SIMS microscopy: a tool to measure the intracellular concentration of carbon 14-labelled molecules.

Monolayer cultures of human fibroblasts were incubated for 24 h with 14C-arginine and observed by means of SIMS microscopy (ion microscopy). Carbon 14 imaging showed the intracellular distribution of labelled arginine which featured high nuclear incorporation. The local concentration of this amino acid in different cells and intracellular structures was assessed through local isotopic 14C/12C ratio measurement. This relates the signal intensity of the labelling isotope carbon 14 to that of the corresponding natural isotope (carbon 12) of known tissular concentration. Using this method we were able to measure minor variations in the molecular concentration of arginine (expressed as mumol/g of tissue) between different fibroblasts. Results of this study indicate that SIMS microscopy is well adapted to carbon 14 detection and can provide quantitative maps of the cellular and subcellular distribution of 14C-labelled molecules.     

Mapping the cellular distribution of labelled molecules by SIMS microscopy.

We took advantage of one of the main possibilities of ion microscopy, ie isotopic analysis, to study the cellular distribution of molecules labelled either with carbon 14 or with stable isotopes of low natural abundance such as nitrogen 15 and deuterium. The surface of the sample is bombarded with an ion beam (O2+, Cs+ etc). Secondary ions emitted from the sample are filtered by a mass spectrometer and the distribution of the labelling isotope is recorded. In this way, we obtained images showing the characteristic distribution of 14C-thymidine and D-arginine in human fibroblasts, and of 15N-adenine in organotypic cultures of human breast cancer cells. The spatial resolution on the acquired images was close to 0.1 micron when using the UPS-ONERA ion microprobe. The sensitivity of the method for detecting carbon 14 is far greater than that of autoradiography and the technique is both fast and quantitative. On the other hand, the capacity of ion microscopy for studying the tissular distribution of molecules labelled with stable isotopes, opens the way for biological and pharmacological tracer studies of human diseases.     

SIMS microscopy in the biomedical field.

We attempted to indicate the requirements for biomedical applications of SIMS microscopy. Sample preparation methodology should preserve both the structural and the chemical integrity of the tissue. Furthermore, it is often necessary to correlate ionic and light microscope images. This implies a common methodological approach to sample preparation for both microscopes. The use of low or high mass resolution depends on the elements studied and their concentrations. To improve the acquisition and processing of images, digital imaging systems have to be designed and require both ionic and optical image superimposition. However, the images do not accurately reflect element concentration; a relative quantitative approach is possible by measuring secondary ion beam intensity. Using an internal reference element (carbon) and standard curves the results are expressed in micrograms/mg of tissue. Despite their limited lateral resolution (0.5 microns) the actual SIMS microscopes are very suitable for the resolution of biomedical problems posed by action modes and drug localization in human pathology. SIMS microscopy should provide a new tool for metabolic radiotherapy by facilitating dose evaluation. The advent of high lateral resolution SIMS imaging (less than 0.1 microns) should open up new fields in biomedical investigation.     

Sample preparation of animal tissues and cell cultures for secondary ion mass spectrometry (SIMS) microscopy.

Sample preparation is a critical step in the elemental analysis of animal tissues and cell cultures with ion microscopy. Since live cells cannot be analyzed with ion microscopy, a careful sample fixation is necessary which preserves the native structural and chemical integrity of a specimen. The evaluation of morphological and chemical integrity of a fixed specimen is necessary before any physiological explanation of ion fluxes is interpreted based on ion microscopy. For diffusible ion localization studies, strict cryogenic procedures are recommended. Examples are shown for diffusible ion microanalysis in frozen-freeze-dried tissues and cell cultures. Ion microscopy studies of tightly bound elements/molecules may be conducted in chemically fixed and/or plastic embedded specimens. Since it is not generally known which elements/molecules are tightly bound to the tissue matrix, a confirmation of elemental distribution with cryogenic procedures is desirable. A recent approach of combining laser scanning confocal fluorescence microscopy and ion microscopy on the same frozen freeze-dried cell is also discussed for recognizing smaller cytoplasmic structures in ion microscopy images.     

Subcellular localization of two neurotropic drugs in three varieties of central nervous system cells by secondary ion mass spectroscopy (SIMS) microscopy.

