaging techniques. Based on this, we identify steps in the current image registration processes where there is potential for improvement. Finally, we propose a roadmap for community efforts to address these challenges in order to increase registration quality and help MSI to fully exploit its multi-modal potential.
... At present, the only option available for coregistration of MALDI MS and LSCM images of this size and shape seems to be the application of fiducial markers discernible in either modality. Chughtai et al. (2012) described coregistration of MALDI MS and fluorescence images of breast cancer cells genetically modified to express a red fluorescent protein (tdTomato), which were injected and grown in athymic nude mice and, following tumor removal, embedded in gelatin. Three cresyl violet fiducial markers were injected inside the block next to the tumor. ...
... Optical images of the gelatin block sections and the ion images were coregistered based on the position of the fiducial markers using the Biomap software. As Chughtai et al. (2012) note, the selection of suitable fiducial markers that can be visualized by both optical microscopy and MSI is not trivial. Good markers must fulfill a number of requirements, such as intense color for microscopic bright-field imaging applications, or absorption/emission at selected wavelengths for fluorescence imaging, and good ionization for MSI. ...
... For the creation of the fiducial markers, we developed a novel approach which is simple, inexpensive, does not require special chemical compounds or equipment, and yields excellent contrast in both MALDI MS and LSCM images. Unlike Chughtai et al. (2012), we do not use fluorescent dyes for the fiducials. Instead, we take advantage of the Leica LSCM transmission bright-field scanning mode in which optical markers are perfectly visible. ...
Article
Spheroids—three-dimensional aggregates of cells grown from a cancer cell line—represent a model of living tissue for chemotherapy investigation. Distribution of chemotherapeutics in spheroid sections was determined using the matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Proliferating or apoptotic cells were immunohistochemically labeled and visualized by laser scanning confocal fluorescence microscopy (LSCM). Drug efficacy was evaluated by comparing coregistered MALDI MSI and LSCM data of drug-treated spheroids with LSCM only data of untreated control spheroids. We developed a fiducial-based workflow for coregistration of low-resolution MALDI MS with high-resolution LSCM images. To allow comparison of drug and cell distribution between the drug-treated and untreated spheroids of different shapes or diameters, we introduced a common diffusion-related coordinate, the distance from the spheroid boundary. In a procedure referred to as “peeling”, we correlated average drug distribution at a certain distance with the average reduction in the affected cells between the untreated and the treated spheroids. This novel approach makes it possible to differentiate between peripheral cells that died due to therapy and the innermost cells which died naturally. Two novel algorithms—for MALDI MS image denoising and for weighting of MALDI MSI and LSCM data by the presence of cell nuclei—are also presented.
... Care should be taken that the distance between the analyzed serial sections is appropriate for the tissue structure to be reconstructed, and also to process the sections under identical conditions [6]. The details of serial sectioning approach to create 3D MALDI images of different biomolecules was described for a variety of different samples [134][135][136]. This includes 3D imaging of protein and peptide distribution in rat midbrain [137], and a reconstructed 3D view of mouse brain using myelin basic protein [138]. ...
... In a different study, an interesting approach developed a coregistration technique using fiducial markers (cresyl violet, Ponceau S, and bromophenol blue), which possess a combination of optical and molecular properties. This allowed accurate coregistration of fluorescent protein expression images with MSI images and reconstruction of 3D images of molecules distribution from a breast tumor model [134]. ...
Thesis
Massenspektrometrisches Imaging (MSI) ist unverzichtbar für die Untersuchung der räumlichen Verteilung von Molekülen in einer Vielzahl von biologischen Proben. Seit seiner Einführung hat sich MALDI zu einer dominierenden Bildgebungsmethode entwickelt, die sich als nützlich erwiesen hat, um die Komplexität von Lipidstrukturen in biologischen Geweben zu bestimmen. Einerseits ist die Rolle von Cisplatin bei der Behandlung von menschlichen malignen Erkrankungen gut etabliert, jedoch ist Nephrotoxizität eine limitierende Nebenwirkung, die Veränderungen des renalen Lipidprofils beinhaltet. Dies führte zu der Motivation, die Lipidzusammensetzung des Nierengewebes in mit Cisplatin behandelten Ratten zu untersuchen, um die involvierten Lipid-Signalwege aufzuklären. Es wurde eine Methode zur Kartierung der Lipidzusammensetzung in Nierenschnitten unter Verwendung von MALDI MSI entwickelt. Die Verteilung von Nierenlipiden in Cisplatin-behandelten Proben zeigte deutliche Unterschiede in Bezug auf die Kontrollgruppen. Darüber hinaus wurde die Beurteilung der Ionenbilder von Lipiden in Cisplatin-behandelten Nieren meist als qualitative Aspekte betrachtet. Relative quantitative Vergleiche wurden durch den variablen Einfluss von experimentellen und instrumentellen Bedingungen begrenzt. Daher bestand die Notwendigkeit, ein Normalisierungsverfahren zu entwickeln, das einen Vergleich der Lipidintensität verschiedener Proben ermöglicht. Das Verfahren verwendete einen Tintenstrahldrucker, um eine Mischung der MALDI-Matrix und der internen internen Lipid-Metall-Standards aufzubringen. Unter Verwendung von ICP-MS erlaubte der interne Metallstandard, die Konsistenz der Matrix und der internen Standards zu bestätigen. Die Anwendung der Methode zur Normalisierung von Ionenintensitäten von Nierenlipiden zeigte eine ausgezeichnete Bildkorrektur und ermöglichte einen relativen quantitativen Vergleich von Lipidbildern in Cisplatin-behandelten Proben.
... MSI employing MAL-DI-MSI visualized the distribution of intact phospholipid molecular ions. All modalities were combined through the presence of fi ducial markers visible in all imaging modalities ( 45 ). Figure 1 presents results obtained from a representative breast tumor xenograft using our multimodal imaging approach. ...
... Tissue sections were stained using a modifi ed H and E staining protocol as previously described (43)(44)(45). Briefl y, 10 µm sections attached to Superfrost Slides (VWR International, Cat. ...
Article
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The lipid compositions of different breast tumor microenvironments are largely unknown due to limitations in lipid imaging techniques. Imaging lipid distributions would enhance our understanding of processes occurring inside growing tumors such as cancer proliferation and metastasis. Recent developments in MALDI mass spectrometry imaging (MSI) enable rapid and specific detection of lipids directly from thin tissue sections. In this study, we performed multimodal imaging of acylcarnitines, phosphatidylcholines (PC), a lysophosphatidylcholine (LPC) and a sphingomyelin (SM) from different microenvironments of breast tumor xenograft models, which carried tdTomato red fluorescent protein as a hypoxia-response element driven reporter gene. The MSI molecular lipid images revealed spatially heterogeneous lipid distributions within tumor tissue. Five of the most abundant lipid species, namely PC(16:0/16:0), PC(16:0/18:1), PC(18:1/18:1) PC(18:0/18:1) and SM(d18:1/16:0) sodium adduct were localized in viable tumor regions, while PC(18:0/22:1) and LPC(16:0/0:0) were detected in necrotic tumor regions. We identified a heterogeneous distribution of palmitoyl- and stearoylcarnitine, which mostly colocalized with hypoxic tumor regions. For the first time, we have applied a multimodal imaging approach that has combined optical imaging and MALDI-MSI with ion mobility separation to spatially localize and structurally identify acylcarnitines and a variety of lipid species present in breast tumor xenograft models.
