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In situ microanalysis of solid samples is often performed using secondary ion mass spectrometry (SIMS) with a submicron ion probe. The destructive nature of the method makes it mandatory to prevent information loss by using instruments combining efficient collection of secondary ions and a mass spectrometer with parallel detection capabilities. The NanoSIMS meets those requirements with a magnetic spectrometer but its mass selectivity has to be improved for accessing opportunities expected from polyatomic secondary ions. We show here that it is possible to perform D/H ratio measurement images using 12CD-/12CH-, 16OD-/16OH-, or 12C2D-/12C2H- ratios. These polyatomic species allow simultaneous recording of D/H ratios and isotopic compositions of heavier elements like 15N/14N (via 12C15N-/12C14N-) and they provide a powerful tool to select the phase of interest (e.g., mineral versus organics). We present high mass resolution spectra and an example of isotopic imaging where D/H ratios were obtained via the 12C2D-/12C2H- ratio with 12C2D- free from neighboring mass interferences. Using an advanced mass resolution protocol, a "conventional" mass resolving power of 25,000 can be achieved. Those results open many perspectives for isotopic imaging at a fine scale in biology, material science, geochemistry, and cosmochemistry.

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 12C15N- and 13C14N-, 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 14C 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 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes 12C, 16O, 14N and 31P; and to track a few 15N-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.

Unlabelled: Thyroid cancer markedly increased in children exposed to iodine radioisotopes following the Chernobyl accident. This increase exceeded predictions based on dose estimates to the whole organ. We sought to investigate whether iodine deficiency may have influenced the pattern of microscopic distribution of radioiodines, which may be important to interpretation of the observed effects. Iodine-deficient new-born rats were injected with iodine-129 (129I) and the microscopic distribution in the thyroid tissue was studied at 24 hr and at one week after administration, using secondary ion mass spectrometry (SIMS). Twenty-four hr after administration, SIMS images showed large differences in 129I uptake among thyroid follicles, with more than a factor ten variation in the local concentration. In addition, the distribution of 129I inside follicles varied with time. At 24 hr, the highest concentration was found at the periphery of the colloid, close to the thyroid cells. There also was enhanced concentration of 129I at one pole of follicles. Distribution inside follicles was homogeneous at 7 days., Conclusions: 1/Dosimetric models, which assume uniform iodine uptake by thyroid follicles, give an oversimplified picture of radiation dosimetry in cases involving iodine deficiency, which induces patchy tissue irradiation. 2/The dynamic pattern of iodine distribution within thyroid follicles suggests that decay events from short-lived iodines will occur closer to thyroid cells than events resulting from iodine-131.

Low light level fluorescence microscopy studies have been carried out on MCF7-P human mammary tumor cells to localize the intracellular distribution of two new anticancer drugs, Pazelliptine and Intoplicine, which are currently under clinical evaluation. These two molecules are thought to act at the nuclear level, through DNA topoisomerase interactions. Because fluorescence of these compounds appears strongly quenched by intercalation in double strand DNA, secondary ion mass spectrometry (SIMS) imaging was used to check the presence of the drugs in the nuclear compartment. In spite of chemical structure similitudes, pazelliptine and intoplicine appear to be distributed in quite different ways within the cells. Incubation for 1 and 24 hours also allowed us to bring to light strong differences in the distribution kinetics. Pazelliptine quickly enters into the nucleoli but is no longer present in the nucleus after 24 hours incubation. Intoplicine was not detected by fluorescence in the nucleus, however SIMS microscopy allowed us to show its accumulation within this cellular compartment as a function of time of exposure. This study shows the complementarity of fluorescence and SIMS microscopies.

Tris-hydroxymethyl-amino-methane telomers bearing a fluorinated end have recently been proposed as potential drug carriers. Using ion microscopy, we have investigated the cell uptake and subcellular distribution of a perfluorinated telomere, called F-TAC, in two cell lines, malignant murine B16 melanoma and normal rat skin fibroblasts. Single layer cell cultures on gold plates were incubated with F-TAC at different concentrations. Ion microscopy using mass spectrometry enabled the detection of Fluorine 19 atoms entering into F-TAC constitution. This microanalytical study showed an elective cytoplasmic localization of the molecule, wherein the distribution is relatively homogeneous. Within same culture and incubation conditions, intercellular variations in F-TAC content were very low. In the malignant line, the intracellular concentration remains practically identical when increasing F-TAC concentration in the culture medium above 0.2 mg/ml, indicating that the uptake phenomenon is saturable. In conclusion, the F-TAC telomer easily crosses the plasma membrane, however, it has difficulties in crossing the nuclear membrane. It is likely that intracellular penetration is essentially due to rapid endocytosis of the telomer.

The effects of selenium and silver salts was studied by scanning ionic microscopy during experimental argyria mapping of the different basement membrane elements. The ionic microscope (IMS 4F) was equipped with a high resolution spectrometer giving high spatial resolution on the image obtained. After long-term treatment with silver salt alone, silver and sulphur deposits were observed in the membranes. After administration of selenium and silver salt, it was possible to map nitrogen, sulphur, selenium and silver to the glomerular basement membrane as well as to the wall of the kidney arterioles. In the latter, sulphur, selenium and silver were localized only in the elastic laminae of the walls. This process of precipitation of silver deposits in the membrane can be interpreted as process of selenium "detoxification" of the organism.

Due to the presence of fluorine atoms in its molecule, the antimalarial drug mefloquine (MQ) can be easily detected in normal and Plasmodium falciparum infected red blood cells (RBC) by scanning ion microscopy and mass spectrometry. The P falciparum infected RBC exhibited intense distribution of MQ inside the parasite. The main compartments of the parasite which accumulate the drug were the food vacuole and the cytoplasm. The correlation between fluorine (19F-) and phosphorus (31P-) as well as probes for the DNA synthesis (BrdU and IdU) emissions shows that the parasite nucleus is also accessible to the drug. This study demonstrates that SIMS technique on smear preparations is an efficient approach for the direct detection and cartography of fluorinated antimalarial drugs in normal and P falciparum infected RBC, without radioactive labelling.

The intra-cellular distribution of eight halogen glucocorticoids was investigated by ion microscopy in two cellular varieties of cultured non-cancer cells (fibroblast 3T3) and cancer cells (human breast tumor cells MCF-7). Two types of ion microscopy helped to determine this distribution, a direct imaging ion microscope (SMI 300) with low spatial resolution, and a scanning ion microscope (IMS4F), featuring high resolution, serving to obtain maps representing the intra-cellular distribution of the fluorine elements and drugs present in these monolayer cultured cells. The fluorine images representative of the drugs containing fluorine showed that these drugs are essentially concentrated in the cell nuclei. In these nuclei, the distribution of these drugs is different from that of heterochromatin and of the nucleolus.

The secondary ion microscope described here allows to obtain the simultaneous registration of chemical and isotopic distribution maps of several elements composing the sample. The instrument has been specially designed to optimize both sensitivity and selectivity; bombardment with primary Cs+ ions to increase the ionization yields of negative secondary ions, efficient collection of secondary ions at the target surface, matching of the secondary ion beam etendue with the acceptance of the mass spectrometer working at high mass resolution, spectrometer with parallel detection capabilities. The probe diameter can be made as low as 30 nm and ion induced electron images registered at the same time as ion images. Presently, four ion micrographs are obtained simultaneously over a field of view up to 20 x 20 micro m2 containing up to 512 x 512 pixels. Examples are shown with an ion probe diameter of 0.1 microm.