High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry

Background: Secondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials. Results: The new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as ^{12}C^{15}N^{-} and ^{13}C^{14}N^{-}, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of ^{14}C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using ^{13}C-oleic acid; to examine nitrogen fixation in bacteria using ^{15}N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using ^{15}N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using ^{15}N-uridine and ^{81}Br of bromodeoxyuridine or ^{14}C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes ^{12}C, ^{16}O, ^{14}N and ^{31}P; and to track a few ^{15}N-labeled donor spleen cells in the lymph nodes of the host mouse. Conclusion: MIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments

Microanalyseur ionique

Here we describe a method of local analysis based on the emission of characteristic ions ejected from a solid sample bombarded by an ion beam of a few keV. It is possible either to get a distribution image of one micron resolution or to perform an analysis in depth with a resolution of 100 Å by a gradual sputtering of the sample. All the elements can be analysed.La méthode d'analyse locale décrite ci-dessous repose sur le phénomène d'émission d'ions caractéristiques arrachés à une cible solide par le bombardement d'un faisceau de particules de quelques kiloélectron-volts d'énergie. On peut soit obtenir directement des images de distribution avec une résolution d'un micron, soit, en pulvérisant graduellement l'objet, l'analyser tranche par tranche avec une résolution en profondeur de l'ordre de 100 Å. Tous les éléments peuvent être analysés

From direct ion images to ion probe scanning

Direct imaging with secondary ions versus scanning with an ion probe have been considered from the transmission and lateral resolution standpoints. Ions emitted from an object point are focused by an immersion lens into an aberration spot. The illumination in the spot (spread function) has been computed, taking into account the energy distribution of secondary ions and the upper limit Φom the lateral initial energy fixed by a material stop in the plane of the crossover. Transmission and spatial resolving limit have been evaluated versus Φ om for a given energy bandwidth. The results are compared to those obtained with an ion probe when the objective lens is working simply as a collecting system. The gains in transmission are evaluated in relation with limitations introduced by the spectrometer according to the mass resolving power being used.L'opposition entre "image ionique directe" et "image par balayage avec une sonde" a été reconsidérée du point de vue de la transmission et de la résolution spatiale. Pour cela, l'éclairement dans la tache d'aberration donnée par l'objectif à immersion d'un objet ponctuel (répon se percusionnelle) a été calculé en tenant compte de la distribution énergétique des ions secondaires et de la valeur maximale Φom de l'énergie latérale fixée par un diaphragme placé au niveau de la pupille. La transmission de l'objectif et sa limite spatiale de résolution ont été évaluées en fonction de Φom et pour une bande d'énergie donnée. Les résultats sont comparés à ceux obtenus avec une sonde lorsque l'objectif est utilisé simplement comme un sytème de collecte des ions. Les gains de transmission ont été évalués en tenant compte des limites imposées à l'étendue du faisceau par le pouvoir séparateur en masse du spectromètre

Achieving high mass resolution with the NanoSIMS 50 while preserving signal transmission from submicron probe impact areas

International audienceElemental and isotopic local analysis using secondary ion emission induced by the impact of a submicron ion probe (SIMS) requires: (i) efficient collection of ejected ions (sensitivity) (ii) high mass resolving power (MRP) to discard interfering species (selectivity). Matching the extracted secondary beam with the acceptance of the spectrometer is then an essential issue. The NanoSIMS 50 fits those requirements fairly well. Nonetheless, a thoughtful study of its mass spectrometer has led to higher performance and suggested further improvements. 12C2D and 12C2H2 with similar peak amplitudes were separated, MRP ≅ 16 800, while preserving half of the maximum signal. MRP of about 9 000, with flat top peaks and a few percent valley between 16OD and 17OH, were obtained while maintaining about 2/3 of maximum signals. Such complete separation should allow D/H ratio measurements via 16OD/16OH with much better sensitivity than that obtained with monoatomic species

Utilisation du microanalyseur ionique en géochimie. Application à la météorite de Juvinas