... When tumors reached a volume of approximately 500-mm 3 , mice were sacrificed and tumors were removed. Each tumor was embedded into a gelatin block prepared using 15-mm×15-mm×5-mm cryomolds (Sakura Finetek, Torrance, CA, USA), 10 % gelatin, cooled to 30°C in order to prevent tissue degradation, and three cresyl violet fiducial markers, which were injected inside the gelatin block next to the tumor as previously described [12][13][14]. This block was sectioned into serial 2mm thick fresh tumor sections using an acrylic adjustable tissue slicer (12-mm depth up to 25-mm width; Braintree Scientific, Inc., Braintree, MA, USA) and tissue slicer blades (Braintree Scientific, Inc.). ...
... A human breast tumor xenograft model expressing tdTomato red fluorescent protein in the hypoxic regions was embedded in a gelatin block containing fiducial markers as previously published [12][13][14]. The gelatin blocks were sectioned into three 2-mm thick sections for ex vivo microscopic visualization of the red fluorescent protein. ...
Article
Full-text available
Mass spectrometric imaging (MSI) in combination with electrospray mass spectrometry (ESI-MS) is a powerful technique for visualization and identification of a variety of different biomolecules directly from thin tissue sections. As commonly used tools for molecular reporting, fluorescent proteins are molecular reporter tools that have enabled the elucidation of a multitude of biological pathways and processes. To combine these two approaches, we have performed targeted MS analysis and MALDI-MSI visualization of a tandem dimer (td)Tomato red fluorescent protein, which was expressed exclusively in the hypoxic regions of a breast tumor xenograft model. For the first time, a fluorescent protein has been visualized by both optical microscopy and MALDI-MSI. Visualization of tdTomato by MALDI-MSI directly from breast tumor tissue sections will allow us to simultaneously detect and subsequently identify novel molecules present in hypoxic regions of the tumor. MS and MALDI-MSI of fluorescent proteins, as exemplified in our study, is useful for studies in which the advantages of MS and MSI will benefit from the combination with molecular approaches that use fluorescent proteins as reporters. Figure
... Therefore, the improvement of strategies for 3D model reconstruction has been crucial in MSI studies (Vos et al., 2019). The most straightforward path to 3D image assembly is to embed fiducial markers onto the tissue sections as reference points (Chughtai et al., 2012). The anatomical features of the optical or MS images have also been used to align 2D MS images. ...
Article
Full-text available
Mass spectrometry (MS) has become a central technique in cancer research. The ability to analyze various types of biomolecules in complex biological matrices makes it well suited for understanding biochemical alterations associated with disease progression. Different biological samples, including serum, urine, saliva, and tissues have been successfully analyzed using mass spectrometry. In particular, spatial metabolomics using MS imaging (MSI) allows the direct visualization of metabolite distributions in tissues, thus enabling in‐depth understanding of cancer‐associated biochemical changes within specific structures. In recent years, MSI studies have been increasingly used to uncover metabolic reprogramming associated with cancer development, enabling the discovery of key biomarkers with potential for cancer diagnostics. In this review, we aim to cover the basic principles of MSI experiments for the nonspecialists, including fundamentals, the sample preparation process, the evolution of the mass spectrometry techniques used, and data analysis strategies. We also review MSI advances associated with cancer research in the last 5 years, including spatial lipidomics and glycomics, the adoption of three‐dimensional and multimodal imaging MSI approaches, and the implementation of artificial intelligence/machine learning in MSI‐based cancer studies. The adoption of MSI in clinical research and for single‐cell metabolomics is also discussed. Spatially resolved studies on other small molecule metabolites such as amino acids, polyamines, and nucleotides/nucleosides will not be discussed in the context.
... We have spatially localized a number of lipid species in two dimensions in this breast tumor xenograft model. 22 It is now possible to perform 3D reconstruction and rendering of MSI tissue volumes by using block-face optical imaging methods 23 or fiducial marker strategies 24 to accurately align successive 2D MSI experiments of tissue sections that are cut with welldefined spacing throughout a biological sample, such as a tumor or an organ. 3D reconstruction and rendering of MSI data is useful for visualizing the characteristics of a tissue volume in 3D, and it also enables quantitative mining of 3D MSI volume data, for example, for quantifying correlations between spectral and spatial features, by using multivariate statistical analysis approaches. ...
Article
Hypoxic areas are a common feature of rapidly growing malignant tumors and their metastases, and are typically spatially heterogeneous. Hypoxia has a strong impact on tumor cell biology and contributes to tumor progression in multiple ways. To date, only a few molecular key players in tumor hypoxia, such as for example hypoxia-inducible factor-1 (HIF-1), have been discovered. The distribution of biomolecules is frequently heterogeneous in the tumor volume, and may be driven by hypoxia and HIF-1α. Understanding the spatially heterogeneous hypoxic response of tumors is critical. Mass spectrometric imaging (MSI) provides a unique way of imaging biomolecular distributions in tissue sections with high spectral and spatial resolution. In this paper, breast tumor xenografts grown from MDA-MB-231-HRE-tdTomato cells, with a red fluorescent tdTomato protein construct under the control of a hypoxia response element (HRE)-containing promoter driven by HIF-1α, were used to detect the spatial distribution of hypoxic regions. We elucidated the 3D spatial relationship between hypoxic regions and the localization of small molecules, metabolites, lipids, and proteins by using principal component analysis - linear discriminant analysis (PCA-LDA) on 3D rendered MSI volume data from MDA-MB-231-HRE-tdTomato breast tumor xenografts. In this study we identified hypoxia-regulated proteins active in several distinct pathways such as glucose metabolism, regulation of actin cytoskeleton, protein folding, translation/ribosome, splicesome, the PI3K-Akt signaling pathway, hemoglobin chaperone, protein processing in endoplasmic reticulum, detoxification of reactive oxygen species, aurora B signaling/apoptotic execution phase, the RAS signaling pathway, the FAS signaling pathway/caspase cascade in apoptosis and telomere stress induced senescence. In parallel we also identified co-localization of hypoxic regions and various lipid species such as PC(16:0/18:1), PC(16:0/18:2), PC(18:0/18:1), PC(18:1/18:1), PC(18:1/18:2), PC(16:1/18:4), PC(18:0/20:3), PC(16:0/22:1), among others. Our findings shed light on the biomolecular composition of hypoxic tumor regions, which may be responsible for a given tumor's resistance to radiation or chemotherapy.
... Sometimes, it is necessary to embed delicate tissue in the supporting material to assist further cryosections. For this reason, gelatin might be used (Chen et al., 2009), with additional, fiducial markers (see Section VI.C, Chughtai et al., 2012a). It is not recommended to use any optimal cutting temperature (OTC) polymers, since they ionize easily and act as potent ion suppressors (Schwartz, Reyzer, & Caprioli, 2003). ...
Article
Imaging Mass Spectrometry (IMS) is strengthening its position as a valuable analytical tool. It has unique ability to identify structures and to unravel molecular changes that occur in the precisely defined part of the sample. These unique features open new possibilities in the field of various aspects of biological research. In this review we briefly discuss the main imaging mass spectrometry techniques, as well as the nature of biological samples and molecules, which might be analyzed by such methodology. Moreover, a novel approach, where different analytical techniques might be combined with the results of IMS study, is emphasized and discussed. With such a fast development of IMS and related methods, we can foresee the promising future of this technique. © 2015 Wiley Periodicals, Inc. Mass Spec Rev. © 2015 Wiley Periodicals, Inc.
... The multimodal fusion and its interrogation with histology evaluation represents a current standard approach in medically important areas. And it brings a complex overview of the analyzed (patho)physiological features within the tissue together with the precise information of local molecular composition of the tissue surface (Chughtai et al., 2012). For the combination of hyperspectral data obtained from molecular or elemental imaging mass spectrometry and optical microscopy, all performed on consecutive sections from one tissue sample, the application of fiducial markers (points of reference) is needed to implement a correct image co-registration (Fig. 4). ...
