Semiquantitative Analysis for High-Speed Mapping Applications of Biological Samples Using LA-ICP-TOFMS.

Laser ablation (LA) in combination with inductively coupled plasma time-of-flight mass spectrometry (ICP-TOFMS) enables monitoring of elements from the entire mass range for every pixel, regardless of the isotopes of interest for a certain application. This provides nontargeted multi-element (bio-)imaging capabilities and the unique possibility to screen for elements that were initially not expected in the sample. Quantification of a large range of elements is limited as the preparation of highly multiplexed calibration standards for bioimaging applications by LA-ICP-(TOF)MS is challenging. In this study, we have developed a workflow for semiquantitative analysis by LA-ICP-TOFMS based on multi-element gelatin micro-droplet standards. The presented approach is intended for the mapping of biological samples due to the requirement of matrix-matched standards for accurate quantification in LA-ICPMS, a prerequisite that is given by the use of gelatin-based standards. A library of response factors was constructed based on 72 elements for the semiquantitative calculations. The presented method was evaluated in two stages: (i) on gelatin samples with known elemental concentrations and (ii) on real-world samples that included prime examples of bioimaging (mouse spleen and tumor tissue). The developed semiquantification approach was based on 10 elements as calibration standards and provided the determination of 136 nuclides of 63 elements, with errors below 25%, and for half of the nuclides, below 10%. A web application for quantification and semiquantification of LA-ICP(-TOF)MS data was developed, and a detailed description is presented to easily allow others to use the presented method.

Quantification anomalies in single pulse LA-ICP-MS analysis associated with laser fluence and beam size.

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has undergone major improvements in recent years which have led to reduction of the analysis time, higher spatial resolution, and better sensitivity. However, quantification and accurate analysis remain one of the bottlenecks in LA-ICP-MS analysis and so far satisfactory calibration solutions are restricted to well-documented matrices and suitable internal standards. Additional uncertainties associated with laser fluence and beam size via various ablation cells and interfaces make quantification even more challenging. This work is focused on the influence of fluence, beam size and aerosol transport on quantification in single pulse LA-ICP-MS analysis via approaches based on pulse intensity, LA spot volumes, noise characteristics, etc. for different elements (As, Gd, La, Ni, Te and Zn), concentrations (between 10 and 1000 µg g-1), and matrices (gelatin standards and NIST SRM 612). The findings indicate that selection of the appropriate laser fluence, just above the ablation threshold, and beam size, depending on the interface of LA and ICP-MS, are critical for reliable quantification and should be properly adjusted to avoid excessive Poisson and Flicker noise, achieve maximum sensitivity, and prevent the formation of double peaks in single pulses.

Biofortification of common buckwheat microgreens and seeds with different forms of selenium and iodine.

BACKGROUND: The biofortification of crops can counteract human diseases, including selenium (Se) and iodine (I) deficiencies in the diet. Little is known about the effects of combinations of Se and I on microgreens and seeds, or on their accumulation in these tissues. The present study aimed to evaluate Se (SeO3 2- , SeO4 2- ) and I (I- , IO3 - ) biofortification of common buckwheat microgreens and seeds with respect to the effects of the addition of Se, I and Se + I on yield and on physiological and biochemical characteristics. RESULTS: In combination treatments, microgreens yield (600-800 g m-2 ) was 50-70% higher than for Se and I alone. The respiratory potential also increased by 60-120%. Fv /Fm was close to 0.8 in all samples. Se content [0.24 µg g-1 dry weight (DW)] was 50% higher for combination treatments than for Se and I alone. I content was highest for IO3 - treatment (216 µg g-1 DW) and decreased in combination treatments with Se by 50%. CONCLUSION: Biofortification of buckwheat microgreens with Se and I should be performed with care because there are synergistic and antagonistic effects of these elements with respect to their accumulation. IO3 - for the biofortification of microgreens should be kept low to prevent exceeding the recommended daily intake of I. © 2019 Society of Chemical Industry.

Iodine determination in animal feed by inductively coupled plasma mass spectrometry - results of a collaborative study.

A collaborative study was conducted in order to fully validate the performance characteristics and to evaluate the suitability of a method for determination of iodine in animal feed. The method consists of an alkaline extraction in tetramethylammonium hydroxide (TMAH) solution followed by the determination of iodine by inductively coupled plasma-mass spectrometry (ICP-MS). The method was validated for different types of feed and feed materials with a broad concentration range of 0.65-622 mg I/kg. Good agreement was found between the overall mean mass fraction values from the collaborative trial (13.8 ± 1.3 mg I/kg and 0.657 ± 0.228 mg I/kg) and the values previously determined in proficiency tests for two of the test materials (12.65 ± 2.47 mg I/kg and 0.72 ± 0.22 mg I/kg) indicating satisfactory accuracy of the method. Reproducibility standard deviations were between 7.85% and 34.65% and the HorRat values were under the acceptable limit of 2 so the between-laboratory precision was considered acceptable. Based on the statistical evaluation of the results it was concluded that the method is suitable for its intended purpose; it has been accepted as European Standard EN17050:2017 by the European Committee for Standardisation (CEN).

Significant Accumulation of Iodine and Selenium in Chicory (Cichorium intybus L. var. foliosum Hegi) Leaves after Foliar Spraying.

