Mass spectrometry images acylcarnitines,phosphatidylcholines, and sphingomyelin in MDA-MB-231 breast tumor models

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 cell proliferation, invasion, 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. Four of the most-abundant lipid species, namely PC(16:0/16:0), PC(16:0/18:1), PC(18:1/18:1), and PC(18:0/18:1), were localized in viable tumor regions, whereas LPC(16:0/0:0) was detected in necrotic tumor regions. We identified a heterogeneous distribution of palmitoylcarnitine, stearoylcarnitine, PC(16:0/22:1), and SM(d18:1/16:0) sodium adduct, which colocalized primarily 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.     

Spatial mapping of lipids at cellular resolution in embryos of cotton

Advances in mass spectrometry (MS) have made comprehensive lipidomics analysis of complex tissues relatively commonplace. These compositional analyses, although able to resolve hundreds of molecular species of lipids in single extracts, lose the original cellular context from which these lipids are derived. Recently, high-resolution MS of individual lipid droplets from seed tissues indicated organelle-to-organelle variation in lipid composition, suggesting that heterogeneity of lipid distributions at the cellular level may be prevalent. Here, we employed matrix-assisted laser desorption/ionization-MS imaging (MALDI-MSI) approaches to visualize lipid species directly in seed tissues of upland cotton (Gossypium hirsutum). MS imaging of cryosections of mature cotton embryos revealed a distinct, heterogeneous distribution of molecular species of triacylglycerols and phosphatidylcholines, the major storage and membrane lipid classes in cotton embryos. Other lipids were imaged, including phosphatidylethanolamines, phosphatidic acids, sterols, and gossypol, indicating the broad range of metabolites and applications for this chemical visualization approach. We conclude that comprehensive lipidomics images generated by MALDI-MSI report accurate, relative amounts of lipid species in plant tissues and reveal previously unseen differences in spatial distributions providing for a new level of understanding in cellular biochemistry.     

MALDI-MS-imaging of whole human lens capsule

The ocular lens capsule is a smooth, transparent basement membrane that encapsulates the lens and is composed of a rigid network of interacting structural proteins and glycosaminoglycans. During cataract surgery, the anterior lens capsule is routinely removed in the form of a circular disk. We considered that the excised capsule could be easily prepared for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-MSI) analysis. MALDI-MSI is a powerful tool to elucidate the spatial distribution of small molecules, peptides, and proteins within tissues. Here, we apply this molecular imaging technique to analyze the freshly excised human lens capsule en face. We demonstrate that novel information about the distribution of proteins by MALDI-MSI can be obtained from this highly compact connective tissue, having no evident histo-morphological characteristics. Trypsin digestion carried out on-tissue is shown to improve MALDI-MSI analysis of human lens capsules and affords high repeatability. Most importantly, MALDI-MSI analysis reveals a concentric distribution pattern of proteins such as apolipoprotein E (ApoE) and collagen IV alpha-1 on the anterior surface of surgically removed lens capsule, which may indicate direct or indirect effects of environmental and mechanical stresses on the human ocular lens.     

Three-dimensional visualization of membrane phospholipid distributions in Arabidopsis thaliana seeds: A spatial perspective of molecular heterogeneity

Arabidopsis thaliana has been widely used as a model plant to study acyl lipid metabolism. Seeds of A. thaliana are quite small (approximately 500 × 300 μm and weigh ~ 20 μg), making lipid compositional analyses of single seeds difficult to achieve. Here we have used matrix assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to map and visualize the three-dimensional spatial distributions of two common membrane phospholipid classes, phosphatidylcholine (PC) and phosphatidylinositol (PI), in single A. thaliana seeds. The 3D images revealed distinct differences in distribution of several molecular species of both phospholipids among different seed tissues. Using data from these 3D reconstructions, the PC and PI mol% lipid profiles were calculated for the embryonic axis, cotyledons, and peripheral endosperm, and these data agreed well with overall quantification of these lipids in bulk seed extracts analyzed by conventional electrospray ionization-mass spectrometry (ESI-MS). In addition, MALDI-MSI was used to profile PC and PI molecular species in seeds of wild type, fad2–1, fad3–2, fad6–1, and fae1–1 acyl lipid mutants. The resulting distributions revealed previously unobserved changes in spatial distribution of several lipid molecular species, and were used to suggest new insights into biochemical heterogeneity of seed lipid metabolism. These studies highlight the value of mass spectrometry imaging to provide unprecedented spatial and chemical resolution of metabolites directly in samples even as small as a single A. thaliana seeds, and allow for expanded imaging of plant metabolites to improve our understanding of plant lipid metabolism from a spatial perspective.     

