Sylvain Halpern

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 μg/mg of tissue. Despite their limited lateral resolution (0.5 μm) 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 (< 0.1 μm) should open up new fields in biomedical investigation.

Localization of chemical elements and isotopes in the leaf of soybean (Glycine max) by secondary ion mass spectrometry microscopy: critical choice of sample preparation procedure

Secondary ion mass spectrometry (SIMS) is one of the few microscopical methods that potentially can detect and in situ localize the various isotopes of virtually all elements. Recent work with SIMS has demonstrated the possibility of imaging the distribution of various elements in plant cell and tissues. However, in these studies, the elements were incorporated in cell macromolecules or associated with structural polymers, precipitated or immobilized in dry seeds. The localization of mineral ions is of particular significance for the physiology of higher plants owing to their quantitative importance and the impact of their cellular distribution on metabolic regulation. Here we analyse the possibility of mapping different elements (K, Ca, Mg, P, S, 15N and 14N) present as soluble and/or bound forms in highly vacuolated leaf cells. Cryoprocedures to prepare samples for SIMS detection are described and discussed. The quality of the results is assessed at each step of the sample preparation and analysis. Various methodologies are used, including photonic and electronic microscopies, and the agreement of the observed ion distribution with current knowledge of ion compartmentalization in plant cells. The K/Ca emission ratio is proposed as an index of the degree of preservation of the natural ion distribution to critically evaluate the results and identify where artefacts are likely to occur.

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.

Microcomputer system for ion microscopy digital imaging and processing

Analytical ion microscopy is a powerful tool for biological tissue analysis as it allows direct chemical distribution imaging, even at low element concentrations. A microcomputer based digital imaging system achieving acquisition at low light level is presented. It includes a high sensitivity video camera connected to a specialized image processor subsystem. Acquired images consist of 512 times 512 pixels with 8 bits accuracy. Real‐time image processing software has been implemented so that image processing may be performed on‐line. Image processing software allows off‐line image manipulation and correlation for biological interpretation of elemental mapping images. System capabilities are illustrated by a study of stable and radio iodine mapping in rat thyroid tissue.

Changes in iodine mapping in rat thyroid during the course of iodine deficiency: imaging and relative quantitation by analytical ion microscope

The analytical ion microscope (AIM) makes possible imaging and relative quantitation of multiple stable or labeled elements on an even tissue section, according to their mass. The purpose of this work was to follow at the rat thyroid follicle level the changes in 127I mapping during low iodine diet (LID) in relation to the ability of thyroid to pick up radioiodine (129I) and to synthesize Tg from its precursor, 2H‐labeled leucine. The overall picture of images and countings of 127I shows a progressive decrease of the luminal iodine concentration which on day 80 was 10‐fold lower than that of control value. In control rat thyroid cell, concentration was 10‐fold lower than that of follicular lumina and was unchanged until 35 days, but the size of the cytoplasmic compartment increased, suggesting a redistribution of iodine stores between thyroid cells and follicular lumina. 129I was always found in colloid as well as in cells at all stages. After 35 days of LID, cytoplasmic and luminal radioiodine concentrations decreased. In control rats, [2H]leucine was found mainly in the cells. During LID its localization was evidenced progressively in most of the lumina. The most striking fact was the presence up to 35 days of some large residual follicles with high 127I concentration and low 129I and 2H incorporation. These data demonstrate the follicular heterogeneity of thyroid response to progressive chronic TSH stimulation induced by LID.

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.

Image registration and distortion correction in ion microscopy

We present a method whereby the geometric registration of a series of ion microscopic images is performed by applying a two‐step procedure. After applying a global linear transformation that corrects for geometric differences, a non‐linear elastic transform is used in order to match local properties and structures of the images. Transformation parameters are computed on the basis of shape‐specific points in the images. Distortion correction is achieved by relating ion images to the optical image of the same field and by using the two‐step algorithm to register the images. This methodology is evaluated on synthetic misaligned objects and on thyroid tissue images.

