Imaging lipids with secondary ion mass spectrometry

This review discusses the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and magnetic sector SIMS with high lateral resolution performed on a Cameca NanoSIMS 50(L) to imaging lipids. The similarities between the two SIMS approaches and the differences that impart them with complementary strengths are described, and various strategies for sample preparation and to optimize the quality of the SIMS data are presented. Recent reports that demonstrate the new insight into lipid biochemistry that can be acquired with SIMS are also highlighted. This article is part of a Special Issue entitled Tools to study lipid functions.     

Imaging of metabolites using secondary ion mass spectrometry

This article provides an overview of the technique of secondary ion mass spectrometry imaging and highlights some current and future areas of application relevant to the field of metabolomics. The approach benefits from label-free analysis of molecular species up to ~1500 Da with minimal sample preparation. Offering the highest spatial resolution of current mass spectrometry imaging methodologies, the technique is well-suited to metabolite imaging in both biological tissue and cells, in both 2D and 3D.     

High resolution imaging by organic secondary ion mass spectrometry

Secondary-ion mass spectrometry (SIMS) is based on the acceleration of high-energy primary ions onto a target. Secondary electrons, neutrals and ions are emitted from the target, reflecting its chemical composition. This enables simultaneous analysis and localization of target molecules, giving valuable information that is difficult or impossible to obtain with other analytical methods. The secondary ions can be extracted and detected by any type of mass analyzer. SIMS is unique in its ability to detect several target molecules simultaneously in small samples and to image their localization at subcellular resolution. The recent development of bioimaging SIMS opens up new possibilities in biotechnology and biological research with applications in biomedicine and pathology. The current development of this technique has the potential to become as important for biotechnology as the advent of the electron microscope, confocal microscope or in situ hybridization.     

Visualization of acetaminophen-induced liver injury by time-of-flight secondary ion mass spectrometry

Time-of-flight secondary ion mass spectrometry (MS) provides secondary ion images that reflect distributions of substances with sub-micrometer spatial resolution. To evaluate the use of time-of-flight secondary ion MS to capture subcellular chemical changes in a tissue specimen, we visualized cellular damage showing a three-zone distribution in mouse liver tissue injured by acetaminophen overdose. First, we selected two types of ion peaks related to the hepatocyte nucleus and cytoplasm using control mouse liver. Acetaminophen-overdosed mouse liver was then classified into three areas using the time-of-flight secondary ion MS image of the two types of peaks, which roughly corresponded to established histopathological features. The ion peaks related to the cytoplasm decreased as the injury became more severe, and their origin was assumed to be mostly glycogen based on comparison with periodic acid–Schiff staining images and reference compound spectra. This indicated that the time-of-flight secondary ion MS image of the acetaminophen-overdosed mouse liver represented the chemical changes mainly corresponding to glycogen depletion on a subcellular scale. In addition, this technique also provided information on lipid species related to the injury. These results suggest that time-of-flight secondary ion MS has potential utility in histopathological applications.     