Article
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This feature article discusses two modern mass spectrometry abbreviations in their clinical applications. Rapid evaporative ionization mass spectrometry (REIMS) is reported as a molecular classification tool useful for spectral features definition prior to mass spectrometry imaging (MSI). REIMS is appreciated not only as an ionization technique coupled with a surgical device but particularly as a biomarker discovery tool. For more complex understanding of pathological processes at cellular and molecular levels, the importance of multimodal approach in imaging applications is documented in the context of fiducial markers needed for hyperspectral data fusion collected by optical microscopy, elemental and molecular MSI. Finally, pathogen inactivation needed prior to the sectioning of the infected tissue is reported, and the impact of formaldehyde crosslinking to signal reduction is discussed.
... Within this picture, MALDI instruments have a unique position when solid samples or surfaces are involved. These include primarily tissue imaging (52,53) and the coupling of protein (and other) microarrays with mass spectrometry (54). Figure 21 shows mass spectra of the peptides captured on four 200 μm spots on a protein microarray containing (A) anti-HA, (B) anti-cmyc, (C) anti-V5 and (D) no antibody, respectively. ...
Article
Long before the introduction of matrix-assisted laser desorption/ionization (MALDI), electrospray ionization (ESI), Orbitraps, and any of the other tools that are now used ubiquitously for proteomics and metabolomics, the highest performance mass spectrometers were sector instruments, providing high resolution mass measurements by combining an electrostatic energy analyzer (E) with a high field magnet (B). In its heyday, the four sector mass spectrometer (or EBEB) was the crown jewel, providing the highest performance tandem mass spectrometry using single, high energy collisions to induce fragmentation. During a time in which quadrupole and tandem triple quadrupole instruments were also enjoying increased usage and popularity, there were, nonetheless, some clear advantages for sectors over their low collision energy counterparts. Time-of-flight (TOF) mass spectrometers are high voltage, high vacuum instruments that have much in common with sectors and have inspired the development of tandem instruments exploiting single high energy collisions. In this retrospective, we recount our own journey to produce high performance TOFs and tandem TOFs, describing the basic theory, problems, and the advantages for such instruments. An experiment testing impulse collision theory (ICT) underscores the similarities with sector mass spectrometers where this concept was first developed. Applications provide examples of more extensive fragmentation, side chain cleavages, and charge-remote fragmentation, also characteristic of high energy sector mass spectrometers. Moreover, the so-called curved-field reflectron has enabled the design of instruments that are simpler, collect and focus all of the ions, and may provide the future technology for the clinic, for tissue imaging, and the characterization of microorganisms.
... Finally, it is clear that 2D images of a tissue section are a just a snap shot of what is occurring in biological samples. Big efforts are being made to obtain not only 3D MSI images (Trede et al., 2012;Norris and Caprioli, 2013) but also to combine different imaging modalities, as fluorescent microscopy or histological staining, by using multimodal molecular imaging to provide more detailed anatomical information (Chughtai et al., 2012;Thiele et al., 2014). ...
Article
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These are definitively exciting times for membrane lipid researchers. Once considered just as the cell membrane building blocks, the important role these lipids play is steadily being acknowledged. The improvement occurred in mass spectrometry techniques (MS) allows the establishment of the precise lipid composition of biological extracts. However, to fully understand the biological function of each individual lipid species, we need to know its spatial distribution and dynamics. In the past 10 years, the field has experienced a profound revolution thanks to the development of MS-based techniques allowing lipid imaging (MSI). Images reveal and verify what many lipid researchers had already shown by different means, but none as convincing as an image: each cell type presents a specific lipid composition, which is highly sensitive to its physiological and pathological state. While these techniques will help to place membrane lipids in the position they deserve, they also open the black box containing all the unknown regulatory mechanisms accounting for such tailored lipid composition. Thus, these results urges to different disciplines to redefine their paradigm of study by including the complexity revealed by the MSI techniques.
... MS-acquired and the histology-based optical images are typically co-registered for an accurate and efficient comparison. So far this has mostly been done manually with the guidance of fiducial markers or using clear anatomic detail [9, 10]. Matusch et al. took advantage of a commercially available software (Pmod) for alignment of histology and MS images [11] . ...
Article
Mass spectrometry imaging (MSI) is a powerful tool for the molecular characterization of specific tissue regions. Histochemical staining provides anatomic information complementary to MSI data. The combination of both modalities has been proven to be beneficial. However, direct comparison of histology based and mass spectrometry-based molecular images can become problematic because of potential tissue damages or changes caused by different sample preparation. Curated atlases such as the Allen Brain Atlas (ABA) offer a collection of highly detailed and standardized anatomic information. Direct comparison of MSI brain data to the ABA allows for conclusions to be drawn on precise anatomic localization of the molecular signal. Here we applied secondary ion mass spectrometry imaging at high spatial resolution to study brains of knock-out mouse models with impaired peroxisomal β-oxidation. Murine models were lacking D-multifunctional protein (MFP2), which is involved in degradation of very long chain fatty acids. SIMS imaging revealed deposits of fatty acids within distinct brain regions. Manual comparison of the MSI data with the histologic stains did not allow for an unequivocal anatomic identification of the fatty acids rich regions. We further employed an automated pipeline for co-registration of the SIMS data to the ABA. The registration enabled precise anatomic annotation of the brain structures with the revealed lipid deposits. The precise anatomic localization allowed for a deeper insight into the pathology of Mfp2 deficient mouse models. Graphical Abstract ᅟ Electronic supplementary material The online version of this article (doi:10.1007/s13361-015-1146-6) contains supplementary material, which is available to authorized users.
... Details of these steps have been described for a variety of sample types. 73,[169][170][171][172][173][174][175][176][177] The different ion planes can be displayed in three dimensions in order to create a volume that can be correlated with other types of 3D imaging data such as MRI. Figure 17 shows the rendering of a 3-dimensional MALDI IMS data set measured in the substantia nigra of a mouse brain, highlighting the distribution of two ions. 169 This example demonstrates that the volume can be viewed from virtually any angle to gain a better appreciation of the distribution of the molecules in the sample. ...
Article
An analysis of tissue specimens by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) in biological and clinical research is studied. In a profiling mode, data are collected from regions of interest defined by tissue pathology, while in imaging mode, the entire sample surface is raster sampled to reproduce an image of the specimen-based ion-specific ion intensities. The complete data set is comprised of many hundreds or thousands of m/z values, and the intensities of each ion can be plotted in a false color display. The resulting set of ion images is used to accurately portray the spatial distribution of the molecules that comprise the sample. After spotting with matrix, the coordinates of the matrix spots are registered to the mass spectrometer and spectra are automatically acquired from each location. Careful planning, attention to detail, and cleanliness in laboratory practices are required to avoid unwanted loss of sensitivity, loss of spatial resolution, or problems with sample stability.
... To perform image registration using algorithms and software developed in other fields, such as elastix, 33 representative images must be selected, which highlight features present in both modalities. These features can be either intentionally incorporated, such as the use of fiducial markers, 36 or aspects of the sample visible in both modalities. The registration process itself is separate from the selection of representative images and can either be performed manually (where an expert selects matching control points in both modalities) or by using automated methods (which search for the optimal transformation to reduce some overlapping information criterion). ...
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An ever-increasing array of imaging technologies are being used in the study of complex biological samples, each of which provides complementary, occasionally overlapping information at different length scales and spatial resolutions. It is important to understand the information provided by one technique in the context of the other to achieve a more holistic overview of such complex samples. One way to achieve this is to use annotations from one modality to investigate additional modalities. For microscopy-based techniques, these annotations could be manually generated using digital pathology software or automatically generated by machine learning (including deep learning) methods. Here, we present a generic method for using annotations from one microscopy modality to extract information from complementary modalities. We also present a fast, general, multimodal registration workflow [evaluated on multiple mass spectrometry imaging (MSI) modalities, matrix-assisted laser desorption/ionization, desorption electrospray ionization, and rapid evaporative ionization mass spectrometry] for automatic alignment of complex data sets, demonstrating an order of magnitude speed-up compared to previously published work. To demonstrate the power of the annotation transfer and multimodal registration workflows, we combine MSI, histological staining (such as hematoxylin and eosin), and deep learning (automatic annotation of histology images) to investigate a pancreatic cancer mouse model. Neoplastic pancreatic tissue regions, which were histologically indistinguishable from one another, were observed to be metabolically different. We demonstrate the use of the proposed methods to better understand tumor heterogeneity and the tumor microenvironment by transferring machine learning results freely between the two modalities.
