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.     

纳米二次离子质谱技术(NanoSIMS)在微生物生态学研究中的应用

超高分辨率显微镜成像技术与同位素示踪技术相结合的纳米二次离子质谱技术(NanoSIMS)具有较高的灵敏度和离子传输效率、极高的质量分辨率和空间分辨率(＜ 50 nm),代表着当今离子探针成像技术的最高水平.利用稳定性或者放射性同位素在原位或者微宇宙条件下示踪目标微生物,然后将样品进行固定、脱水、树脂包埋或者导电镀膜处理,制备成可供二次离子质谱分析的薄片,进一步通过NanoSIMS成像分析,不仅能够在单细胞水平上提供微生物的生理生态特征信息,而且能够准确识别复杂环境样品中的代谢活跃的微生物细胞及其系统分类信息,对于认识微生物介导的元素生物地球化学循环机制具有重要意义.介绍了纳米二次离子质谱技术的工作原理和技术路线,及其与同位素示踪技术、透射电子显微镜(TEM)、扫描电子显微镜(SEM)、荧光原位杂交技术(FISH)、催化报告沉积荧光原位杂交技术(CARD-FISH)、卤素原位杂交技术(Halogen In Situ Hybridization,HISH)等联合使用在微生物生态学研究方面的应用.     

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.     

Lipid mapping in human dystrophic muscle by cluster-time-of-flight secondary ion mass spectrometry imaging

Human striated muscle samples, from male control and Duchenne muscular dystrophy-affected children, were subjected to cluster-time-of-flight secondary ion mass spectrometry (cluster-ToF-SIMS) imaging using a 25 keV Bi(3)(+) liquid metal ion gun under static SIMS conditions. Spectra and ion density maps, or secondary ion images, were acquired in both positive and negative ion mode over several areas of 500 x 500 microm(2) (image resolution, 256 x 256 pixels). Characteristic distributions of various lipids were observed. Vitamin E and phosphatidylinositols were found to concentrate within the cells, whereas intact phosphocholines accumulated over the most damaged areas of the dystrophic muscles, together with cholesterol and sphingomyelin species. Fatty acyl chain composition varied depending on the region, allowing estimation of the local damage extent.     

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.     

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.     

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.

     

Evaluating the challenges associated with time-of-fight secondary ion mass spectrometry for metabolomics using pure and mixed metabolites

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is potentially well placed to contribute to metabolomic analysis while bringing the added benefit of high resolution, label free imaging. The focused ion beams used to desorb species from the sample can be focused below 1 μm allowing chemical imaging on a sub-cellular scale. In this study we test the capability of ToF-SIMS to generate mass spectrometry and MSMS spectra from a set of standard metabolites that can be compared with open access metabolite databases containing ESI-CID MSMS spectra. The influence of the chemical environment, the matrix effect, on the observed mass spectra is assessed using a mixed metabolite sample and the data discussed in terms of compound identification and quantification. Radical ions and small fragment ions seem to be less sensitive to ion suppression or enhancement and may provide a route to quantification. Understanding such parameters will be key for the successful application of the technique for in situ metabolomics with ToF-SIMS.     

Exploration of inorganic C and N assimilation by soil microbes with time-of-flight secondary ion mass spectrometry

Stable C and N isotopes have long been used to examine properties of various C and N cycling processes in soils. Unfortunately, relatively large sample sizes are needed for accurate gas phase isotope ratio mass spectrometric analysis. This limitation has prevented researchers from addressing C and N cycling issues on microbially meaningful scales. Here we explored the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) to detect 13C and 15N assimilation by individual bacterial cells and to quantify N isotope ratios in bacterial samples and individual fungal hyphae. This was accomplished by measuring the relative abundances of mass 26 (12C14N-) and mass 27 (13C14N- and 12C15N-) ions sputtered with a Ga+ probe from cells adhered to an Si contact slide. TOF-SIMS was successfully used to locate and quantify the relative 15N contents of individual hyphae that grew onto Si contact slides in intimate contact with a model organomineral porous matrix composed of kaolin, straw fragments, and freshly deposited manure that was supplemented with 15NO3-. We observed that the 15N content of fungal hyphae grown on the slides was significantly lower in regions where the hyphae were influenced by N-rich manure than in regions influenced by N-deficient straw. This effect occurred over distances of tens to hundreds of microns. Our data illustrate that TOF-SIMS has the potential to locate N-assimilating microorganisms in soil and to quantify the 15N content of cells that have assimilated 15N-labeled mineral N and shows promise as a tool with which to explore the factors controlling microsite heterogeneities in soil.     

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.     