Application of nanoscale secondary ion mass spectrometry to plant cell research

Imaging resource flow in soil-plant systems remains central to understanding plant development and interactions with the environment. Typically, subcellular resolution is required to fully elucidate the compartmentation, behavior, and mode of action of organic compounds and mineral elements within plants. For many situations this has been limited by the poor spatial resolution of imaging techniques and the inability to undertake studies in situ. Here we demonstrate the potential of Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS), which is capable of the quantitative high-resolution spatial imaging of stable isotopes (e.g., ¹²C, ¹³C, ¹⁴N, ¹⁵N, ¹⁶O, ¹⁸O, ³¹P, ³⁴S) within intact plant-microbial-soil systems. We present examples showing how the approach can be used to investigate competition for ¹⁵N-labelled nitrogen compounds between plant roots and soil microorganisms living in the rhizosphere and the spatial imaging of ³¹P in roots. We conclude that NanoSIMS has great potential to elucidate the flow of isotopically-labelled compounds in complex media (e.g., soil) and opens up countless new opportunities for studying plant responses to abiotic stress (e.g., ¹⁸O₃, elevated ¹³CO₂), signal exchange, nutrient flow and plant-microbial interactions.Key words: mass spectrometry, NanoSIMS, rhizosphere, isotope labelling, soil, nitrogen, carbon, phosphorus, ¹⁵N, ¹³C, ³¹PWe have used the NanoSIMS technique to investigate the flow of nutrients between microbial and plant cells within the rhizosphere. Secondary Ion Mass Spectrometry (SIMS) involves bombarding a sample with a high-energy ion beam, which sputters atoms, molecules and electrons from the sample surface. Ionized species (secondary ions) are extracted to a mass spectrometer, sorted according to their energy and their mass-to-charge ratio, and counted. NanoSIMS, a recent development in SIMS, combines high sensitivity with high spatial resolution (typically 100 nm) to allow elemental and isotopic imaging of secondary ions, such as ¹²C-, ¹⁶O- and ¹²C¹⁴N-, on a range of biological materials at the sub-cellular scale (Fig. 1A and B). An element map is obtained by scanning the primary ion beam over the sample surface and measuring the secondary ion intensities of any given ion species, at each pixel in the image. The intrinsically high mass resolution allows the separation of different ion species at the same nominal atomic mass (e.g., ¹²C¹⁵N- from ¹³C¹⁴N- at mass 27), while the multi-collection capability allows the simultaneous measurement of up to five ion species. This makes it possible to obtain images of different isotopes from the same area simultaneously, from which quantitative isotope ratios from individual components can then be extracted. As such, NanoSIMS offers a means of elucidating processes involved in the transport of ions and molecules into cells and their distribution within cells, at scales and sensitivities not attainable by other methods.^1–5Open in a separate windowFigure 1(A) ¹²C¹⁴N^- and (B) ³¹P^- images of a wheat root cell nucleus from NanoSIMS illustrating the potential to map different elements at the sub-cellular scale; (C) TEM image of two bacteria attached to a cortical cell wall; (D) corresponding ¹⁵N/¹⁴N ratio image from NanoSIMS of the same bacteria. The differential uptake of ¹⁵N is illustrated by the color scale; ranging from natural abundance (blue) to a ¹⁵N/¹⁴N ratio = 1.0 (i.e., 50 at% ¹⁵N) (pink) for the plant cell and bacteria, respectively; (E) Linescan (3.5 µm) illustrating the variation in ¹⁵N/¹⁴N across an enriched bacterium and an un-enriched plant cell wall (line in D). Error bars are based on the Poisson counting statistics for each pixel.We previously demonstrated the use of NanoSIMS to image and map the location of ¹⁵N-labelled bacterial communities artificially introduced into soil microhabitats.^6,7 We extended this approach to a natural ecosystem, by examining the differential partitioning of ¹⁵N-labelled ammonium (¹⁵NH₄⁺) between plant roots and soil microbial communities at the nanometer scale (Fig. 1C and D).⁸ It was shown that introduced ¹⁵N could be detected, and more importantly, mapped, in individual bacterial cells found in the soil matrix, within the rhizosphere, within root hairs, and intra-cellular within the root. The ¹⁵N/¹⁴N ratio data (determined as the ratio between the ¹²C¹⁵N- and the ¹²C¹⁴N- signals) could then be extracted from specific regions of interest—groups of pixels bounding a particular feature, such as a bacterium or a root cell wall, or linescans (Fig. 1E). This unique approach allows the visualization of nutrient flows and metabolic pathways through complex, multi-component ecosystems. Here we consider further the application of the technique to study nutrient availability in plant cell research.     

Biofilms and corrosion interactions on stainless steel in seawater

Biofouling and biocorrosion lead to an important modification of the metal/ solution interface inducing changes in the type and concentration of ions, pH values, oxygen levels, flow velocity, etc. Metal dissolution in seawater is mainly conditioned by two different processes: (a) biofouling settlement and (b) corrosion products formation.Corrosion-resistant alloys such as stainless steel present an ideal substratum for microbial colonization, rather similar to inert non-metallic surfaces, due to the lack of corrosion products. Stainless steels are sensitive to pitting and other types of localized corrosion in chloride-containing media such as seawater. Biofilms and bacterial metabolism may accelerate the initiation of crevice attack by depletion of oxygen in the crevice solution due to microbial respiration. Bacterial colonization occurs within a period of 24–72 h on stainless steel samples exposed to natural seawater and, depending on environmental conditions, a copious and patchy biofilm is generally formed.Different interpretations of biofilms' effects on corrosion are critically discussed. A practical case, involving polluted harbour seawater, is reported to illustrate biofilm and corrosion interactions on stainless steel samples.     

