Franklin expedition lead exposure: New insights from high resolution confocal x-ray fluorescence imaging of skeletal microstructure.

In the summer of 1845, under the command of Sir John Franklin, 128 officers and men aboard Royal Navy ships HMS Erebus and HMS Terror sailed into Lancaster Sound and entered the waters of Arctic North America. The goal of this expedition was to complete the discovery of a northwest passage by navigating the uncharted area between Barrow Strait and Simpson Strait. Franklin and his crew spent the first winter at Beechey Island, where three crewmen died and were buried. In September 1846, the ships became stranded in ice off the northwest coast of King William Island, where they remained until April 1848. At that time, the crew, reduced to 105, deserted the ships and retreated south along the island's western and southern shores in a desperate attempt to reach the mainland and via the Back River, to obtain aid at a Hudson's Bay Company Post. Sadly, not one individual survived. Previous analyses of bone, hair, and soft tissue samples from expedition remains found that crewmembers' tissues contained elevated lead (Pb) levels, suggesting that Pb poisoning may have contributed to their demise; however, questions remain regarding the timing and degree of exposure and, ultimately, the extent to which the crewmembers may have been impacted. To address this historical question, we investigated three hypotheses. First, if elevated Pb exposure was experienced by the crew during the expedition, we hypothesized that those sailors who survived longer (King William Island vs. Beechey Island) would exhibit more extensive uptake of Pb in their bones and vice versa. Second, we hypothesized that Pb would be elevated in bone microstructural features forming at or near the time of death compared with older tissue. Finally, if Pb exposure played a significant role in the failure of the expedition we hypothesized that bone samples would exhibit evidence of higher and more sustained uptake of Pb than that of a contemporary comparator naval population from the 19th century. To test these hypotheses, we analyzed bone and dental remains of crew members and compared them against samples derived from the Royal Navy cemetery in Antigua. Synchrotron-based high resolution confocal X-ray fluorescence imaging was employed to visualize Pb distribution within bone and tooth microstructures at the micro scale. The data did not support our first hypothesis as Pb distribution within the samples from the two different sites was similar. Evidence of Pb within skeletal microstructural features formed near the time of death lent support to our second hypothesis but consistent evidence of a marked elevation in Pb levels was lacking. Finally, the comparative analysis with the Antigua samples did not support the hypothesis that the Franklin sailors were exposed to an unusually high level of Pb for the time period. Taken all together our skeletal microstructural results do not support the conclusion that Pb played a pivotal role in the loss of Franklin and his crew.

A new method to image heme-Fe, total Fe, and aggregated protein levels after intracerebral hemorrhage.

An intracerebral hemorrhage (ICH) is a devastating stroke that results in high mortality and significant disability in survivors. Unfortunately, the underlying mechanisms of this injury are not yet fully understood. After the primary (mechanical) trauma, secondary degenerative events contribute to ongoing cell death in the peri-hematoma region. Oxidative stress is thought to be a key reason for this delayed injury, which is likely due to free-Fe-catalyzed free radical reactions. Unfortunately, this is difficult to prove with conventional biochemical assays that fail to differentiate between alterations that occur within the hematoma and peri-hematoma zone. This is a critical limitation, as the hematoma contains tissue severely damaged by the initial hemorrhage and is unsalvageable, whereas the peri-hematoma region is less damaged but at risk from secondary degenerative events. Such events include oxidative stress mediated by free Fe presumed to originate from hemoglobin breakdown. Therefore, minimizing the damage caused by oxidative stress following hemoglobin breakdown and Fe release is a major therapeutic target. However, the extent to which free Fe contributes to the pathogenesis of ICH remains unknown. This investigation used a novel imaging approach that employed resonance Raman spectroscopic mapping of hemoglobin, X-ray fluorescence microscopic mapping of total Fe, and Fourier transform infrared spectroscopic imaging of aggregated protein following ICH in rats. This multimodal spectroscopic approach was used to accurately define the hematoma/peri-hematoma boundary and quantify the Fe concentration and the relative aggregated protein content, as a marker of oxidative stress, within each region. The results revealed total Fe is substantially increased in the hematoma (0.90 µg cm(-2)), and a subtle but significant increase in Fe that is not in the chemical form of hemoglobin is present within the peri-hematoma zone (0.32 µg cm(-2)) within 1 day of ICH, relative to sham animals (0.22 µg cm(-2)). Levels of aggregated protein were significantly increased within both the hematoma (integrated band area 0.10 AU) and peri-hematoma zone (integrated band area 0.10 AU) relative to sham animals (integrated band area 0.056 AU), but no significant difference in aggregated protein content was observed between the hematoma and peri-hematoma zone. This result suggests that the chemical form of Fe and its ability to generate free radicals is likely to be a more critical predictor of tissue damage than the total Fe content of the tissue. Furthermore, this article describes a novel approach to colocalize nonheme Fe and aggregated protein in the peri-hematoma zone following ICH, a significant methodological advancement for the field.