The intracellular localization of two neurotropic drugs, flunitrazepam (benzodiazepine) and triflupromazine (phenothiazine), was studied by secondary ion mass spectrometry microscopy (SIMS) in three varieties of cells. The images of the intracellular distributions of the two drugs are easily obtained by selecting the fluorine atom of the molecules. These images show that the drug from the benzodiazepine group is mainly located in the nuclei, whereas the phenothiazine is exclusively located inside the cytoplasm.     

Detection on liver tissue sections of S-phase markers in synchronized cycling rat hepatocytes by SIMS microscopy

Summry— The aim of this study was to localise two ionic S-phase markers in tissue sections using SIMS microscopy: aluminium as a potential endogenous marker and bromine as an exogenous marker after in vivo injection of bromodeoxyuridine (BrdU). This study was performed in an experimental model of hyperplastic proliferation after partial hepatectomy in rat. Aluminium was never detected in nuclei which were positive or negative for tritiated thymidine uptake, as determined by autoradiography in tissue prepared by cryotechniques. In contrast, bromine of BrdU was found in hepatocyte nuclei. However, there was a discrepancy between SIMS bromine images and BrdU immunohistochemistry detection which appears more sensitive. This is probably due to problems of stereology intrinsic to the correlation method which requires serial sections for this multi-instrumental approach.     

Significance of SIMS microscopy for the radioiodine detection in animal and human thyroid tissue.

We defined the SIMS conditions for radioiodine detection in animal and man thyroid follicles, in tissue sections (3 microns) chemically fixed and resin embedded. Two radioisotopes were tested: 125I and 129I, of high (14 mCi 125I micrograms-1) and low specific activity (1.07 10(-6) mCi 129I micrograms-1). In animal study, Wistar rats fed a normal iodine diet (10 micrograms 127I day-1) were injected ip 24 h before sacrifice either with 125I (7 10(-3) micrograms) or with 129I at a dose identical to iodine diet (10 micrograms) or 3 times higher (30 micrograms). No SIMS signal of 125I was obtained in vivo due to its too low concentration, while radioiodine distribution was evidenced with both doses of 129I. Local concentration of previously stored 127I in follicular lumen was not modified, when compared to control (4.14 +/- 0.03 micrograms/mg, m +/- SE), by 125I or 129I at a dose of 10 micrograms, but was nearly doubled with 129I at a dose of 30 micrograms, proof of a pharmacological effect on thyroid iodine regulation. In human study 129I was excluded due to its long half-life (1.6 10(7) years), and 125I was tested only in vitro on two surgical specimens of normal perinodular thyroid tissue maintained in mini-organ culture for 48 h in presence of 100 microCi/ml of 125I. The 125I was detectable, its concentration was 1,000-fold higher than that of 127I (1.5 +/- 0.004 micrograms/mg). For both in vivo and in vitro studies, a positive correlation exists between newly organified radioiodine (125I or 129I) and previously stored iodine (127I).(ABSTRACT TRUNCATED AT 250 WORDS)     

Preservation of the diffusible cations for SIMS microscopy. I. A problem related to the state of water in the cell.

The notion of diffusible ions is reviewed in the light of recent knowledge on the stage of water in biological matrices. It appears that ion distributions would be little affected as long as water-macromolecular equilibrium is maintained, but they risk to be significantly modified during dehydration, because the transformation of bound water into highly solvating free water can produce ion displacements. In addition, differently hydrated areas may undergo unequal volume variations. The principal modes of preparing material for SIMS (secondary ion mass spectrometry) microscopy are envisaged from this viewpoint.     

Cytogenetic applications of high resolution secondary ion imaging microanalysis: detection and mapping of tracer isotopes in human chromosomes.

Analytical imaging by secondary ion mass spectrometry (SIMS) using a state-of-the-art scanning ion microprobe enables the detection and mapping of tracer isotopes in human metaphase chromosomes. The stimulated mitosis of cells cultured in media containing labelled nucleosides, typically 14C-labelled thymidine or adenosine, and BrDU, yields chromosomes that have incorporated the labelled molecule in their constituent DNA. The label is subsequently detected and localized by SIMS imaging. The relative label signal intensities of sister chromatids can be quantified. The occurrence of sister chromatid exchanges (SCE) can be detected. The distribution of specific nucleosides can be directly mapped. This is non-uniform along the chromatids, giving rise to characteristic banding patterns (SIMS bands) that seem to correspond to the well known G- or Q-bands resulting from conventional staining methods. The study of a number of cytogenetic problems is expected to benefit from the use of this new method of approach, similar in principle, but potentially more sensitive and capable of higher spatial resolution than autoradiography.     

