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)     

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.     

Mapping of intracellular halogenous molecules by low and high resolution SIMS microscopy.

The subcellular distribution of halogenous molecules has been studied by SIMS microscopy in cultured cells of a human breast carcinoma (MCF-7 cell line). Two instruments of microanalysis were used. A low lateral resolution ion microscope (SMI 300 CAMECA) and a prototype scanning ion microscope equipped with a cesium gun that gives high lateral resolution images. This apparatus has been developed by G Slodzian, in Onera Laboratories (Office National d'Etudes et de Recherches Aérospatiales). Molecules studied by low lateral resolution ion microscope were halogenous steroids: fluorometholone, triamcinolone, bromocriptine and bromoandrosterone. Analytical images show that the first two compounds are mainly localized in the nuclear structure of MCF-7 cells whereas the last two molecules are localized in cytoplasm of these cells. Images were obtained with a resolution of 1 micron. With the scanning ion microscope, it is now possible to obtain images at the ultrastructural level. Four analytical images can be simultaneously obtained by a single scan of the imaged area, corresponding to a depth of erosion of the section of ten nm. The intranuclear distributions of three pyrimidine analogs, 5-bromo-2'-deoxyuridine, 5-iodo-2'-deoxyuridine and 5-fluorouracil have been studied in phase S and M of MCF-7 cells and these images have been compared to the distribution of sulfur, nitrogen and phosphorus. All these images have been obtained with a lateral resolution better than 100 nm.     

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.     

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.     

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.     

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.     

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.     

14N and 15N imaging by SIMS microscopy in soybean leaves

The distribution of ¹⁵N and ¹⁴N compounds in cryofixed and resin embedded sections of soybean (Glycine max L) leaves was studied by SIMS microscopy. The results indicate that, with a mass resolution M/ΔM higher than 6000, images of the nitrogen distribution can be obtained from the mapping of the two secondary cluster ions ¹²C¹⁴N^? and ¹²C¹⁵N^?, in samples of both control and ¹⁵N-labeled leaves. The ionic images were clearly related to the histological structure of the organ, and allow the detection of ¹⁴N and ¹⁵N at the subcellular level. Furthermore, relative measurements of the ¹²C¹⁴N^? and ¹²C¹⁵N^? beams made possible the quantification of the ¹⁵N atom% in the various tissues of the leaf.     

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.     

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.     

Anaplastic thyroid cancer: outcomes of trimodal therapy

BackroundThe purpose of this study is to assess the impact of trimodal therapy [surgery, chemotherapy and external beam radiotherapy (EBRT)] in patients with anaplastic thyroid cancer (ATC) treated with curative intent.Materials and methodsRetrospective review of patients with ATC treated at a tertiary referral centre between January 2009 and June 2020. Data were collected regarding demographics, histology, staging, treatment and outcomes.ResultsSeven patients (4 female) were identified. Median age was 58 years (range 52–83 years). All patients received EBRT with concurrent doxorubicin. Six patients received surgery followed by chemoradiotherapy (CRT), and one underwent neoadjuvant CRT followed by surgery. Median radiological tumour size was 50mm (range 40–90 mm). Six patients had gross extrathyroidal extension and three had N1b disease. Prescribed radiotherapy schedules were 46.4 Gy in 29 bidaily fractions (n = 2, treated 2010), 60 Gy in 30 daily fractions (n = 2), 66 Gy in 30 fractions (n = 2) and 70 Gy in 35 fractions (n = 1; patient received neoadjuvant CRT). CRT was discontinued early for two patients due to toxicities. At median follow up of 5.8 months, 42.9% (3/7) patients were alive and disease-free. Only one patient developed a local failure. Three patients died from distant metastases without locoregional recurrence.ConclusionsDespite poor prognosis of ATC, selected patients with operable tumours may achieve high locoregional control rates with trimodal therapy, with possibility of long-term survival in select cases.     

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.     

Determination of the elemental composition of mature wheat grain using a modified secondary ion mass spectrometer (SIMS)

An imaging secondary ion mass spectrometry system has been developed that allows the distribution of elements or ions to be superimposed on an image of the plant cell or tissue generated by ion-induced secondary electrons. This system has been evaluated by analysing the aleurone and sub-aleurone cells of mature wheat grain, showing high spatial resolution (100-200 nm) images of O-, PO(2)-, Mg+, Ca+, Na+ and K+ within the phytate granules of the aleurone, with CN- being diagnostic for proteins and C(2)- being diagnostic for starch in the starchy endosperm cells. This system should provide improved localization of elements in a range of other plant systems.     

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.     

近场扫描光学显微镜及其在生物学领域的应用

评述了近场扫描光学显微镜（near-field scanning optical microscopy,NSOM）的仪器构造、工作原理及其在生物学领域的应用成果。对NSOM目前存在的主要问题进行了讨论,并展望了NSOM在该领域的发展潜力。     

Shedding light on fibered confocal fluorescence microscopy: Applications in biomedical imaging and therapies

Discoveries of major importance in life sciences and preclinical research are linked to the invention of microscopes that enable imaging of cells and their microstructures. Imaging technologies involving in vivo procedures using fluorescent dyes that permit labelling of cells have been developed over the last two decades. Fibered confocal fluorescence microscopy (FCFM) is an imaging technology equipped with fiber‐optic probes to deliver light to organs and tissues of live animals. This enables not only in vivo detection of fluorescent signals and visualization of cells, but also the study of dynamic processes, such cell proliferation, apoptosis and angiogenesis, under physiological and pathological conditions. This will allow the diagnosis of diseased organs and tissues and the evaluation of the efficacy of new therapies in animal models of human diseases. The aim of this report is to shed light on FCFM and its potential medical applications and discusses some factors that compromise the reliability and reproducibility of monitoring biological processes by FCFM. This report also highlights the issues concerning animal experimentation and welfare, and the contributions of FCFM to the 3Rs principals, replacement, reduction and refinement.