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 μm) chemically fixed and resin embedded. Two radioisotopes were tested: 125I and 129I, of high (14 mCi 125I μg−1) and low specific activity (1.07 10−6 mCi 129I μg−1). In animal study, Wistar rats fed a normal iodine diet (10 μg 127I day−1) were injected ip 24 h before sacrifice either with 125I (7 10−3 μg) or with 129I at a dose identical to iodine diet (10 μg) or 3 times higher (30 μg). 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 μg/mg, m ± SE), by 125I or 129I at a dose of 10 μg, but was nearly doubled with 129I at a dose of 30 μg, proof of a pharmacological effect on thyroid iodine regulation. In human study 129I was excluded due to its long half‐life (1.6 107 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 μCi/ml of 125I. The 125I was detectable, its concentration was 1000‐fold higher than that of 127I (1.5 ± 0.004 μg/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). In conclusion, the radioisotope 129I used at a dose of 10 μg is well adapted for SIMS detection in vivo of thyroid exchangeable iodine, but only in animal, while 125I isotope is especially adapted for in vitro study in man.

In vivo drug intracellular localisation by analytical ion microscopy: preliminary study in gastric adenocarcinomas treated with 5-fluorouracil.

THE PRECISE antitumour activity of 5-fluorouracil (5-FLJ) remains uncertain but at least two mechanisms of action are involved causing cell injury: inhibition of thymidylate synthetase and incorporation into RNA [ 11. Although the penetration of this drug into the cells of neoplastic tissue is critical for this activity, the in viva distribution between normal and tumour cells is unknown. Recently, analytical ion microscopy (AIM), the first available method capable of mapping chemical elements in tissue sections [2,3], has been employed to detect the fluorine (F) content of 5 fluor-2’deoxyuridine in cultured cells [4]. The aim of this preliminary study was to localise and quantify the fluorine of 5-FU found in cell nuclei of human biopsies. 11 patients with gastric adenocarcinomas were included. Biopsies of gastric mucosa were obtained during endoscopy before the initiation of treatment (6 patients, group I), 15 min after the beginning of chemotherapy with 5-FU at a dose of 1 g/m2/day (4 patients with oral consent, group II) and 23 days after 5-FU perfusion (2 patients, group III). Fragments first chemically fixed were embedded in methacrylate resin. Semithin sections (1 micron in thickness) were used as histological controls and serial semithin sections (3 microns) were deposited on ultrapure gold holders for ion analysis. Mass resolution (M/AM 2000) was used in order to eliminate interferences between cluster ions and specific ions studied. The elemental analytical images were displayed on a fluorescent screen connected to an image analysis system [5]. The fluorine beam intensity was also measured, using an electron multiplier, on nuclei selected with a special aperture (1.5 micron). The main advantage of AIM is its ability to map fluorine (Fig. 1, lower) in relation to the histological structure which can be

Signal standardization of the secondary ion mass spectrometry (SIMS) microscope for quantification of halogens and calcium in biological applications

The secondary ion mass spectrometry (SIMS) microscope is able to map chemical elements in tissue sections. Although absolute quantification of an element remains difficult, a relative quantitative approach is possible for soft tissue by using carbon (12C) as an internal reference present at large homogeneous and constant concentration in specimen and embedding resin. In this study, this approach is used to standardize the signal of an SIMS microscope for the quantification of halogens (19F—, 35Cl— and 79Br—) and calcium (40Ca+). Standard preparation was determined based on homogeneity and stability criteria by molecular incorporation (halogens) or mixing (calcium) in methacrylate resin. Standard measurements were performed by depth analysis on areas of 8 μm (halogens) and 150 μm (calcium) in diameter for 10–30 min, under Cs+ (halogens) or O2+ (calcium) bombardment. Results obtained from 100–120 measurements for each standard dilution show that the relationship between the signal intensity measured and the elemental concentration (μg/mg of wet tissue or mm) is linear in the range of biological concentrations. This quantitative approach was applied firstly to bromine of the 5‐bromo‐2′‐deoxyuridine (BrdU) used as nuclear marker of rat hepatocytes in proliferation. The second model concerns depletion of calcium concentration in cortical compartment in Paramecium tetraurelia during exocytosis. Then signal standardization in SIMS microscopy allows us to correlate quantitative results with those obtained from other methods.