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.     

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.     

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.     

Label free biochemical 2D and 3D imaging using secondary ion mass spectrometry

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) provides a method for the detection of native and exogenous compounds in biological samples on a cellular scale. Through the development of novel ion beams the amount of molecular signal available from the sample surface has been increased. Through the introduction of polyatomic ion beams, particularly C(60), ToF-SIMS can now be used to monitor molecular signals as a function of depth as the sample is eroded thus proving the ability to generate 3D molecular images. Here we describe how this new capability has led to the development of novel instrumentation for 3D molecular imaging while also highlighting the importance of sample preparation and discuss the challenges that still need to be overcome to maximise the impact of the technique.     

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.     

Progress in analytical imaging of the cell by dynamic secondary ion mass spectrometry (SIMS microscopy)

This paper reviews the most recent methodological advances in the field of biological imaging using dynamic secondary ion mass spectrometry (SIMS). After a short reminder of the basic principle of SIMS imaging, the latest high-resolution dynamic SIMS equipment is briefly described. This new ion nanoprobe (CAMECA NanoSIMS 50) has a lateral resolution of less than 50 nm with primary Cs+ ion, the ability to detect simultaneously 5 different ions from the same micro-volume and a very good transmission even at high mass resolution (60% at M/DeltaM=5000). Basic considerations related to sample preparation, mass resolution and primary ion implantation are given. The decisive capability of this new instrument, and more generally of high-resolution dynamic SIMS imaging in biology, are illustrated with the most recent examples of utilization.     

High resolution spatial distribution of neuropeptides by MALDI imaging mass spectrometry in the terrestrial snail, Helix pomatia

Imaging mass spectrometry (IMS) is a powerful technique that combines the chemical and spatial analysis of surface materials. It allows spatial localization of peptides, proteins or lipids that are recorded in parallel without the need of a label. It is currently one of the most rapidly developing techniques in the proteomics toolbox. In the present study, accurate mass matrix-assisted laser desorption/ionization imaging mass spectrometry (MALD IMS) was used for direct molecular mapping of nervous tissue at micrometer spatial resolution. Cryosections of the whole brain of the terrestrial snail, Helix pomatia, were placed on indium-tin-oxide (ITO)-coated conductive glass slides and covered with a thin layer of α-cyano-4-hydroxycinnamic acid (CHCA) matrix by electro spray deposition. High-resolution molecular ion maps of well-known neuropeptides, such as FMRFamide were constructed. FMRFamide is known to exert powerful modulatory effect on synaptic transmission in molluscs. FMRFamide was predominantly localized in the cluster of neurons in the pro-, meso- and postcerebral regions of cerebral ganglia, pedal ganglia and right parietal ganglia of the central nervous system. Our present study, using MALDI IMS confirmed the distribution of FMRFamide containing cells in the Helix central nervous system previously detected by antibody dependent immunohistochemistry.     

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.     

Mass spectrometry imaging with high resolution in mass and space

Mass spectrometry (MS) imaging links molecular information and the spatial distribution of analytes within a sample. In contrast to most histochemical techniques, mass spectrometry imaging can differentiate molecular modifications and does not require labeling of targeted compounds. We have recently introduced the first mass spectrometry imaging method that provides highly specific molecular information (high resolution and accuracy in mass) at cellular dimensions (high resolution in space). This method is based on a matrix-assisted laser desorption/ionization (MALDI) imaging source working at atmospheric pressure which is coupled to an orbital trapping mass spectrometer. Here, we present a number of application examples and demonstrate the benefit of ‘mass spectrometry imaging with high resolution in mass and space.’ Phospholipids, peptides and drug compounds were imaged in a number of tissue samples at a spatial resolution of 5–10 μm. Proteins were analyzed after on-tissue tryptic digestion at 50-μm resolution. Additional applications include the analysis of single cells and of human lung carcinoma tissue as well as the first MALDI imaging measurement of tissue at 3 μm pixel size. MS image analysis for all these experiments showed excellent correlation with histological staining evaluation. The high mass resolution (R = 30,000) and mass accuracy (typically 1 ppm) proved to be essential for specific image generation and reliable identification of analytes in tissue samples. The ability to combine the required high-quality mass analysis with spatial resolution in the range of single cells is a unique feature of our method. With that, it has the potential to supplement classical histochemical protocols and to provide new insights about molecular processes on the cellular level.     

Direct analysis of lipids on thin layer plates by matrix-assisted secondary ion mass spectrometry

A simple and rapid method for the analysis of lipids on a thin layer chromatography (TLC) plate by matrix-assisted secondary ion mass spectrometry (SI-MS) is reported. Analysis was performed without elution of the sample from the TLC plate. Mass spectra obtained by this method are free from interference due to the TLC plate absorbent and reagents used for the detection of the spots. About 1 micrograms of lipids applied on a TLC plate can be analyzed by this method. On scanning the plate, mass chromatograms of each lipid were obtained based on its migration distance along the plate.     

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.     

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.]     