MALDI-imaging mass spectrometry - An emerging technique in plant biology

Recent advances in instrumentation and sample preparation have facilitated the mass spectrometric (MS) imaging of a large variety of biological molecules from small metabolites to large proteins. The technique can be applied at both the tissue and the single-cell level, and provides information regarding the spatial distribution of specific molecules. Nevertheless, the use of MS imaging in plant science remains far from routine, and there is still a need to adapt protocols to suit specific tissues. We present an overview of MALDI-imaging MS (MSI) technology and its use for the analysis of plant tissue. Recent methodological developments have been summarized, and the major challenges involved in using MALDI-MSI, including sample preparation, the analysis of metabolites and peptides, and strategies for data evaluation are all discussed. Some attention is given to the identification of differentially distributed compounds. To date, the use of MALDI-MSI in plant research has been limited. Examples include leaf surface metabolite maps, the characterization of soluble metabolite translocation in planta, and the profiling of protein/metabolite patterns in cereal grain cross-sections. Improvements to both sample preparation strategies and analytical platforms (aimed at both spectrum acquisition and post-acquisition analysis) will enhance the relevance of MALDI-MSI technology in plant research.     

Imaging lipids in biological samples with surface-assisted laser desorption/ionization mass spectrometry: A concise review of the last decade

Knowing the spatial location of the lipid species present in biological samples is of paramount importance for the elucidation of pathological and physiological processes. In this context, mass spectrometry imaging (MSI) has emerged as a powerful technology allowing the visualization of the spatial distributions of biomolecules, including lipids, in complex biological samples. Among the different ionization methods available, the emerging surface-assisted laser desorption/ionization (SALDI) MSI offers unique capabilities for the study of lipids. This review describes the specific advantages of SALDI-MSI for lipid analysis, including the ability to perform analyses in both ionization modes with the same nanosubstrate, the detection of lipids characterized by low ionization efficiency in MALDI-MS, and the possibilities of surface modification to improve the detection of lipids. The complementarity of SALDI and MALDI-MSI is also discussed. Finally, this review presents data processing strategies applied in SALDI-MSI of lipids, as well as examples of applications of SALDI-MSI in biomedical lipidomics.     

Spatially resolved analysis of small molecules by matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI)

? Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) of tissues provides the means to analyse the spatial distributions of small molecules and proteins within tissues. This imaging technique is commonplace in medicinal and pharmaceutical research, but its application in plant science is very recent. Broader introduction requires specific adaptations for plant tissues. Sample preparation is of paramount importance in order to obtain high-quality spectra providing sufficient spatial resolution for compounds. Optimization is required for sectioning, choice of matrix and means of matrix deposition. ? Here, we present our current protocols for the detection of small molecules in cryodissected immature barley (Hordeum vulgare) grains and tobacco (Nicotiana tabacum) roots. ? Examples of MALDI-MSI measurements are provided, and the level of reproducibility across biological replicates is addressed. Furthermore, our approaches for the validation of distribution patterns and for the identification of molecules are described. ? Finally, we discuss how MALDI-MSI can contribute to applied plant research.     

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.     

Advances in the characterization of supported lipid films with the atomic force microscope

During the past decade, the atomic force microscope (AFM) has become a key technique in biochemistry and biophysics to characterize supported lipid films, as testified by the continuous growth in the number of papers published in the field. The unique capabilities of AFM are: (i) capacity to probe, in real time and in aqueous environment, the surface structure of lipid films; (ii) ability to directly measure physical properties at high spatial resolution; (iii) possibility to modify the film structure and biophysical processes in a controlled way. Such experiments, published up to June 2000, are the focus of the present review. First, we provide a general introduction on the preparation and characterization of supported lipid films as well as on the principles of AFM. The section 'Structural properties' focuses on the various applications of AFM for characterizing the structure of supported lipid films: visualization of molecular structure, formation of structural defects, effect of external agents, formation of supported films, organization of phase-separated films (coexistence region, mixed films) and, finally, the use of supported lipid bilayers for anchoring biomolecules such as DNA, enzymes and crystalline protein arrays. The section 'Physical properties' introduces the principles of force measurements by AFM, interpretation of these measurements and their recent application to supported lipid films and related structures. Finally, we highlight the major achievements brought by the technique and some of the current limitations.     

Usual and Unusual Methods for Detection of Lipid Peroxides as Indicators of Tissue Injury in Cerebral Ischemia: What Is Appropriate and Useful?

1. Free radical-dependent lipid peroxidation processes have long been thought to contribute to brain damage following stroke or cerebral ischemia/reperfusion.2. The preponderance of evidence for this belief has been derived indirectly, through diminution of tissue injury indices (e.g., brain infarct volume) facilitated by application of free radical scavenger substances.3. Direct, unequivocal evidence for lipid peroxidation in terms of classical assays (detection of conjugated diene absorbance or thiobarbituric acid-reactive substances) is considerably less common, and its validity can be questioned.4. Correlations of treatment-induced diminishment of brain injury indices with reductions in lipid peroxidation level are rarer still.5. Reasons underlying the disparity between the belief that lipid peroxidation contributes to ischemic brain injury and direct evidence for this contribution (at least acutely) are proposed, along with evidence that new methods are being developed which should provide the basis for obtaining a definitive answer.     