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.     

Confocal microscopy as a tool to reveal the tridimensional organization of intracellular lumens and intercellular cysts in a human colon adenocarcinoma cell line

Adenocarcinoma cells often form intracellular lumens and intercellular cysts. In order to study the structural relationships between these lumens and the apical domain of normal enterocytes, we have applied electron microscopy and confocal microscopy to a cloned cell line derived from the human colon adenocarcinoma cell line LoVo which express a high number of intracellular lumens and intercellular cysts. Microvilli reminiscent of those detected in the brush border of small intestinal cells are formed in the two types of compartments. By immunofluorescence, we found that a 135 kDa membrane glycoprotein characterized by a monoclonal Ab and normally associated with the brush-border of enterocytes is expressed at the surface of the intracellular lumens and intercellular cysts present in the adenocarcinoma cells. Comparison of fluorescence and reflection contrast micrographs obtained by confocal microscopy demonstrate the presence of spherical intracellular lumens in the juxtanuclear region of single cells, and of more complex shaped intercellular cysts located within clusters of cells. The later cells form junctional complexes limiting an apical plasma membrane domain in contact with the intercellular cyst. It is suggested that the intracellular lumens may represent the abortive form of an apical plasma membrane due to the lack of components required to establish epithelial cell contacts. As opposed to conventional fluorescence microscopy, confocal microscopy allows rapid inspection of the tridimensional organization of intracellular lumens and intercellular cysts even when they are located in cell multilayers.     

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.     

The effect of primycin on the intracellular monovalent ion and water contents of rat hepatocytes as revealed by energy dispersive X-ray microanalysis and interference microscopy

Using energy-dispersive X-ray microanalytic and interference microscopic techniques, the intracellular concentration of the monovalent ions (Na+, K+, Cl+) as well as the intracytoplasmic and intracellular water contents were studied in normal and adrenalectomized rat hepatocytes with and without primycin treatment. Although primycin influenced significantly only the intracellular potassium content of the adrenalectomized group, it exerted a marked influence on the intranuclear water content in both the normal and adrenalectomized rats. The intranuclear water content increased significantly in the primycin-treated animals. The conclusion is drawn that the increased level of hydration of the nuclear substances reflects a 'decondensation' of the chromatin which on the other hand, may represent the basis for the various effects of primycin on the induction of certain hepatic enzymes.     

Detection and cartography of the fluorinated antimalarial drug mefloquine in normal and Plasmodium falciparum infected red blood cells by scanning ion microscopy and mass spectrometry

Summary— 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.     

Imaging of plant microtubules with high resolution scanning electron microscopy

Summary High resolution scanning electron microscopy was used to obtain images of cortical microtubules and associated structures in onion root tips. Specimens were prepared using a modified quick-freeze deep-etch technique utilising cytosolic extraction with saponin and conductive staining with osmium.Abbreviations DMSO dimethylsulfoxide - HRSEM high resolution scanning electron microscope/microscopy - MTSB microtubule stabilising buffer - TEM transmission electron microscope/microscopy     

Microanalysis and image processing of stable and radioactive elements in ecotoxicology. Current developments using SIMS microscope and electron microprobe

Ecotoxicological investigations were performed on two sets of biological models. The first one concerns marine pollution and was composed of invertebrates (molluscs and crustaceans) contaminated by stable or radioactive elements originating from wastes discharged into sea water. The second one concerns freshwater pollution and was composed of vertebrates (fish) contaminated by aluminium which was dissolved in rivers, as a consequence of an atmospheric pollution by acid rain. Mechanisms involved in the uptake, storage and elimination processes of these toxicants were studied, with a special emphasis on cellular and subcellular aspects of concentration sites. Two microanalytical methods were employed: secondary ion mass spectrometry (SIMS), using the ion microscope and the ion microprobe, and X-ray spectrometry using the electron microprobe (EMP). SIMS, which enables the visualization of trace elements, was associated with an image processing system using a highly sensitive television camera connected to an image computer. Polychromatic images were obtained, allowing to establish the cellular distribution of metal contaminants. In marine organisms, the target organs and tissues of Al, rare earth elements (Tm and La) and radionuclides (U, Pu, Am) were shown to be mainly digestive gland and exoskeleton. The target organelles were shown to be spherocrystals and lysosomes where the enzymatic lysosomal coprecipitation with phosphorus was observed. Amoebocytes, which are enzymatically equipped with lysosomal phosphatase, were involved in the phagocytic clearance of metal pollutants. In trout, two processes appeared to be involved in Al accumulation. The first one corresponds to the well known insolubilisation of Al phosphate, within lysosomes of organs devoted to uptake and excretion such as gill and kidney. The second one demonstrates that organs and tissues which cannot eliminate, such as bone, heart and brain, retain Al, exhibiting a high intracellular metal concentration; moreover, large Al deposits inducing nervous tissue destruction have been observed. Data have been discussed in connection with the relationship between man and his environment.     