Biodegradation of BTE in soil by indigenous microbial populations with and without biogenic substrates

Degradation of benzene, toluene, and ethylbenzene (BTE) by microbial populations indigenous to the soil and populations proliferated from the indigenous using biogenic substrates were compared. The reaction system consisted of aerobic microcosms representing an unsaturated soil. Microcosms supplemented with glucose and citrate, when compared to the unsupplemented microcosms, showed increases in bacterial counts, but the overall degradation rates for B, T, or E were reduced in spite of shorter lag times. Both biogenic substrate supplements were non-beneficial for BTE degradation due largely to the preferential and healthy growth of the indigenous populations on the biogenic substrates, and thus the urgency of developing a favorable amount of BTE degraders was reduced.     

Lipid imaging by gold cluster time-of-flight secondary ion mass spectrometry: application to Duchenne muscular dystrophy

Imaging with time-of-flight secondary ion mass spectrometry (TOF-SIMS) has expanded very rapidly with the development of gold cluster ion sources (Au(3+)). It is now possible to acquire ion density maps (ion images) on a tissue section without any treatment and with a lateral resolution of few micrometers. In this article, we have taken advantage of this technique to study the degeneration/regeneration process in muscles of a Duchenne muscular dystrophy model mouse. Specific distribution of different lipid classes (fatty acids, triglycerides, phospholipids, tocopherol, coenzyme Q9, and cholesterol) allows us to distinguish three different regions on a mouse leg section: one is destroyed, another is degenerating (oxidative stress and deregulation of the phosphoinositol cycle), and the last one is stable. TOF-SIMS imaging shows the ability to localize directly on a tissue section a great number of lipid compounds that reflect the state of the cellular metabolism.     

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.     

The imaging and quantification of aluminium in the human brain using dynamic secondary ion mass spectrometry (SIMS).

Dynamic secondary ion mass spectrometry (SIMS) has been utilised to study the post-mortem distribution of aluminium in air-dried frozen sections from unfixed, unstained human brain in order to minimise contamination of the tissue and avoid redistribution and extraction of endogenous tissue aluminium. Substrates, sputter-coated with silver, were found to be free of focal aluminum surface contamination and thus minimised substrate induced artefacts in the tissue aluminium ion image. SIMS imaging of aluminium secondary ions at a mass resolution that eliminated the major molecular interferences, combined with a photomontage technique provided a unique strategy for studying aluminium distribution in tissue unrivalled by other spatially resolved microanalytical techniques such as laser microprobe mass spectrometry or X-ray microanalysis. Using this strategy, high densities of focal aluminium accumulations have been demonstrated in the cerebral cortex of the majority of chronic renal dialysis patients studied. In contrast, such aluminium accumulations were absent in control patients. SIMS imaging of aluminium appeared to provide much better discrimination between the dialysis patient group and the control group than one of the most widely used techniques for measuring aluminium in bulk samples, graphite furnace atomic absorption spectrometry. Preliminary studies have shown the feasibility of quantifying focal aluminium SIMS images obtained from brain tissue using aluminium-loaded brain homogenates as reference standards.     

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.     

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.     

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.     

Comparative study of acidic glycosphingolipids by field desorption and secondary ion mass spectrometry

Acidic glycosphingolipids were analyzed by field desorption (FD-MS) and secondary ion mass spectrometry (SI-MS) using the primary ion Xe+ with a glycerol matrix. In the analysis of underivatized gangliosides by FD-MS, the fragment corresponding to the asialo residue resulting from the cationized cluster ion (M + Na)+ was the base peak, and ions due to cleavage at the glycosidic linkages were detected, as in the neutral glycosphingolipids. In the case of sulfatide, the ceramide fragment showed the highest intensity in the spectrum. In SI-MS spectra of acidic glycosphingolipids, (M + Na)+, (M + 2Na-H)+, and (M + K)+ were continuously detected as relatively high intensity ions during analysis of gangliosides and sulfatide. Other ions were mostly similar to those obtained by FD-MS. In FD-MS spectra of permethylated gangliosides, the cationized molecular ion (M + Na)+ was the base peak, and fragment ions due to asialo gangliosides were prominent. Other peaks were hard to detect. In SI-MS, molecular ions (M + H)+ and (M + H-32)+ and other ions due to cleavage of the glycosidic linkages were clearly detected. In this case, the sensitivity was greatly improved. Ions due to the non reducing end sugars were clearly detected, because of the relatively low intensity of ion peaks due to the glycerol matrix. It is concluded that the combination with FD-MS and SI-MS is particularly useful for the determination of molecular weight, sugar sequence and ceramide structure with sample amounting to only a few micrograms order.     

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.