... Since its introduction by Crecelius et al. [38] 3D MALDI imaging MS experiments have grown in popularity, but at a much slower pace than 2D imaging MS experiments owing to the much greater demands placed on experimental reproducibility and data handling [39]. Andersson et al. have published an experimental workflow [30], and alignment procedures have been reported to combine 3D imaging MS with 3D magnetic resonance imaging [31] and 3D optical imaging techniques [40]. These studies have focused on reproducing the histo-architectures of the tissues, such as mouse or crustacean brain architecture [30,38,41] and localizing tumors [31]. ...
Article
MALDI mass spectrometry can simultaneously measure hundreds of biomolecules directly from tissue. Using essentially the same technique but different sample preparation strategies, metabolites, lipids, peptides and proteins can be analyzed. Spatially correlated analysis, imaging MS, enables the distributions of these biomolecular ions to be simultaneously measured in tissues. A key advantage of imaging MS is that it can annotate tissues based on their MS profiles and thereby distinguish biomolecularly distinct regions even if they were unexpected or are not distinct using established histological and histochemical methods e.g. neuropeptide and metabolite changes following transient electrophysiological events such as cortical spreading depression (CSD), which are spreading events of massive neuronal and glial depolarisations that occur in one hemisphere of the brain and do not pass to the other hemisphere , enabling the contralateral hemisphere to act as an internal control. A proof-of-principle imaging MS study, including 2D and 3D datasets, revealed substantial metabolite and neuropeptide changes immediately following CSD events which were absent in the protein imaging datasets. The large high dimensionality 3D datasets make even rudimentary contralateral comparisons difficult to visualize. Instead non-negative matrix factorization (NNMF), a multivariate factorization tool that is adept at highlighting latent features, such as MS signatures associated with CSD events, was applied to the 3D datasets. NNMF confirmed that the protein dataset did not contain substantial contralateral differences, while these were present in the neuropeptide dataset.
... In most cases, the samples lack known or visible spatial details, which makes correct alignment impossible without markers. 46 Generally, embedded fiducial markers can be used as reference points to accurately co-register and stack the sections. 47 However, for microscopic samples such as single cells, traditional markers are too large to obtain a high correction accuracy. ...
Article
Mass spectrometry imaging (MSI) is a label free technique capable of providing simultaneous identification and localization of biomolecules. A multimodal approach is required that allows for the study of the complexity of biological tissue samples to overcome the limitations of a single MSI technique. Secondary Ion Mass Spectrometry (SIMS) allows for high spatial resolution imaging while Matrix-Assisted Laser Desorption (MALDI) offers a significantly wider mass range. The combination of co-registered SIMS and MALDI images results in detailed and unique biomolecular information. In this technical note we describe how gold sputtered/implanted fiducial markers (FM) are created and can be used to insure a proper overlay and co-registration of the two dimensional images provided by the two MSI modalities.
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Imaging mass spectrometry (IMS) is still a relatively young imaging technique that allows molecular mapping of diverse biomolecules in their natural environment. Furthermore, IMS allows for the direct correlation of tissue histology and proteomic, metabolomic or lipidomic information. In recent years, increasing efforts have been made in the development and improvement of IMS, which aid its application in clinical research. In this article, current frontiers of clinical research applications of IMS are discussed in the context of recent developments of IMS technology. Critical stages in planning and realizing clinical studies are highlighted. Finally, a selection of recent prominent examples for successful clinical applications of IMS is presented.
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Fiducial markers are necessary for the accurate overlay of images obtained by different methods from small areas of a sample. Thus far, fiducial markers have been used for biological sample analysis by mass spectrometry imaging. In this work, spatial distributions of excipient and active drug substance at the surfaces of drug tablets produced under different pressures are observed by multimodal desorption electrospray ionization–mass spectrometry (DESI-MS) imaging with two novel fiducial marker systems being used to achieve accurate overlay of ion and optical images. DESI-MS ion images in the negative and positive ion modes as well as ion and optical images obtained from circa 3 mm2 areas have been precisely superimposed, allowing observing the particulate composition of the tablet's ingredients, a feature that is remarkably conserved in spite of the pressure used to produce the tablet. This approach can be applied broadly for product development, quality control, and counterfeit detection.[Supplementary materials are available for this article. Go to the publisher's online edition of Analytical Letters for the following free supplemental resource(s): Experimental details and supplementary Figures (S1-S4).]
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Mass spectrometry imaging holds great potential for understanding the molecular basis of neurological disease. Several key studies have demonstrated its ability to uncover disease-related biomolecular changes in rodent models of disease, even if highly localized or invisible to established histological methods. The high analytical requirements for the biomedical application of mass spectrometry imaging means it is widely developed in mass spectrometry laboratories. However many lack the expertise to correctly annotate the complex anatomy of brain tissue, or have the capacity to analyze the number of animals required in pre-clinical studies, especially considering the significant variability in sizes of brain regions. To address this issue we have developed a pipeline to automatically map mass spectrometry imaging datasets of mouse brains to the Allen Brain Reference Atlas, which contains publically available data combining gene expression with brain anatomical locations. Our pipeline enables facile and rapid inter-animal comparisons by first testing if all each animal's tissue section was sampled at a similar location, and enabling the extraction of the biomolecular signatures from specific brain regions.
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A novel sample mount design with integrated fiducial marks and software for assisting operators in easily and efficiently locating points of interest established in previous analytical sessions is described. The sample holder and software were evaluated with experiments to demonstrate the utility and ease of finding the same points of interest in two different microscopy instruments. Also, numerical analysis of expected errors in determining the same position with errors unbiased by a human operator was performed. Based on the results, issues related to acquiring reproducibility and best practices for using the sample mount and software were identified. Overall, the sample mount methodology allows data to be efficiently and easily collected on different instruments for the same sample location.
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With a rapidly growing number of biomedical applications of mass-spectrometry imaging (MSI) and expansion of the technique into the clinic, spectrum annotation is an increasingly pressing issue in MSI. Although identification of the species of interest is the key to answering biomedical research questions, only few of the hundreds of observed biomolecular signals in each MSI spectrum can be easily identified or interpreted. So far, no standardized protocols resolve this issue.Present strategies for protein identification in MSI, their limitations and future developments are the scope of this review. We discuss advances in MSI technology, workflows and bioinformatic tools to improve the confidence and the number of protein identifications within MSI studies.
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Imaging Mass Spectrometry (IMS) has been a useful tool for investigating protein, peptide, drug and metabolite distributions in human and animal tissue samples for almost 15 years. The major advantages of this method include a broad mass range, the ability to detect multiple analytes in a single experiment without the use of labels and the preservation of biologically relevant spatial information. Currently the majority of IMS experiments are based on imaging animal tissue sections or small tumor biopsies. An alternative method currently being developed is the application of IMS to three-dimensional cell and tissue culture systems. With new advances in tissue culture and engineering, these model systems are able to provide increasingly accurate, high-throughput and cost-effective models that recapitulate important characteristics of cell and tissue growth in vivo. This review will describe the most recent advances in IMS technology and the bright future of applying IMS to the field of three-dimensional cell and tissue culture.