The ion microanalyzer has been used to obtain images of the distribution of elements on a polished section of the Juvinas achondritic meteorite. This specimen which is mainly composed of silicates has been chosen to study a few characteristics of secondary ion emission in geological materials. In the article is presented a critical examination of some problems inherent to this method when used to obtain quantitative measurements. Discussed are the risks of interferences during the determination of trace-elements, the dectection limit, the mass resolving power and the spatial resolution. The use of this analytical technique in investigating the Juvinas eucrite permits to ascertain previous observations made with the optical microscope, in particular on the nature of micron-size inclusions in the minerals pyroxene and plagioclase. A titanium rich mineral — probably rutile — has been found in some pyroxene crystals. This would be the first occurence of a titanium oxide in this type of meteorite. Thus, a few applications are given of the use of the ion micro-analyzer in the fields of mineralogy and geochemistry.On a utilisé le microanalyseur ionique pour obtenir des images de distribution d'éléments sur une section polie de la météorite achondritique de Juvinas. Cet échantillon composé principalement de silicates a été choisi afin d'étudier quelques caractéristiques de l'émission ionique secondaire dans les matériaux géologiques. On présente un examen critique de certains problèmes soulevés par l'analyse quantitative au moyen de ce type de source, en particulier les risques d'interférences lors du dosage d'éléments en traces, et l'on discute la limite de détection, le pouvoir séparateur en masse et la résolution spatiale. L'application de cette technique d'analyse à l'eucrite de Juvinas permet de préciser des observations antérieures effectuées au microscope optique, notamment sur la nature d'inclusions de l'ordre du micron dans les minéraux pyroxène et plagioclase. Un minéral riche en titane — probablement du rutile — a été trouvé dans certains cristaux de pyroxène. Ce serait la première fois qu'un oxyde de titane est observé dans ce type de météorite. On illustre ainsi quelques possibilités du microanalyseur ionique pour les études de minéralogie et de géochimie.Jérôme Dominique Yves, Slodzian Georges. Utilisation du microanalyseur ionique en géochimie. Application à la météorite de Juvinas. In: Bulletin de la Société française de Minéralogie et de Cristallographie, volume 94, 5-6, 1971. pp. 538-548

Dynamic transfer applied to secondary ion imaging over large scanned fields with the nanoSIMS 50 at high mass resolution

International audienceDynamic transfer is an adaptive optical approach used for coupling a scanning ion probe with the mass spectrometer designed for analyzing sputtered ions emanating from the probe impact. Its tuning is of crucial importance for getting uniform signal collection over large scanning fields and therefore scanning images free of vignetting in a context of high mass resolution. Revisiting the optical design of the NanoSIMS 50 instrument, where the same set of lenses focuses the primary ion probe on the sample and collects secondary ions from the sample, led us to develop novel experimental procedures to achieve dynamic transfer tuning and overcome instrumental imperfections. It is the case for scanning distortion that may be induced by the octopole used for correcting probe astigmatism and may cause irreducible vignetting on scanning images. We show that it is possible to develop complete tuning procedures by compromising temporarily on the sharpness of the probe focus. Most importantly, we show that, in a context of high mass resolution, the transfer does not significantly disturb isotopic ratios over large scanned fields provided external coils are properly adjusted to compensate ambient magnetic fields.Deepening the procedures led us to demonstrate that the scanning center of the probe may not coincide with the imaging center of COOL, Coaxial Objective Lenses forming the probe and extracting secondary ions. We have checked that bringing those two centers into coincidence resulted in a better image quality over large fields.In the present work, we show how to handle the secondary beam in order to position it before it enters the spectrometer. That capability is essential for optimizing transmission at high mass resolution by aligning the secondary beam axis on a given entrance axis of the spectrometer.These results led us to propose several instrumental improvements including the crucial interest of an additional octopole upstream in the primary ion probe column to prevent scanning distortion when performing astigmatism correction and the possibility of offsetting primary beam deviating plates to bring scanning and imaging centers in coincidence

On the origin of organics in ultracarbonaceous Antarctic micrometeorites (UCAMMs), Irradiation of N-rich ices in the outer Solar System

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