Molecular markers applied to plant tissue culture

Summary The last decade has witnessed successful applications of plant tissue culture techniques in several crops. During that same period, studies in plant molecular genetics have also grown exponentially. Molecular markers (isozymes, RFLPs, and PCR-based markers such as RAPDs) are now used to study many of the current limitations of tissue culture. They have been used to investigate mechanisms that underlie somaclonal variation in the nuclear, mitochondrial, and chloroplast genomes. One recurrent problem with several tissue culture systems has been the difficulty of determining the origin of regenerants. Molecular markers represent powerful tools to determine precisely the origin of plants derived from microspore or anther culture, protoplast fusion, and other tissue culture studies where this information is important. With improvements in tissue culture techniques, populations of doubled haploid lines have been produced in several major crop species. Doubled haploid populations have proven useful in the production of molecular maps and in tagging important agronomic traits. This review describes the use of molecular markers to address fundamental and practical questions of plant tissue culture, and discusses the potential of improvements in molecular techniques and new molecular markers such as SCAR and STS along with high-resolution mapping strategies.     

Time-dependent evolution of tissue markers by MALDI-MS imaging

We have used MALDI-MS imaging (MALDI-MSI) to monitor the time dependent appearance and loss of signals when tissue slices are brought rapidly to room temperature for short to medium periods of time. Sections from mouse brain were cut in a cryostat microtome, placed on a MALDI target and allowed to warm to room temperature for 30 s to 3 h. Sections were then refrozen, fixed by ethanol treatment and analysed by MALDI-MSI. The intensity of a range of markers were seen to vary across the time course, both increasing and decreasing, with the intensity of some markers changing significantly within 30 s and markers also showed tissue location specific evolution. The markers resulting from this autolysis were compared directly to those that evolved in a comparable 16 h on-tissue trypsin digest, and the markers that evolved in the two studies were seen to be substantially different. These changes offer an important additional level of location-dependent information for mapping changes and seeking disease-dependent biomarkers in the tissue. They also indicate that considerable care is required to allow comparison of biomarkers between MALDI-MSI experiments and also has implications for the standard practice of thaw-mounting multiple tissue sections onto MALDI-MS targets.     

Layered electrophoretic transfer - A method for pre-analytic processing of histological sections

Current technologies for measuring protein expression across a tissue section are based on MS or in situ detection such as immunohistochemistry. However, due to the inherent molecular complexity of tissue samples and the large dynamic range of protein expression in cells, current approaches are often unable to measure moderate- and low-abundant proteins. In addition, they do not provide information on the physico-chemical properties of the proteins studied. To address these problems, we are developing a new pre-analytic methodology termed layered electrophoretic transfer (LET) that selectively separates and processes proteins from an intact tissue section without compromising important two-dimensional histological information. LET offers two potential advantages over standard techniques: (i) A reduced complexity of the tissue proteome for subsequent analysis; (ii) An opportunity to assess the biochemical status of proteins as they exist in situ. As an initial proof-of-concept, we demonstrate here that the protein content from a mixture of molecular weight standards, human tissue lysates, and tissue sections can be successfully transferred and separated using LET, and further demonstrate that the method can be coupled with immunoblotting or MS for downstream measurements. LET technology represents a new pre-analytic tool for interrogating the proteome in tissue sections while preserving valuable spatial information.     

Shedding new light on lipid biology with coherent anti-Stokes Raman scattering microscopy

Despite the ubiquitous roles of lipids in biology, the detection of lipids has relied on invasive techniques, population measurements, or nonspecific labeling. Such difficulties can be circumvented by a label-free imaging technique known as coherent anti-Stokes Raman (CARS) microscopy, which is capable of chemically selective, highly sensitive, and high-speed imaging of lipid-rich structures with submicron three-dimensional spatial resolution. We review the broad applications of CARS microscopy to studies of lipid biology in cell cultures, tissue biopsies, and model organisms. Recent technical advances, limitations of the technique, and perspectives are discussed.     

