Using EDXRF for Elemental Micro-Analysis in Biological Applications

X-ray fluorescence (XRF) is a useful method in characterizing sample element compositions down to parts per million (ppm) levels; however, biologists oftentimes ignore such technique because it traditionally requires destructive vacuum conditions, making bio-samples containing with large water content easily dehydrate under these conditions. The emergence of the groundbreaking XGT-5000, concerns regarding the XRF method could be resolved.

The machine analyzes samples at normal atmospheric pressure in order for qualitative and quantitative data to represent an accurate, healthy sample. This article presents a unique 10 µm spatial resolution of the XGT-5000 coupled with atmospheric pressure analysis to be used in the exploration of leaf structure and behavior. The combination of single-point analysis and mapped imaging would yield more information regarding the growth of leaves and their behavior in the presence of pollutants.

Calcium Modules in Mulberry Leaves

Transmitted x-ray and XRF composite images (Transmission + Ca) acquired from mulberry leaves in different growth stages: (A) young, (B) middle aged, and (C) old.

Figure 1. Transmitted x-ray and XRF composite images (Transmission + Ca) acquired from mulberry leaves in different growth stages: (A) young, (B) middle aged, and (C) old.

Mulberry plants are well-known in many parts of the world, with species native to America and China. This type of plant is commonly utilized as a food source for silk work; however, as its varied and expansive functionality is being recognized in modern times, the mulberry fruit is now being harvested and turned into a bottled fruit juice product, even gaining popularity in many parts of Asia. Apart from this, the mulberry fruit is also used in Chinese herbal medicine. An analysis of mulberry plant leaves shows the possibility of imaging the main leaf structure using transmitted x-rays; meanwhile, the elemental information of XRF could identify the accumulation of calcium-containing nodules.

These nodules, typically in the order of 50-150 µm in diameter, are virtually non-existent in new leaf growth; yet, analysis of older leaves in the mulberry plant illustrates a dramatic increase in their concentration. Plants usually develop and adapt strategies in order to defend themselves from herbivores. Following this argument, it may be possible that mineral incorporation in mulberry leaves were mechanisms automatically developed against plant destruction through silkworm feeding. A micro-analysis of the type utilized in the study may provide useful information in further discussing the nature and structure of calcium accumulation in mulberry plants.

Pollutant Uptake in Plants

The industrial revolution catalyzed the problem of pollution. Along with this heavy challenge are the imposing health risks of heavy metals such as lead and cadmium.

An emerging method to cope with such pollution is phytoremediation, a method wherein plants to naturally absorb and accumulate heavy metals within the soil. With the heavy metals fixed within the plant foliage, the plants are then harvested and disposed of in a suitable manner, leaving the soil clean and safe.

An investigation into this modern-day solution requires fundamental research into the pathways of pollutants within plants. The XGT-5000 is an ideal mechanism that could perform such function. Leaves could be analyzed non-destructively in terms of element concentration and distribution, ultimately allowing uptake pathways to be characterized.

Analysis of leaf prior to introduction of pollutant to root system. Transmitted x-ray (TX), potassium (K) and calcium (Ca) mapped images are shown.

Figure 2. Analysis of leaf prior to introduction of pollutant to root system. Transmitted x-ray (TX), potassium (K) and calcium (Ca) mapped images are shown.

The preliminary results presented in the above figure illustrate how leaves can be quickly and easily analyzed for element distribution; simultaneously, additional information provided by simultaneous transmission x-ray analysis picks out the major leaf veins.

A 1% lead solution was introduced to the root system of a living plant; once lead introduction is successful, uptake leaves were analyzed again using the XGT-5000. The machine showed that the presence of lead within the veins is unambiguous. Based on the success of the experiment, it can be noted that such technique for following heavy metal pathways in plants is feasible.

Analysis of leaf after introduction of pollutant to root system. Transmitted x-ray (TX), potassium (K), calcium (Ca) and lead (Pb) mapped images are shown

Figure 3. Analysis of leaf after introduction of pollutant to root system. Transmitted x-ray (TX), potassium (K), calcium (Ca) and lead (Pb) mapped images are shown.

Spectra acquired from (A) high and (B) low lead concentration regions in the leaf shown in Figure 3.

Figure 4. Spectra acquired from (A) high and (B) low lead concentration regions in the leaf shown in Figure 3.

Spectra presented in Figure 4 were acquired from high and low lead concentration regions within the leaf. Clearly visible in the high concentration spectrum were the lead Lα and Lβ lines. With limits of detection below 0.1% for the heavy metals, depending upon x-ray beam diameter and sample, it would be possible for even trace amounts of pollutants to be analyzed.

Conclusions

The non-destructive nature of micro-XRF analysis with the XGT system makes it possible to apply elemental analysis in biological studies. The capability for large area, high spatial resolution analysis would mean that even whole leaves can be analyzed without the need for a damaging vacuum.

This information has been sourced, reviewed and adapted from materials provided by HORIBA Scientific.

For more information on this source, please visit HORIBA Scientific.

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