Dynamic secondary ion mass spectrometry imaging of microbial populations utilizing C-labelled substrates in pure culture and in soil

We demonstrate that dynamic secondary ion mass spectrometry (SIMS)-based ion microscopy can provide a means of measuring (13)C assimilation into individual bacterial cells grown on (13)C-labelled organic compounds in the laboratory and in field soil. We grew pure cultures of Pseudomonas putida NCIB 9816-4 in minimal media with known mixtures of (12)C- and (13)C-glucose and analysed individual cells via SIMS imaging. Individual cells yielded signals of masses 12, 13, 24, 25, 26 and 27 as negative secondary ions indicating the presence of (12)C(-), (13)C(-), (24)((12)C(2))(-), (25)((12)C(13)C)(-), (26)((12)C(14)N)(-) and (27)((13)C(14)N)(-) ions respectively. We verified that ratios of signals taken from the same cells only changed minimally during a approximately 4.5 min period of primary O(2)(+) beam sputtering by the dynamic SIMS instrument in microscope detection mode. There was a clear relationship between mass 27 and mass 26 signals in Pseudomonas putida cells grown in media containing varying proportions of (12)C- to (13)C-glucose: a standard curve was generated to predict (13)C-enrichment in unknown samples. We then used two strains of Pseudomonas putida able to grow on either all or only a part of a mixture of (13)C-labelled and unlabelled carbon sources to verify that differential (13)C signals measured by SIMS were due to (13)C assimilation into cell biomass. Finally, we made three key observations after applying SIMS ion microscopy to soil samples from a field experiment receiving (12)C- or (13)C-phenol: (i) cells enriched in (13)C were heterogeneously distributed among soil populations; (ii) (13)C-labelled cells were detected in soil that was dosed a single time with (13)C-phenol; and (iii) in soil that received 12 doses of (13)C-phenol, 27% of the cells in the total community were more than 90% (13)C-labelled.     

Fluorinated colloidal gold immunolabels for imaging select proteins in parallel with lipids using high-resolution secondary ion mass spectrometry

The local abundance of specific lipid species near a membrane protein is hypothesized to influence the protein's activity. The ability to simultaneously image the distributions of specific protein and lipid species in the cell membrane would facilitate testing these hypotheses. Recent advances in imaging the distribution of cell membrane lipids with mass spectrometry have created the desire for membrane protein probes that can be simultaneously imaged with isotope labeled lipids. Such probes would enable conclusive tests to determine whether specific proteins colocalize with particular lipid species. Here, we describe the development of fluorine-functionalized colloidal gold immunolabels that facilitate the detection and imaging of specific proteins in parallel with lipids in the plasma membrane using high-resolution SIMS performed with a NanoSIMS. First, we developed a method to functionalize colloidal gold nanoparticles with a partially fluorinated mixed monolayer that permitted NanoSIMS detection and rendered the functionalized nanoparticles dispersible in aqueous buffer. Then, to allow for selective protein labeling, we attached the fluorinated colloidal gold nanoparticles to the nonbinding portion of antibodies. By combining these functionalized immunolabels with metabolic incorporation of stable isotopes, we demonstrate that influenza hemagglutinin and cellular lipids can be imaged in parallel using NanoSIMS. These labels enable a general approach to simultaneously imaging specific proteins and lipids with high sensitivity and lateral resolution, which may be used to evaluate predictions of protein colocalization with specific lipid species.     

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.     

Detection of fatty acids from intact microorganisms by molecular beam static secondary ion mass spectrometry

We report the use of a surface analysis approach, static secondary ion mass spectrometry (SIMS) equipped with a molecular (ReO(4)(-)) ion primary beam, to analyze the surface of intact microbial cells. SIMS spectra of 28 microorganisms were compared to fatty acid profiles determined by gas chromatographic analysis of transesterfied fatty acids extracted from the same organisms. The results indicate that surface bombardment using the molecular primary beam cleaved the ester linkage characteristic of bacteria at the glycerophosphate backbone of the phospholipid components of the cell membrane. This cleavage enables direct detection of the fatty acid conjugate base of intact microorganisms by static SIMS. The limit of detection for this approach is approximately 10(7) bacterial cells/cm(2). Multivariate statistical methods were applied in a graded approach to the SIMS microbial data. The results showed that the full data set could initially be statistically grouped based upon major differences in biochemical composition of the cell wall. The gram-positive bacteria were further statistically analyzed, followed by final analysis of a specific bacterial genus that was successfully grouped by species. Additionally, the use of SIMS to detect microbes on mineral surfaces is demonstrated by an analysis of Shewanella oneidensis on crushed hematite. The results of this study provide evidence for the potential of static SIMS to rapidly detect bacterial species based on ion fragments originating from cell membrane lipids directly from sample surfaces.     

Salamander retina phospholipids and their localization by MALDI imaging mass spectrometry at cellular size resolution

Salamander large cells facilitated identification and localization of lipids by MALDI imaging mass spectrometry. Salamander retina lipid extract showed similarity with rodent retina lipid extract in phospholipid content and composition. Like rodent retina section, distinct layer distributions of phospholipids were observed in the salamander retina section. Phosphatidylcholines (PCs) composing saturated and monounsaturated fatty acids (PC 32:0, PC 32:1, and PC 34:1) were detected mainly in the outer and inner plexiform layers (OPL and IPL), whereas PCs containing polyunsaturated fatty acids (PC 36:4, PC 38:6, and PC 40:6) composed the inner segment (IS) and outer segment (OS). The presence of PCs containing polyunsaturated fatty acids in the OS layer implied that these phospholipids form flexible lipid bilayers, which facilitate phototransduction process occurring in the rhodopsin rich OS layer. Distinct distributions and relative signal intensities of phospholipids also indicated their relative abundance in a particular cell or a cell part. Using salamander large cells, a single cell level localization and identification of biomolecules could be achieved by MALDI imaging mass spectrometry.