Lipid imaging with time-of-flight secondary ion mass spectrometry (ToF-SIMS)

Fundamental advances in secondary ion mass spectrometry (SIMS) now allow for the examination and characterization of lipids directly from biological materials. The successful application of SIMS-based imaging in the investigation of lipids directly from tissue and cells are demonstrated. Common complications and technical pitfalls are discussed. In this review, we examine the use of cluster ion sources and cryogenically compatible sample handling for improved ion yields and to expand the application potential of SIMS. Methodological improvements, including pre-treating the sample to improve ion yields and protocol development for 3-dimensional analyses (i.e. molecular depth profiling), are also included in this discussion. New high performance SIMS instruments showcasing the most advanced instrumental developments, including tandem MS capabilities and continuous ion beam compatibility, are described and the future direction for SIMS in lipid imaging is evaluated.     

Geobiological investigations using secondary ion mass spectrometry: microanalysis of extant and paleo-microbial processes

The application of secondary ion mass spectrometry (SIMS) has tremendous value for the field of geobiology, representing a powerful tool for identifying the specific role of micro-organisms in biogeochemical cycles. In this review, we highlight a number of diverse applications for SIMS and nanoSIMS in geobiological research. SIMS performs isotope and elemental analysis at microscale enabling the investigation of the physiology of individual microbes within complex communities. Additionally, through the study of isotopic or chemical characteristics that are common in both living and ancient microbial communities, SIMS allows for direct comparisons of potential biosignatures derived from extant microbial cells and their fossil equivalents.     

Positive and negative liquid secondary ion mass spectrometry of chloro- and nitrophenylglucuronides

Mass spectra of a series of chloro- and nitrophenylglucuronides by liquid secondary ion (LSI) mass spectrometry were obtained. In the positive ion mode class characteristic fragmentations and adduct ions are observed only in the presence of alkali salt additives. No additives were necessary in the negative ion mode to see abundant class characteristic [M-H]- and aglycone fragment ions. Cluster ion formation was found to be prominent but only in the negative ion mode.     

Analysis of lung surfactant model systems with time-of-flight secondary ion mass spectrometry

An often-used model lung surfactant containing dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and the surfactant protein C (SP-C) was analyzed as Langmuir-Blodgett film by spatially resolved time-of-flight secondary ion mass spectrometry (TOF-SIMS) to directly visualize the formation and composition of domains. Binary lipid and lipid/SP-C systems were probed for comparison. TOF-SIMS spectra revealed positive secondary ions (SI) characteristic for DPPC and SP-C, but not for DPPG. SI mapping results in images with domain structures in DPPC/DPPG and DPPG/SP-C, but not in DPPC/SP-C films. We are able to distinguish between the fluid and condensed areas probably due to a matrix effect. These findings correspond with other imaging techniques, fluorescence light microscopy (FLM), scanning force microscopy (SFM), and silver decoration. The ternary mixture DPPC/DPPG/SP-C transferred from the collapse region exhibited SP-C-rich domains surrounding pure lipid areas. The results obtained are in full accordance with our earlier SFM picture of layered protrusions that serve as a compressed reservoir for surfactant material during expansion. Our study demonstrates once more that SP-C plays a unique role in the respiration process.     

The use of secondary ion mass spectrometry (SIMS) to evaluate internal contamination.

Secondary ion mass spectrometry (SIMS) permits the detection of stable and radioactive elements in microvolume. Based on the ablation of specimens by ion bombardment, this mass spectrometry method allows a rapid assessment of trace elements in biological samples and enables accurate isotopic ratio determination. In this work, an application of SIMS in studies involving element microdistribution is illustrated on the basis of analyses of duodenal tissue sections from rats contaminated with either cerium or thorium. For this purpose, tests are performed with SIMS to analyze tissue sections obtained 12, 24 and 48 hr after contamination. In this report, strengths and limitations of SIMS are pointed out as an important tool in biological research.     

The role of secondary ion mass spectrometry (SIMS) in biological microanalysis: technique comparisons and prospects.

The virtues and limitations of SIMS ion microscopy are compared with other spectroscopic techniques applicable to biological microanalysis, with a special emphasis on techniques for elemental localization in biological tissue (electron, X-ray, laser, nuclear, ion microprobes). Principal advantages of SIMS include high detection sensitivity, high depth resolution, isotope specificity, and possibilities for three-dimensional imaging. Current limitations, especially in comparison to X-ray microanalysis, center on lateral spatial resolution and quantification. Recent SIMS instrumentation advances involving field emission liquid metal ion sources and laser post-ionization will help to minimize these limitations in the future. The molecular surface analysis capabilities of static SIMS, especially with the new developments in commercial time-of-flight spectrometers, are promising for application to biomimetic, biomaterials, and biological tissue or cell surfaces. However, the direct microchemical imaging of biomolecules in tissue samples using SIMS will be hindered by limited concentrations, small analytical volumes, and the inefficiencies of converting surface molecules to structurally significant gas phase ions. Indirect detection using elemental or isotopically tagged molecules, however, shows considerable promise for molecular imaging studies using SIMS ion microscopy.     