The interactions between the uptake of selenium (as selenite and selenate) and iodine (as iodate and iodide) by red chicory (Cichorium intybus L. var. foliosum Hegi) and their effects on selected morphological and physiological characteristics were investigated. Seedlings were transplanted to the field, and at the onset of head formation, the plants were foliar-sprayed with the following solutions: Milli-Q water (control), Se (IV), Se (VI), I (-I), I (V), Se (IV) + I (-I), Se (IV) + I (V), Se (VI) + I (-I) and Se (VI) + I (V). The different treatments had no significant effects on the yield (39.8-51.5 t ha-1) and mass (970-1200 g) of the chicory heads. The selenium content in Se-treated plants was up to 5.5-times greater than the control plants. The iodine content in the chicory leaves enriched with I was 3.5-times greater than the control plants. Iodide or iodate, applied together with selenite in the spray solution, increased the uptake of Se by chicory plants, while both forms of iodine, applied together with selenate, reduced the uptake of Se. Plants treated with I (V) had lower amounts of chlorophyll a and carotenoids than the control, while respiratory potential was higher than the control, which indicated the possible presence of stress in I (V)-treated plants. However, the potential photochemical efficiency of photosystem II was similar and close to the theoretical maximum (0.83) in the control and treated groups, which indicated that all of the plants were in good condition. Furthermore, the plant mass and yield were comparable in the control and treated groups. Molecular studies, like gene expression analysis, would represent a major upgrade of the present study by defining the mechanisms of Se and I uptake and their interactions and by enhancing the knowledge of the Se and I transporters.

Biofortification with selenium and iodine changes morphological properties of Brassica oleracea L. var. gongylodes) and increases their contents in tubers.

Kohlrabi (Brassica oleracea L. var. gongylodes L.) was biofortified with selenium (Se), as selenite and selenate, and iodine (I), as iodide and iodate, and their combinations through foliar spraying, to study absorption of these elements by the plants, separately and in combination. The effects on selected physiological and morphological traits and optical characteristics were monitored. Treatments with Se positively affected total chlorophylls and carotenoids, and leaf stomata dimensions. Addition of I decreased total chlorophylls and increased anthocyanins. In reflectance spectra of the leaves, specific colour regions differed significantly due to the different treatments. Reflectance in the UV correlated positively with Se and I contents of the leaves, which indicated lower demand for production of phenolic compounds. Differences in reflectance in UV part of the spectra could be a consequence of changes in the cuticle. The Se and I levels increased markedly in leaves and tubers, without loss of biomass or yield. Se had antagonistic effects on accumulation of I in leaves. The similar levels of Se and I in the leaves and tubers suggest that the transport of both elements in these plants occurs from the leaves to the tubers through the phloem. According to the Se and I contents in the kohlrabi tubers, biofortification with both elements simultaneously is feasible for human nutrition.

Response of Pumpkin to Different Concentrations and Forms of Selenium and Iodine, and their Combinations.

The elements selenium (Se) and iodine (I) are both crucial for the normal functioning of the thyroid. Biofortification with these elements is particularly feasible in areas where they show a deficit. Iodine and selenium can have positive effects on different plants when applied at the correct concentrations. The effects of their simultaneous addition on plant physiology and biochemistry, as well as on seed germination and sprout biomass, were studied in pumpkin (Cucurbita pepo L. ssp. pepo). To study the effect of Se and I on sprouts, sprouts were grown from seeds soaked in solutions of different forms of Se, I and their combination in the growth chamber experiment. In the field experiment, pumpkins plants were foliarly treated with the same concentrations and forms of Se and I. The combination of Se and I treatments enhanced the germination of the soaked seeds, with no significant differences between Se and I treatments for sprout mass. The yield of pumpkins and seed production were unaffected by Se and I foliar application. The anthocyanin levels and respiratory potential measured via the electron transport system's activity showed different patterns according to treatments and plant parts (sprouts, leaves, seeds). The redistribution of Se and I from seeds to sprouts was significant. The accumulation of Se was higher in sprouts from the seeds treated with Se together with I, compared to sprouts from the seeds treated with Se alone. Interactions between Se and I were also noted in the seeds, which developed in the treated plants.

Is foliar enrichment of pea plants with iodine and selenium appropriate for production of functional food?

Pea (Pisum sativum L.) plants were sown in a field and foliar sprayed at blooming stage with solutions of different forms of iodine (I) - I- and IO3- and selenium (Se) - SeO32- and SeO42-. The possibility of enrichment of pea seeds to nutritionally important levels of both elements and their distribution through the plant parts were studied. To evaluate stress caused by application of I and Se, some morphological, physiological and biochemical characteristics were determined. The results showed elevated concentrations of both elements in all parts of pea plants. In seeds, I content was more than 6-fold higher, while Se content was up to 12-fold higher than in control plants. Although the plants were in good condition, some differences in pod characteristics and electron transport system activity were observed. Glutathione content was not affected by any treatment and only the I- + SeO42- combination decreased the amount of anthocyanins in plants.

Seed soaking in I and Se solutions increases concentrations of both elements and changes morphological and some physiological parameters of pea sprouts.

Pea (Pisum sativum L., cv. 'Petit Provencal') seeds were soaked in solutions of different iodine (I) and selenium (Se) forms (1000 mg I L-1 and 10 mg Se L-1). Iodine and selenium content in different parts of pea sprouts, as well as morphological, biochemical and physiological characteristics were measured in sprouts. The results showed increased concentrations of both elements in sprouts grown from treated seeds. Soaking influenced the biomass and height of the sprouts. Significant differences between plants grown from treated seeds in comparison with control plants were also observed for electron transport system activity and concentrations of tocopherol and glutathione. On the other hand, the content of photosynthetic pigments and anthocyanins remained similar as in control plants. Potential photochemical efficiency of photosystem II was close to theoretical maximum 0.8 in all samples. From the pattern of changes of stress indicators we suppose that plants adapted to the stress earlier in the experiment, i.e. before they were sampled for physiological measurements.