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We have developed standard-free methods for PIXE analysis of hair, nail, urine and serum, and they have widely been applied to studies on human exposure to some toxic elements caused by pollution in many countries. Although these samples are known to be quite suited for evaluating human exposure to toxic elements, there is a case where it is required to quantitatively analyze other samples taken from people. We have examined sweat, saliva, snivel and tear, which are expected to be useful for estimating concentration of various elements in a human body. However, sufficient quantities of them are difficult to be taken from a human body and it is required to establish standard-free methods for these samples. In the present work, standard-free methods for sweat and saliva were established and its accuracy and reproducibility are confirmed. It becomes possible to quantitatively analyze only a drop of sweat and saliva by means of this method. For snivel and tear, potassium concentration is obtained by the internal-standard method for a small-spot sample and the conversion coefficient required for the standard-free method was evaluated. It is found that analyses of these samples give us useful information about some specific elements. As the standard-free methods are free from dryness of samples, which becomes a serious problem in the case where the internal-standard method is applied, it is expected to give more essential information about elemental concentration in a human body.

We developed and reported standard-free methods for various samples such as hair, nail, urine, serum, sweat, snivel, tear and saliva, and they have been confirmed to be effective for addressing global environmental issues. It is expected that analyses of beard samples give us useful information about daily changes of elemental concentration in the body in comparison with the other samples. In this work, a quantitative method for untreated beard analysis based on the standard-free method has been developed. By means of the method, it becomes possible to quantitatively analyze extremely small quantity of beard samples, which are daily taken with ordinary electric shaver. The optimum conditions of washing samples have also been examined. It is found that some elements such as potassium, calcium and chlorine are lost by washing in distilled water and it is better to wash the sample in acetone. As a result, the standard-free method for beard samples has been successfully established and good accuracy and reproducibility were achieved.

We developed and reported a standard-free method for beard (including mustache and whiskers) samples that enables us to quantitatively analyze powdered beard samples of extremely small quantity. In order to investigate intake of essential elements and also exposure to toxic elements, daily changes of elemental concentration in the body give us essential information. Firstly, we have measured dependence of elemental concentration in beard, mustache and whiskers on the collecting regions in the face. Then, daily changes of elemental concentration in the body, which reflect daily ingestion of foods and waters, have been estimated by means of quantitative analyses of beard samples. These beard samples were taken from three persons everyday over successive 7, 14 and 33 days, respectively. As a result, some elements show long-term changes and a few toxic elements such as arsenic and lead exhibit changes in a period of a few days. We have also measured daily changes of elemental concentration in urine and beard taken from the same person at the same time every half day over 10 days, and clear correlation of daily changes of the arsenic concentration between urine and beard samples was observed. It is found that the standard-free method is quite effective for beard samples of very small quantities, and that beard analyses are quite suited to studies on daily changes of elemental concentration in the human body in comparison with the other samples depending on the elements.

A standard-free method for small lymph node and spleen samples taken from rats, and lymph node, esophagus, colon etc. taken from real patients has been developed in order to take measure to meet increasing demands for quantitative analyses of small amount of organs. It becomes possible to quantitatively analyze organ samples of less than 1 mg collected from small experimental animals and also from patients by biopsy, and accuracy and sensitivity of the method were examined by comparing the results with those obtained by a powdered-internal-standard method and a chemical-ashing method. It is found that the method is quite effective for estimating therapeutic effect of CDDP (Cisplatin) since it allows us to quantitatively evaluate uptake of CDDP into organs by analyzing small quantity of samples taken from actual patients by biopsy. It is expected that the method will become a powerful tool for studies not only in fundamental and clinical medicines but also on practical diagnosis and treatment.

We developed and reported standard-free methods for various bio-samples in both invacuum and in-air PIXE and they have been applied to quantitative analyses of traces of bio-samples whose weights are less than 0.1 mg. In this study, we established a method of quantitative analysis for bio-samples of nearly $1\mu \text{g}$ in in-vacuum PIXE. In order to improve sensitivity and accuracy of analysis for smaller samples on the basis of the standard-free method, which makes use of continuous X-rays emitted from the sample, it is essential to design appropriate backing materials for supporting the sample. In the present study, we have examined various backing materials such as thinner and threadlike backings. As a result, it was found that a thread-like backing made of extended adhesive, which contains almost no impurity, is the most suitable for bio-samples of extremely small quantities, since it produces no large amount of continuous X-rays. The method has been applied to quantitative analyses of small insects and plants. Moreover, the method was applied to analyses of small marine organisms such as opossum shrimps and squilla shrimps which play important roles in a food chain in marine ecosystem. The results gave us valuable information about regeneration of marine ecosystem in the Sanriku district attacked by the huge tsunami that occurred on 11 March 2011.

In this paper, methods of quantitative elemental analysis of very small quantity of cultured cells were developed. First of all, an internal-standard method for the solution containing cells whose density is more than 1 × 10⁶ cells/mL was established and then a standard-free method for cultured cells was developed. It was confirmed that the method allows us to quantitatively analyze more than 25 elements in the samples containing only 20,000 cells. Also, the methods for removing cultured cells from a flask were examined in order to improve accuracy and sensitivity of analysis since the use of trypsin and PBS sometimes brings a large amount of sodium, phosphorus and potassium, which have direct effect upon accuracy of analysis based on the standard-free method. It was found that the method of removing cells with a scraper without using trypsin and PBS is the best manner. Also, the effects of using thinner backing materials were examined in order to improve sensitivity of analyses. It is expected that accurate analysis of samples containing nearly two thousand cells is possible on the basis of the standard-free method when using a thinner backing material.

A standard-free method developed by ourselves has been applied to bio-medical samples of less than 1 mg or less than 1 μl, to which an ordinary internal standard method can not be accurately applied. As the result, correct values of potassium concentration in NIST-Bovine liver samples of nearly 0.5 mg were obtained. Furthermore, the standard-free method has been applied to small quantity of serum and whole-blood samples, and satisfactory results were obtained. For samples which easily peel off from a backing film, such as serum and whole blood, a carbon tape with an adhesive agent was also used as a backing film, and it is found that this method is also applicable. Moreover, the standard-free method was confirmed to be effective for a case where a specially designed x-ray absorber is used.