Lateral connections between heart muscle cells as revealed by conventional and high voltage transmission electron microscopy

Summary The lateral surfaces of heart muscle cells are interconnected by a varied and extensive network of structures that exist in addition to intercalated discs. Ultrastructural images of this network are vastly improved over those from epoxy-embedded material, particularly for low density components, through the application of a method for removing the embedding matrix from thin or thick sections that are then stereoscopically analyzed with standard or high voltage transmission electron microscopy. The connections include cables, 3–20 nm in diameter, multi-strand cables, 10–40 nm-granules, meshlike mats, and sheets, all extensively interwoven. It is suggested that intercellular connections of varying strength and distribution aid in the integration of mechanical performance of the large population of myocytes during the contractile cycle of the heart.This study was supported by a grant from NIH Biotechnology Resources through the University of Colorado High Voltage E.M. Laboratory, NIH Research Grant HL 24336, a N.Y. Heart Association Grant-in-Aid, and NIH Research Career Development Award HL 00568I thank Dr. E.H. Sonnenblick for continual aid and encouragement and Dr. R. Terry, Ms. Y. Kress, and Ms. J. Fant for use of facilities. I also thank Dr. K.R. Porter for guidance in the use of the HVEM technique, Dr. J.J. Wolosewick and Dr. M. Fotino for valuable suggestions, and Ms. J. Fleming, Mr. G. Wray, and Mr. G. Charlie of HVEM staff at Boulder. I acknowledge Dr. F. Pepe for use of facilities, Dr. R. Bloodgood for comments, and Mrs. L. Cohen-Gould, Ms. T. Downey, Mr. F. Reingold, Mrs. T. Maio, and Mrs. R. Shamoon for excellent assistance     

Imaging of subcellular structures by scanning ion microscopy and mass spectrometry. Advantage of cryofixation and freeze substitution procedure over chemical preparation

With the IMS 4F, a scanning ion microscope and mass spectrometer (SIMS), it is possible to map chemical elements with a lateral resolution of about 250 nm over a field of view of 50 × 50 μm². Such conditions should enable the imaging of subcellular structures with constitutive ionic species such as CN^?, P^?, S^?. The study was performed on heart and renal tissues prepared either by chemical procedure or cryofixation-freeze substitution (CF-FS) prior to embedding. Heart tissue was chosen because cardiocytes display a simple structural organization whereas the structural organization of kidney tubular cells is more complex. Whatever the preparation procedure, nuclei were easily identified due to their high P^? content. The CN^?, P^?, and S^? ion images obtained on heart and renal tissues prepared by chemical procedure showed weak contrasts inside the cytoplasm so that it was difficult to recognize the organelles. After CF-FS, enhanced contrasted images allow organelle (mitochondria, myofibrils, lysosomes, vacuoles, basal lamina, etc) characterization. This work demonstrated that CF-FS is a more suitable preparation procedure than chemical method to reveal organelle structures by their chemical composition. The improvements in the imaging of these structures is an essential step to establish the correlation between the localization of a trace element (or a molecule tagged with isotopes or particular atoms) and its subcellular targets.     

Correlative light and electron microscopy (CLEM) as a tool to visualize microinjected molecules and their eukaryotic sub-cellular targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited. On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM), affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy. This powerful technique has been implemented to study many aspects of cell biology. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from ≤5 s up to several hours, depending on the nature of the microinjected sample.     

Intracellular boron localization and uptake in cell cultures using imaging secondary ion mass spectrometry (ion microscopy) for neutron capture therapy for cancer.

Quantitative ion microscopy of freeze-fractured, freeze-dried cultured cells is a technique for single cell and subcellular elemental analysis. This review describes the technique and its usefulness in determining the uptake and subcellular distribution of the boron from boron neutron capture therapy drugs.