Oxidants and human disease: some new concepts

Oxidant species such as superoxide radical (O.2-), hydrogen peroxide (H2O2), hydroxyl radical (HO.), and lipid peroxides (LOOH) are becoming increasingly implicated in human disease. However, the question of whether such oxidants are a major cause of tissue injury in human disease or are merely produced during such injury has been difficult to answer because of inadequate experimental techniques, and possibly because of an overemphasis on lipid peroxidation as a mechanism of oxidant injury. Recent developments in methodology, in our understanding of the primary mechanism of oxidant toxicity to cells, and in concepts of antioxidant protection are reviewed. Good evidence now exists for some role of oxidant damage to tissues in the pathology of several human diseases, including rheumatoid arthritis, reperfusion injury, immune injury to lung and kidney, and cerebral trauma or ischemia. These have led to promising suggestions for new therapeutic approaches.     

A matter of fat: an introduction to lipidomic profiling methods

In recent years, lipidomics or lipid profiling, an extension of metabolomics where the lipid complement of a cell, tissue or organism is measured, has been the recipient of increasing attention as a research tool in a range of diverse disciplines including physiology, lipid biochemistry, clinical biomarker discovery and pathology. The advancement of the field has been driven by the development of analytical technologies, and in particular advances in liquid chromatography mass spectrometry and chemometric methods. In this review, we give an overview of the current methods with which lipid profiling is being performed. The benefits and shortcomings of mass spectrometry both in the presence and absence of chromatographic separation techniques such as liquid-, gas- and thin layer chromatography are explored. Alone these techniques have their limitations but through a combination many of the disadvantages may be overcome providing a valuable analytical tool for a variety of disease processes.     

Micrometric molecular histology of lipids by mass spectrometry imaging

Time-Of-Flight Secondary Ion Mass Spectrometry is compared to other mass spectrometry imaging techniques, and recent improvements of the experimental methods, driven by biological and biomedical applications, are described and discussed. This review shows that this method that can be considered as a micrometric molecular histology is particularly efficient for obtaining images of various lipid species at the surface of a tissue sample, without sample preparation, and with a routine spatial resolution of 1μm or less.     

New directions in photodynamic therapy.

Photodynamic therapy (PDT), a treatment approach that makes use of a photosensitizer to generate a localized toxic species in diseased tissue, has recently become an approved treatment modality. So far, however, only a handful of photosensitizers have received regulatory approval and for a small number of diseases. This chapter outlines the major limitations of PDT and speculates on the possible improvements that are required in order to advance PDT to a front line therapy. Seven areas of improvements are discussed: drug selectivity, drug delivery, light delivery, combination therapies, pigmented tumors, other potential uses, and protocol optimization. For each area, current limitations are discussed, and further required studies are recommended.     

Quantitative in vivo imaging of embryonic development: opportunities and challenges

Animal models are critically important for a mechanistic understanding of embryonic morphogenesis. For decades, visualizing these rapid and complex multidimensional events has relied on projection images and thin section reconstructions. While much insight has been gained, fixed tissue specimens offer limited information on dynamic processes that are essential for tissue assembly and organ patterning. Quantitative imaging is required to unlock the important basic science and clinically relevant secrets that remain hidden. Recent advances in live imaging technology have enabled quantitative longitudinal analysis of embryonic morphogenesis at multiple length and time scales. Four different imaging modalities are currently being used to monitor embryonic morphogenesis: optical, ultrasound, magnetic resonance imaging (MRI), and micro-computed tomography (micro-CT). Each has its advantages and limitations with respect to spatial resolution, depth of field, scanning speed, and tissue contrast. In addition, new processing tools have been developed to enhance live imaging capabilities. In this review, we analyze each type of imaging source and its use in quantitative study of embryonic morphogenesis in small animal models. We describe the physics behind their function, identify some examples in which the modality has revealed new quantitative insights, and then conclude with a discussion of new research directions with live imaging.     

Nebulized vs. instilled exogenous surfactant in an adult lung injury model.

Three days after subcutaneous injection of N-nitroso-N-methylurethane (NNNMU) to induce lung injury, adult rabbits were mechanically ventilated and lung function was evaluated. Each animal then received either nebulized Survanta (Neb Surv), nebulized saline (Neb Saline), nebulized gas alone (Neb Gas), or tracheally instilled Survanta (Inst Surv). The ventilation efficiency index (VEI) value increased significantly compared with pretreatment values (P less than 0.01) over a 3-h treatment period for the Neb Surv animals, whereas VEI values for the other three groups decreased after treatment (P less than 0.05). Arterial PO2-to-fraction of inspired O2 ratios and dynamic compliance values significantly decreased after treatment for the Inst Surv group (P less than 0.05). Pressure-volume curves demonstrated a significantly greater volume at maximal pressure for the Neb Surv group compared with each of the other groups studied (P less than 0.01). The calculated quantity of surfactant recovered in lung tissue for the Neb Surv group was only 4.9 +/- 1.0 mg lipid/kg compared with 100 mg lipid/kg delivered to the Inst Surv group. Surfactant administered as an aerosol resulted in modest physiological improvements in this model of lung injury and was superior to the tracheal instillation technique.