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The remodeling of the synovial membrane, which normally lubricates the joints by producing synovial fluid, is one of the most characteristic events in the pathology of osteoarthritis (OA). The heterogeneity and spatial distribution of proteins in the synovial membrane are poorly studied and we hypothesized that they constitute excellent molecular disease classifiers for the accurate diagnosis of the disease. Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) allows for the study of the localization and identification of hundreds of different molecules with high sensitivity in very thin tissue sections. In this work, we employed MALDI-MSI in combination with principal component analysis and discriminant analysis to reveal the specific profile and distribution of digested proteins in human normal and OA synovial membranes. Proteins such as hemoglobin subunit alpha 2, hemoglobin subunit beta, actin aortic smooth muscle, biglycan, and fibronectin have been directly identified from human synovial biopsies. In addition, we have determined the location of disease-specific OA markers. Some of them which are located in areas of low inflammation provide valuable information on tissue heterogeneity. Finally, we described the OA molecular protein signatures common to synovial and other articular tissues such as cartilage. For the first time, normal and OA human synovial membranes have been classified by MALDI-MSI, thus offering a new sensitive tool for the study of rheumatic pathologies.
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Abtract In the previous chapters we demonstrated that, over the last 50 years, different analytical methods were developed and refined to link the composition and structure of man-made and natural materials at the nano/microscale to their functional behaviour at the macroscopic scale. These developments came at the price of increasingly complex analytical equipment and procedures of analysis. They also gave rise to a vast increase of information on each element of a 2-D or a 3-D data array resulting from systematic 2-D or 3-D local measurements. In these arrays, each individual element thus becomes a complex integrated set of morphological, structural and compositional information. With imaging analysis and other analytical methodologies that produce massive amounts of data, analytical chemistry is increasingly transformed from a hypothesis-driven targeted methodology into a discovery-driven, shotgun methodology. In the nontargeted approach, one must decide what needs to be measured, the method should be selected and validated and the analysis performed. In a nontargeted approach everything feasible is determined and information is extracted from the collected data. In such conditions, the central concept of analytical chemistry and its relation to standard metrological concepts (uncertainty, validation and/or traceability to fundamental standards, etc.) seem to lose their central guiding role.
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Since biology is by and large a 3-dimensional phenomenon, it is hardly surprising that 3D imaging has had a significant impact on many challenges in the life sciences. Imaging Mass Spectrometry (MS) is a spatially-resolved label-free analytical technique that recently maturated into a powerful tool for in situ localisation of hundreds of molecular species. Serial 3D imaging MS reconstructs 3D molecular images from serial sections imaged with mass spectrometry. As such, it provides a novel 3D imaging modality inheriting the advantages of imaging MS. Serial 3D imaging MS has been steadily developing over the last decade and many of the technical challenges have been met. Essential tools and protocols were developed, in particular to improve the reproducibility of sample preparation, speed up data acquisition, and enable computationally-intensive analysis of the big data generated. As a result, experimental data is starting to emerge that takes advantage of the extra spatial dimension that 3D imaging MS offers. Most studies still focus on method development rather than on exploring specific biological problems. The future success of 3D imaging MS requires it to find its own niche alongside existing 3D imaging modalities through finding applications that benefit from 3D imaging and at the same time utilise the unique chemical sensitivity of imaging mass spectrometry. This perspective article critically reviews the challenges encountered during the development of serial-sectioning 3D imaging MS and discusses the steps needed to tip it from being an academic curiosity into a tool of choice for answering biological and medical questions.
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A simple method for the location and auto-alignment of sample fiducials for sample registration using widely available MATLAB/LabVIEW software is demonstrated. The method is robust, easily implemented, and applicable to a wide variety of experiment types for improved reproducibility and increased setup speed. The software uses image processing to locate and measure the diameter and center point of circular fiducials for distance self-calibration and iterative alignment and can be used with most imaging systems. The method is demonstrated to be fast and reliable in locating and aligning sample fiducials, provided here by a nanofabricated array, with accuracy within the optical resolution of the imaging system. The software was further demonstrated to register, load, and sample the dynamically wetted array.
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Laser desorption/ionization mass spectrometry (LDI MS) is used to image brain lipids in the fruit fly, Drosophila, a common invertebrate model organism in biological and neurological studies. Three different sample preparation methods, including sublimation with two common organic matrices for matrix assisted laser desorption ionization (MALDI) and surface assisted laser desorption ionization (SALDI) using gold nanoparticles are examined for sample profiling and imaging the fly brain. Recrystallization with trifluoroacetic acid following matrix deposition in MALDI is shown to increase the incorporation of biomolecules with one matrix, resulting in more efficient ionization, but not for the other matrix. The key finding here is that the mass fragments observed for the fly brain slices with different surface modifications are significantly different. Thus, these approaches can be combined to provide complementary analysis of chemical composition, particularly for the small metabolites, diacylglycerides, phosphatidylcholines, and triacylglycerides, in the fly brain. Furthermore, imaging appears to be beneficial using modification with gold nanoparticles in place of matrix in this application showing its potential for cellular and subcellular imaging. The imaging protocol developed here with both MALDI and SALDI provides the best and most diverse lipid chemical images of the fly brain to date with LDI.
Chapter
Single-cell imaging is a very important area in biological, medical, and pharmaceutical research, yet it is one of the most challenging fields for mass spectrometry. Imaging mass spectrometry (IMS), which is a powerful label-free analytical technique, has proved its capability to chemically visualize single cells with high sensitivity, chemical selectivity, and in certain cases subcellular spatial resolution. We present here an overview of the capabilities and current progress of IMS, particularly secondary ion mass spectrometry, matrix-assisted laser desorption ionization, and desorption electrospray ionization for single-cell imaging. The principles and technical developments of each technique are introduced. Critical aspects regarding single-cell imaging are addressed, especially sensitivity, spatial resolution, and sample handling. Current achievements in this field of application are also presented for both 2-D imaging and 3-D imaging. Furthermore, we address the potential contribution of IMS single-cell 'omics' and discuss its future development.
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Although tumor hypoxia is associated with tumor aggressiveness and resistance to cancer treatment, many details of hypoxia-induced changes in tumors remain to be elucidated. Mass spectrometry imaging (MSI) is a technique that is well suited to study the biomolecular composition of specific tissue regions, such as hypoxic tumor regions. Here, we investigate the use of pimo-nidazole as exogenous hypoxia marker for matrix-assisted laser desorption/ionization (MALDI) MSI. In hypoxic cells, pimoni-dazole is reduced and forms reactive products that bind to thiol groups in proteins, peptides and amino acids. We show that a reductively activated pimonidazole metabolite can be imaged by MALDI-MSI in a breast tumor xenograft model. Immunohistochemical detection of pimonidazole adducts on adjacent tissue sections confirmed that this metabolite is localized to hypoxic tissue regions. We used this metabolite to image hypoxic tissue regions and their associated lipid and small molecule distributions with MALDI-MSI. We identified a heterogeneous distribution of 1-methylnicotinamide and acetylcarnitine, which mostly co-localized with hypoxic tumor regions. As pimonidazole is a widely used immunohistochemical marker of tissue hypoxia, it is likely that the presented direct MALDI-MSI approach is also applicable to other tissues from pimonidazole-injected animals or humans.