Quantitative secondary ion monitoring gas chromatography/mass spectrometry of diethylstilbestrol in bovine liver

A procedure is described for the extraction of diethylstilbestrol (DES) from animal tissue for quantitative capillary gas chromatography/mass spectrometry (GC/MS). The procedure is based upon use of a strong anion exchange polystyrene divinylbenzene resin for sample purification. The recovery of DES from the resin clean up was 88% in the high parts per trillion (ppt) range. Criteria for identification of DES using selected ion monitoring (SIM) GC/MS are presented. Liquid chromatography/mass spectrometry (LC/MS) was used to investigate altered DES cis/trans ratios observed in biological extracts.     

Lipid specificity of surfactant protein B studied by time-of-flight secondary ion mass spectrometry

One of the key functions of mammalian pulmonary surfactant is the reduction of surface tension to minimal values. To fulfill this function it is expected to become enriched in dipalmitoylphosphatidylcholine either on its way from the alveolar type II pneumocytes to the air/water interface of the lung or within the surface film during compression and expansion of the alveoli during the breathing cycle. One protein that may play a major role in this enrichment process is the surfactant protein B. The aim of this study was to identify the lipidic interaction partner of this protein. Time-of-flight secondary ion mass spectrometry was used to analyze the lateral distribution of the components in two SP-B-containing model systems. Either native or partly isotopically labeled lipids were analyzed. The results of both setups give strong indications that, at least under the specific conditions of the chosen model systems (e.g., concerning pH and lipid composition), the lipid interacting with surfactant protein B is not phosphatidylglycerol as generally accepted, but dipalmitoylphosphatidylcholine instead.     

Analysis of long-chain bases in sphingolipids by positive ion fast atom bombardment or matrix-assisted secondary ion mass spectrometry

The structures of long-chain bases are expressed as [CH2C(NH2) = CHR]+ (Z+) in the positive ion mode spectra obtained on fast atom bombardment (FAB) mass spectrometry or liquid-matrix-assisted secondary ion mass spectrometry (SIMS) [Benninghoven, A., Ed. (1983) Ion Formation from Organic Solids, Springer, Berlin]. This phenomenon is common to sphingolipids in general: glycosphingolipids [see reviews by Sweeley and Nunez [Sweeley, C. C., & Nunez, H. A. (1985) Annu. Rev. Biochem. 54, 765] and Kanfer and Hakomori [Kanfer, J. N., & Hakomori, S. (1983) Handb. Lipid Res. 3]] and phosphonosphingolipids [Hayashi, A., & Matsubara, T. (1982) in New Vistas in Glycolipid Research (Makita, A., Handa, S., Taketomi, T., & Nagai, Y., Eds.) p 103, Plenum, New York], inclusive. Phytosphingosine compounds show the same type of fragmentation without additional dehydration if a neutral matrix is used. A Z+ ion is easily detected in the lower mass region (m/z 200-400) as an even mass number fragment ion, and confirmation is made by means of B/E constant and B2/E constant linked scan techniques [Boyd, R. K., & Beynon, J. H. (1977) Org. Mass Spectrom. 12, 163; Boyd, R. K., & Shushan, B. (1981) Int. J. Mass Spectrom. Ion Phys. 37, 355; Macdonald, C. G., & Lacey, M. J. (1984) Org. Mass Spectrom. 19, 55]. [Principles of linked scannings are explicitly summarized by Jennings and Mason [Jennings, K. R., & Mason, R. S. (1983) in Tandem Mass Spectrometry (McLafferty, F. W., Ed.) p 197, Wiley, New York] besides the cited literature.]     

Pyrolysis mass spectrometry characterisation and numerical taxonomy ofAeromonas spp.