Article
en Fiducial markers are used to correct the microscope drift and should be photostable, be usable at multiple wavelengths and be compatible for multimodal imaging. Fiducial markers such as beads, gold nanoparticles, microfabricated patterns and organic fluorophores lack one or more of these criteria. Moreover, the localization accuracy and drift correction can be degraded by other fluorophores, instrument noise and artefacts due to image processing and tracking algorithms. Estimating mechanical drift by assuming Gaussian distributed noise is not suitable under these circumstances. Here we present a method that uses fluorescent nanodiamonds as fiducial markers and uses an improved maximum likelihood algorithm to estimate the drift with both accuracy and precision within the range 1.55−5.75 nm. Lay Description fr Every microscope drifts can vary depending on the design and surroundings. It is important to estimate the microscope drift with accuracy and precision better than the desired accuracy and precision of imaging applications such as tracking single molecules and cells. For fluorescent imaging, fluorescent nanodiamonds (FNDs) offer excellent advantages compared to other types of fiducial markers such as beads and quantum dots. FNDs have side excitation and emission spectra with stable emission intensity that never dies, and therefore, suitable for single‐ and multicolour imaging. In this paper, the microscope drift has been estimated with precision and accuracy of less than 5 nm using FNDs and an improved maximum likelihood method that works in the presence of Gaussian and non‐Gaussian noise.
Chapter
In multimodal imaging diverse imaging techniques are applied to the same subject usually within a limited time frame in order to capture the same functional, morphologic, and metabolic state of the subject. The historical most compelling need for multimodal imaging stems from the requirement to match functional or metabolic information captured with, e.g., positron emission tomography (PET) or single-photon emission computed tomography (SPECT) with morphological information about the subject obtained from computed tomography (CT) or magnetic resonance imaging (MRI).
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Understanding the biodistribution, metabolism and accumulation of drugs in the body is a fundamental of pharmaceutical research and development. Mass Spectrometry imaging (MSI) has been proven to be a powerful tool to image the unlabelled spatial distribution of exogenous drugs and endogenous metabolites from the surface of tissue sections or small clinical biopsies, aiding the delivery of safe and effective medicines to the market and ultimately benefiting patients. Here we review the current advancements in MSI sample preparation, qualitative and quantitative MSI methodology and drug discovery and development applications. MSI is shown to be supporting R&D from early target identification through to the clinic. In conclusion, we discuss future directions of the technology and the hurdles that need addressing to strengthen its status in multimodal imaging.
Chapter
Mass spectrometry imaging (MSI) is a method for surface analysis that was introduced at the end of the twentieth century, and since then a huge development of this technique can be observed. It is widely used in biological, biochemical, and medical research. MSI technique combines two elements. The first is the ability to obtain the mass spectrum of a given point on the surface, which provides information about the molecules present in this particular point. The second element is the design of the ion source, which enables movement of the examined surface in the x‐ and y‐axis. The most widespread MS techniques for imaging are secondary ion mass spectrometry, matrix‐assisted laser desorption/ionization, and desorption electrospray ionization. Each of them allows for the analysis of slightly different types of molecules. The chapter discusses their advantages and disadvantages in the context of surface imaging as well as exemplary applications.
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In this paper, we present an easy-to-follow procedure for the analysis of tissue sections from 3D cell cultures (spheroids) by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and laser scanning confocal microscopy (LSCM). MALDI MSI was chosen to detect the distribution of the drug of interest, while fluorescence immunohistochemistry (IHC) followed by LSCM was used to localize the cells featuring specific markers of viability, proliferation, apoptosis and metastasis. The overlay of the mass spectrometry (MS) and IHC spheroid images, typically without any morphological features, required fiducial-based coregistration. The MALDI MSI protocol was optimized in terms of fiducial composition and antigen epitope preservation to allow MALDI MSI to be performed and directly followed by IHC analysis on exactly the same spheroid section. Once MS and IHC images were coregistered, the quantification of the MS and IHC signals was performed by an algorithm evaluating signal intensities along equidistant layers from the spheroid boundary to its center. This accurate colocalization of MS and IHC signals showed limited penetration of the clinically tested drug perifosine into spheroids during a 24 h period, revealing the fraction of proliferating and promigratory/proinvasive cells present in the perifosine-free areas, decrease of their abundance in the perifosine-positive regions and distinguishing between apoptosis resulting from hypoxia/nutrient deprivation and drug exposure.
Article
Chemical imaging techniques are increasingly being used in combination to achieve a greater amount of understanding of a sample. This is especially true in the case of mass spectrometry imaging (MSI), where the use of different ionisation sources allows detection of different classes of molecules across a range of spatial resolutions. There has been significant recent effort in the development of data fusion algorithms which attempt to combine the benefits of multiple techniques, such that the output provides additional information that would have not been present or obvious from the individual techniques alone. However, the majority of the data fusion methods currently in use rely on image registration to generate the fused data, and therefore can suffer from artefacts caused by interpolation. Here we present a method for data fusion, which does not incorporate interpolation-based artefacts into the final fused data, applied to data acquired from multiple chemical imaging modalities. The method is evaluated using simulated data and a model polymer blend sample, before being applied to biological samples of mouse brain and lung.
Article
Rationale: Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is routinely employed to monitor the distribution of compounds in tissue sections and generate 2D images. Whilst informative the images do not represent the distribution of the analyte of interest through the entire organ. The generation of 3D images is an exciting field that can provide a deeper view of the analyte of interest throughout an entire organ. Methods: Serial sections of mouse and rat lung tissue were obtained at 120 μm depth intervals and imaged individually. Homogenate registration markers were incorporated in order to aid the final 3D image construction. Using freely available software packages, the images were stacked together to generate a 3D image that showed the distribution of endogenous species throughout the lungs. Results: Preliminary tests were performed on 16 serial tissue sections of mouse lungs. A 3D model showing the distribution of phosphocholine at m/z 184.09 was constructed, which defined the external structure of the lungs and trachea. Later, a second experiment was performed using 24 serial tissue sections of the left lung of a rat. Two molecular markers, identified as [PC (32:1)+K]+ at m/z 770.51 and [PC (36:4)+K]+ at m/z 820.52 were used to generate 3D models of the parenchyma and airways, respectively. Conclusions: A straightforward method to generate 3D MALDI-MS images of selected molecules in lung tissue has been presented. Using freely available imaging software, the 3D distributions of molecules related to different anatomical features were determined.
Article
Because of the heterogeneity and complexity of tumor structure, cancer drugs often cannot effectively reach the tumor, which is one of the reasons for drug resistance and treatment failure. Therefore, the distribution and the metabolism of cancer drugs within the tumor and the body are essential concerns during drug development and cancer therapy. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been demonstrated to be a powerful label-free technique to characterize the spatial biodistributions of cancer drugs and their metabolites, facilitating the delivery of safe and effective drugs to the market and eventually benefiting cancer patients. In this review, recent advances are thoroughly reviewed regarding the MALDI-MSI analysis of cancer drugs in preclinical and clinical settings, including technical aspects and practical applications. In conclusion, the significance and future directions of MALDI-MSI in cancer drug research and development are summarized.
Chapter
Biomedical visualization to infer the relationship between anatomical features and biochemical functions often requires several imaging modalities. Biomedical imaging systems can be divided based on the physical mechanism of the technique (e.g., X-rays, photons, acoustic waves, and ions), spatial resolution (macroscopic or microscopic), information obtained (anatomical, physiological, cellular, or molecular), or by types of samples analyzed or application. A combination of imaging techniques is often referred to as multimodal imaging. Multimodal imaging often gives an improved understanding of biological system by overcoming the limitations of the individual techniques. In this chapter, an overview of multimodal imaging mass spectrometry encompassing several imaging systems is provided.
Chapter
Over the past two decades, multiple new methods have been developed for probing the structure and function of human tissues and organ systems. These innovative methods have paved the road toward a new era in medicine, where diseases are subclassified, not only based on histology but also at the molecular level, and often based on an integrated assessment of clinical signs and symptoms, detailed in vivo imaging, and histologic and molecular evaluation of biopsied tissues. Advances in detection, identification, and quantification of both cell-free and extracellular vesicle-derived biomarkers in the blood are more commonly allowing for noninvasive measures of organ function, precluding the need for invasive biopsies. In addition, stem cells, “organoids,” and “organ-on-a-chip” models have permitted the study of human cells and tissues in greater detail and complexity. The application of these innovative methods to the human placenta is detailed in this chapter, highlighting not only the new information gained about the structure and function of this important transient organ but also the ways in which this information can be translated into, and thus transform, the practice of placental pathology.