Reference strains (2) and 29 isolates ofAeromonas spp. from clinical material and environmental specimens were characterised in traditional biochemical tests, and in pyrolysis mass spectrometry, which gives data reflecting whole-cell composition. Numerical taxonomic analyses of the data sets were compared with conventional identification at species level, and pathogenic potential, as inferred from the origin of the isolates. Clustering with conventional test reaction patterns showed, for each of the species represented, a clearly defined core group of typical isolates, surrounded by a halo of aberrant strains. One further cluster comprised strains intermediate betweenA. caviae andA. hydrophila, and one strain was grossly atypical in both analyses. Clustering from pyrolysis data corresponded less well with species identification. Broadly, the biochemical division between core and halo strains was supported in pyrolysis forA. caviae andA. sobria, but the main group ofA. hydrophila in pyrolysis comprised strains clustering in the core and halo groups of this species, and three strains intermediate betweenA. hydrophila andA. caviae in biochemical tests. Two further pyrolysis clusters comprised core and halo strains ofA. hydrophila. However, pyrolysis clustering correlated well with inferred pathogenicity, showing four clusters of probable pathogens, six clusters of probable nonpathogens, and one two member cluster of doubtful status. Most strains that clustered in the species haloes, or in species-intermediate groups in biochemical tests, were non-human isolates, or were isolated in the absence of symptomatic infection. The correlation of inferred pathogenicity with biochemical clustering was poorer than that with pyrolysis clustering.Abbreviations CTRP conventional test reaction pattern - PyMS pyrolysis mass spectrometry     

Supported membrane composition analysis by secondary ion mass spectrometry with high lateral resolution

The lateral organization of lipid components within membranes is usually investigated with fluorescence microscopy, which, though highly sensitive, introduces bulky fluorophores that might alter the behavior of the components they label. Secondary ion mass spectroscopy performed with a NanoSIMS 50 instrument also provides high lateral resolution and sensitivity, and many species can be observed in parallel without the use of bulky labels. A tightly focused beam (approximately 100 nm) of Cs ions is scanned across a sample, and up to five of the resulting small negative secondary ions can be simultaneously analyzed by a high-resolution mass spectrometer. Thin layers of (15)N- and (19)F-labeled proteins were microcontact-printed on an oxidized silicon substrate and imaged using the NanoSIMS 50, demonstrating the sensitivity and selectivity of this approach. Supported lipid bilayers were assembled on an oxidized silicon substrate, then flash-frozen and freeze-dried to preserve their lateral organization. Lipid bilayers were analyzed with the NanoSIMS 50, where the identity of each specific lipid was determined through detection of its unique secondary ions, including (12)C(1)H(-), (12)C(2)H(-), (13)C(-), (12)C(14)N(-), and (12)C(15)N(-). Steps toward obtaining quantitative composition analysis of lipid membranes that varied spatially in isotopic composition are presented. This approach has the potential to provide a composition-specific analysis of membrane organization that compliments other imaging modalities.     

Probing natural product biosynthetic pathways using Fourier transform ion cyclotron resonance mass spectrometry

Two natural products, diazepinomicin (1) and dioxapyrrolomycin (2), containing stable isotopic labels of ¹⁵N or deuterium, were used to demonstrate the utility of Fourier transform ion cyclotron resonance mass spectrometry for probing natural product biosynthetic pathways. The isotopic fine structures of significant ions were resolved and subsequently assigned elemental compositions on the basis of highly accurate mass measurements. In most instances the mass measurement accuracy is less than one part per million (ppm), which typically makes the identification of stable-isotope labeling unambiguous. In the case of the mono-¹⁵N-labeled diazepinomicin (1) derived from labeled tryptophan, tandem mass spectrometry located this ¹⁵N label at the non-amide nitrogen. Through the use of exceptionally high mass resolving power of over 125,000, the isotopic fine structure of the molecular ion cluster of 1 was revealed. Separation of the ¹⁵N₂ peak from the isobaric ¹³C¹⁵N peak, both having similar abundances, demonstrated the presence of a minor amount of doubly ¹⁵N-labeled diazepinomicin (1). Tandem mass spectrometry amplified this isotopic fine structure (Δm = 6.32 mDa) from mDa to 1 Da scale thereby allowing more detailed scrutiny of labeling content and location. Tandem mass spectrometry was also used to assign the location of deuterium labeling in two deuterium-labeled diazepinomicin (1) samples. In one case three deuterium atoms were incorporated into the dibenzodiazepine core; while in the other a mono-D label was mainly incorporated into the farnesyl side chain. The specificity of ¹⁵N-labeling in dioxapyrrolomycin (2) and the proportion of the ¹⁵N-label contained in the nitro group were determined from the measurement of the relative abundance of the ¹⁴NO₂^1? and ¹⁵NO₂^1? fragment ions.