Article
Mass spectrometry imaging (MSI) as an analytical tool for bio-molecular and bio-medical research targets accurate compound localization and identification. In terms of dedicated instrumentation, this translates into the demand for more detail in the image dimension (spatial resolution) and in the spectral dimension (mass resolution and accuracy), preferably combined in one instrument. At the same time, large area biological tissue samples require fast acquisition schemes, instrument automation and a robust data infrastructure. This review discusses the analytical capabilities of an "ideal" MSI instrument for bio-molecular and bio-medical molecular imaging. The analytical attributes of such an ideal system are contrasted with technological and methodological challenges in MSI. In particular, innovative instrumentation for high spatial resolution imaging in combination with high sample throughput is discussed. Detector technology that targets various shortcomings of conventional imaging detector systems is highlighted. The benefits of accurate mass analysis, high mass resolving power, additional separation strategies and multimodal three-dimensional data reconstruction algorithms are discussed to provide the reader with an insight in the current technological advances and the potential of MSI for bio-medical research.
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Phosphocholine (PC) and total choline (tCho) are increased in malignant breast tumors. In this study, we combined magnetic resonance spectroscopic imaging (MRSI), mass spectrometry (MS) imaging, and pathologic assessment of corresponding tumor sections to investigate the localization of choline metabolites and cations in viable versus necrotic tumor regions in the nonmetastatic MCF-7 and the highly metastatic MDA-MB-231 breast cancer xenograft models. In vivo three-dimensional MRSI showed that high tCho levels, consisting of free choline (Cho), PC, and glycerophosphocholine (GPC), displayed a heterogeneous spatial distribution in the tumor. MS imaging performed on tumor sections detected the spatial distributions of individual PC, Cho, and GPC, as well as sodium (Na+) and potassium (K+), among many others. PC and Cho intensity were increased in viable compared with necrotic regions of MDA-MB-231 tumors, but relatively homogeneously distributed in MCF-7 tumors. Such behavior may be related to the role of PC and PC-related enzymes, such as choline kinase, choline transporters, and others, in malignant tumor growth. Na+ and K+ colocalized in the necrotic tumor areas of MDA-MB-231 tumors, whereas in MCF-7 tumors, Na+ was detected in necrotic and K+ in viable tumor regions. This may be attributed to differential Na+/K+ pump functions and K+ channel expressions. Principal component analysis of the MS imaging data clearly identified different tumor microenvironmental regions by their distinct molecular signatures. This molecular information allowed us to differentiate between distinct tumor regions and tumor types, which may, in the future, prove clinically useful in the pathologic assessment of breast cancers.
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Although the mechanisms through which hypoxia influences several phenotypic characteristics such as angiogenesis, selection for resistance to apoptosis, resistance to radiation and chemotherapy, and increased invasion and metastasis are well characterized, the relationship between tumor hypoxia and components of the extracellular matrix (ECM) is relatively unexplored. The collagen I (Col1) fiber matrix of solid tumors is the major structural part of the ECM. Col1 fiber density can increase tumor initiation, progression, and metastasis, with cancer cell invasion occurring along radially aligned Col1 fibers. Here we have investigated the influence of hypoxia on Col1 fiber density in solid breast and prostate tumor models. Second harmonic generation (SHG) microscopy was used to detect differences in Col1 fiber density and volume between hypoxic and normoxic tumor regions. Hypoxic regions were detected by fluorescence microscopy, using tumors derived from human breast and prostate cancer cell lines stably expressing enhanced green fluorescent protein (EGFP) under transcriptional control of the hypoxia response element. In-house fiber analysis software was used to quantitatively analyze Col1 fiber density and volume from the SHG microscopy images. Normoxic tumor regions exhibited a dense mesh of Col1 fibers. In contrast, fewer and structurally altered Col1 fibers were detected in hypoxic EGFP-expressing tumor regions. Microarray gene expression analyses identified increased expression of lysyl oxidase and reduced expression of some matrix metalloproteases in hypoxic compared with normoxic cancer cells. These results suggest that hypoxia mediates Col1 fiber restructuring in tumors, which may impact delivery of macromolecular agents as well as dissemination of cells.
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We created a method for three-dimensional (3-D) registration of medical images (e.g., magnetic resonance imaging (MRI) or computed tomography) to images of physical tissue sections or to other medical images and evaluated its accuracy. Our method proved valuable for evaluation of animal model experiments on interventional-MRI guided thermal ablation and on a new localized drug delivery system. The method computes an optimum set of rigid body registration parameters by minimization of the Euclidean distances between automatically chosen correspondence points, along manually selected fiducial needle paths, and optional point landmarks, using the iterative closest point algorithm. For numerically simulated experiments, using two needle paths over a range of needle orientations, mean voxel displacement errors depended mostly on needle localization error when the angle between needles was at least 20°. For parameters typical of our in vivo experiments, the mean voxel displacement error was
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The objective of this work was to develop and then validate a stereotactic fiduciary marker system for tumor xenografts in rodents which could be used to co-register magnetic resonance imaging (MRI), PET, tissue histology, autoradiography, and measurements from physiologic probes. A Teflon fiduciary template has been designed which allows the precise insertion of small hollow Teflon rods (0.71 mm diameter) into a tumor. These rods can be visualized by MRI and PET as well as by histology and autoradiography on tissue sections. The methodology has been applied and tested on a rigid phantom, on tissue phantom material, and finally on tumor bearing mice. Image registration has been performed between the MRI and PET images for the rigid Teflon phantom and among MRI, digitized microscopy images of tissue histology, and autoradiograms for both tissue phantom and tumor-bearing mice. A registration accuracy, expressed as the average Euclidean distance between the centers of three fiduciary markers among the registered image sets, of 0.2 +/- 0.06 mm was achieved between MRI and microPET image sets of a rigid Teflon phantom. The fiduciary template allows digitized tissue sections to be co-registered with three-dimensional MRI images with an average accuracy of 0.21 and 0.25 mm for the tissue phantoms and tumor xenografts, respectively. Between histology and autoradiograms, it was 0.19 and 0.21 mm for tissue phantoms and tumor xenografts, respectively. The fiduciary marker system provides a coordinate system with which to correlate information from multiple image types, on a voxel-by-voxel basis, with sub-millimeter accuracy--even among imaging modalities with widely disparate spatial resolution and in the absence of identifiable anatomic landmarks.
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Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.
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We have evaluated the use of the Xenogen IVIS 200 imaging system for real-time fluorescence protein-based optical imaging of metastatic progression in live animals. We found that green fluorescent protein-expressing cells (100 x 10(6)) were not detectable in a mouse cadaver phantom experiment. However, a 10-fold lower number of tdTomato-expressing cells were easily detected. Mammary fat pad xenografts of stable MDA-MB-231-tdTomato cells were generated for the imaging of metastatic progression. At 2 weeks postinjection, barely palpable tumor burdens were easily detected at the sites of injection. At 8 weeks, a small contralateral mammary fat pad metastasis was imaged and, by 13 weeks, metastases to lymph nodes were detectable. Metastases with nodular composition were detectable within the rib cage region at 15 weeks. 3-D image reconstructions indicated that the detection of fluorescence extended to approximately 1 cm below the surface. A combination of intense tdTomato fluorescence, imaging at > or = 620 nm (where autofluorescence is minimized), and the sensitivity of the Xenogen imager made this possible. This study demonstrates the utility of the noninvasive optical tracking of cancer cells during metastatic progression with endogenously expressed fluorescence protein reporters using detection wavelengths of > or = 620 nm.
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We described an interactive method for correcting spatial distortion in histology samples, applied them to a large set of image data, and quantitatively evaluated the quality of the corrections. We demonstrated registration of histology samples to photographs of macroscopic tissue samples and to MR images. We first described methods for obtaining corresponding fiducial and anatomical points, including a new technique for determining boundary correspondence points. We then describe experimental methods for tissue preparation, including a technique for adding color-coded internal and boundary ink marks that are used to validate the method by measuring the registration error. We applied four different transformations with internal and boundary correspondence points, and measured the distance error between other internal ink fiducials. A large number of boundary points, typically 20-30, and at least two internal points were required for accurate warping registration. Interior errors with the transformation methods were ordered: thin plate spline (TPS) approximately non-warping<triangle warping<polynomial warping. Although non-warping surprisingly gave the lowest interior distance error (0.5+/-0.3mm), TPS was more robust, gave an insignificantly greater error (0.6+/-0.3mm) and much better results near boundaries where distortion was more evident, and allowed us to correct torn histology samples, a common problem. Using the method to evaluate RF thermal ablation, we found good zonal correlation between MR images and corrected histology samples. The method can be practically applied to this and other emerging applications such as in vivo molecular imaging.
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We developed a three-dimensional (3D) registration method to align medical scanner data with histological sections. After acquiring 3D medical scanner images, we sliced and photographed the tissue using, a custom apparatus, to obtain a volume of tissue section images. Histological samples from the sections were digitized using a video microscopy system. We aligned the histology and medical images to the reference tissue images using our 3D registration method. We applied the method to correlate in vivo magnetic resonance (MR) and histological measurements for radio-frequency thermal ablation lesions in rabbit thighs. For registration evaluation, we used an ellipsoid model to describe the lesion surfaces. The model surface closely fit the inner (M1) and outer (M2) boundaries of the hyperintense region in MR lesion images, and the boundary of necrosis (H1) in registered histology images. We used the distance between the model surfaces to indicate the 3D registration error. For four experiments, we measured a registration accuracy of 0.96+/- 0.13 mm (mean+/-SD) from the absolute distance between the M2 and H1 model surfaces, which compares favorably to the 0.70 mm in-plane MR voxel dimension. This suggests that our registration method provides sufficient spatial correspondence to correlate 3D medical scanner and histology data.
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To test injectable fiducial markers for magnetic resonance (MR) histological correlation in ex vivo or in vivo animal experiments. A total of 35 potential markers were tested ex vivo in pork muscle. The end-points were: 1) visibility, size, and shape on MR images and at macroscopic examination; 2) 24-hour stability; and 3) microscopic appearance. Selected markers were injected in vivo (rabbit's muscle and breast tumor tissue) to test their three-hour in vivo stability and their potential toxicity. Finally, different dilutions of the two best markers were assessed again through the same screening tests to determine whether their size on MR images could be customized by dilution. Two fluid acrylic paints containing inorganic pigments were found to be potentially interesting markers. On MR images, they created well-defined susceptibility artifacts. The markers made with iridescent bronze paint (iron oxide coated mica particles) were readily visible on microscopy and their size on MR images could be customized by dilution. The iridescent stainless steel paint (iron, chromium, nickel) created ex vivo the smallest markers in tissue but needed colloidal iron staining to be visible on microscopy and could not be easily diluted. Fluid acrylic paints are potentially interesting markers for MR histological correlation. Further studies are needed to assess their long-term properties.
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To investigate the ability of magnetic resonance (MR) to monitor radio-frequency (RF) ablation treatments by comparing MR images of thermal lesions to histologically assayed cellular damage. We developed a new methodology using three-dimensional registration for making spatial correlations. A low-field, open MRI system was used to guide an ablation probe into rabbit thigh muscle and acquire MR volumes after ablation. After fixation, we sliced and photographed the tissue at 3-mm intervals, using a specially designed apparatus, to obtain a volume of tissue images. Histologic samples were digitized using a video microscopy system. For our three-dimensional registration method, we used the tissue images as the reference, and registered histology and MR images to them using two different computer alignment steps. First, the MR volume was aligned to the volume of tissue images by registering needle fiducials placed near the tissue of interest. Second, we registered the histology images with the tissue images using a two-dimensional warping technique that aligned internal features and the outside boundary of histology and tissue images. The MR and histology images were very well aligned, and registration accuracy, determined from displacement of needle fiducials, was 1.32 +/- 0.39 mm (mean +/- SD), which compared favorably to the MR voxel dimensions (0.70 mm in-plane and 3.0 mm thick). A preliminary comparison of MR and tissue response showed that the region inside the elliptical hyperintense rim in MR closely corresponds to the region of necrosis as established by histology, with a mean absolute distance between MR and histology boundaries of 1.17 mm, slightly smaller than the mean registration error. The MR region slightly overestimated the region of necrosis, with a mean signed distance between boundaries of 0.85 mm. Our results suggest that our methodology can be used to achieve three-dimensional registration of histology and in vivo MR images. In MR lesion images, the inner border of the hyperintense region corresponds to the border of irreversible cell damage. This is good evidence that during RF ablation treatments, iMRI lesion images can be used for real-time feedback.
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Imaging mass spectrometry (IMS) that utilizes matrix-assisted laser desorption/ionization (MALDI) technology can provide a molecular ex vivo view of resected organs or whole-body sections from an animal, making possible the label-free tracking of both endogenous and exogenous compounds with spatial resolution and molecular specificity. Drug distribution and, for the first time, individual metabolite distributions within whole-body tissue sections can be detected simultaneously at various time points following drug administration. IMS analysis of tissues from 8 mg/kg olanzapine dosed rats revealed temporal distribution of the drug and metabolites that correlate to previous quantitative whole-body autoradiography studies. Whole-body MALDI IMS is further extended to detecting proteins from organs present in a whole-body sagittal tissue section. This technology will significantly help advance the analysis of novel therapeutics and may provide deeper insight into therapeutic and toxicological processes, revealing at the molecular level the cause of efficacy or side effects often associated with drug administration.
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We have developed a method for integrating three dimensional-volume reconstructions of spatially resolved matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) ion images of whole mouse heads with high-resolution images from other modalities in an animal-specific manner. This approach enabled us to analyze proteomic profiles from MALDI IMS data with corresponding in vivo data provided by magnetic resonance imaging.
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As large genomic and proteomic datasets are generated from homogenates of various tissues, the need for information on the spatial localization of their encoded products has become more pressing. Matrix-assisted laser desorption-ionization (MALDI) imaging mass spectrometry (IMS) offers investigators the means with which to unambiguously study peptides and proteins with molecular specificity, and to determine their distribution in two and three dimensions. In the past few years, several parameters have been optimized for IMS, including sample preparation, matrix application and instrumental acquisition parameters (Box 1). These developments have resulted in a high degree of reproducibility in mass accuracy and peak intensities (Supplementary Fig. 1 online). Recently, we have optimized our protocol to be able to increase the number of molecular species analyzed by collecting two sets of sections, covering one set of sections with sinapinic acid for optimal detection of proteins and adjacent sections with 2,5-dihydroxybenzoic acid (DHB) matrix for the optimal detection of low-mass species, including peptides. Approximately 1,000 peaks can be observed in each dataset (Fig. 1). Furthermore, the sections are collected at an equal distance, 200 mum instead of 400-500 mum used previously, thus enabling the use of virtual z-stacks and three-dimensional (3D) volume renderings to investigate differential localization patterns in much smaller brain structures such as the substantia nigra and the interpeduncular nucleus. Here we present our optimized step-by-step procedure based on previous work in our laboratory, describing how to make 3D volume reconstructions of MALDI IMS data, as applied to the rat brain.
  • N C Shaner
Shaner NC. Nature Biotechnology. 2004; 22:1562-1572.