Optimization of analyzer-based imaging systems for minimal surface absorbed dose.

Analyzer-based imaging has improved tissue X-ray imaging beyond what conventional radiography was able to achieve. The extent of the improvement is dependent on the crystal reflection used in the monochromator and analyzer combination, the imaging photon energy, the geometry of the sample and the imaging detector. These many factors determine the ability of the system to distinguish between various bone tissues or soft tissues with a specified statistical certainty between pixels in a counting detector before any image processing. The following discussion will detail changes in the required number of imaging photons and the resulting surface absorbed dose when the imaging variables are altered. The process whereby the optimal imaging parameters to deliver the minimum surface absorbed dose to a sample while obtaining a desired statistical certainty between sample materials for an arbitrary analyzer-based imaging system will be described. Two-component samples consisting of bone and soft tissue are discussed as an imaging test case. The two-component approach will then be generalized for a multiple-component sample.

Soller slit design and characteristics.

For X-ray absorption spectroscopy, either in transmission mode with concentrated samples or for dilute samples in fluorescence mode, it is advantageous to improve the signal-to-noise ratio by implementing a slit apparatus. Several investigations into the improvement of measurements when slits and filters are employed have been reported; however, these have always been for a particular design and are not transferable between dissimilar systems. A generalized approach to Soller slit design will be presented which enables a target level of noise rejection to be achieved by varying the number, size and placement of the filter and Soller slit assembly. A procedure for determining the reduction in efficiency of the Soller slits with respect to misalignment with the sample will also be discussed.

Development of an x-ray prism for analyzer based imaging systems.

Analyzer crystal based imaging techniques such as diffraction enhanced imaging (DEI) and multiple imaging radiography (MIR) utilize the Bragg peak of perfect crystal diffraction to convert angular changes into intensity changes. These x-ray techniques extend the capability of conventional radiography, which derives image contrast from absorption, by providing large intensity changes for small angle changes introduced from the x-ray beam traversing the sample. Objects that have very little absorption contrast may have considerable refraction and ultrasmall angle x-ray scattering contrast improving visualization and extending the utility of x-ray imaging. To improve on the current DEI technique an x-ray prism (XRP) was designed and included in the imaging system. The XRP allows the analyzer crystal to be aligned anywhere on the rocking curve without physically moving the analyzer from the Bragg angle. By using the XRP to set the rocking curve alignment rather than moving the analyzer crystal physically the needed angle sensitivity is changed from submicroradians for direct mechanical movement of the analyzer crystal to tens of milliradians for movement of the XRP angle. However, this improvement in angle positioning comes at the cost of absorption loss in the XRP and depends on the x-ray energy. In addition to using an XRP for crystal alignment it has the potential for scanning quickly through the entire rocking curve. This has the benefit of collecting all the required data for image reconstruction in a single measurement thereby removing some problems with motion artifacts which remain a concern in current DEI/MIR systems especially for living animals.

Development of a combined K-edge subtraction and fluorescence subtraction imaging system for small animals.

A novel combined imaging system for small animals using dilute concentrations of iodine as a contrast agent was developed for wide and pencil photon beam image acquisitions. This combined imaging system used K-edge subtraction (KES) and fluorescence subtraction imaging (FSI) and was tested at the Hard x-ray Microanalysis beamline at the Canadian Light Source. The initial wide beam KES image acquired with a charge-coupled device camera was used to identify regions of interest for further investigation and determine the location and area of the raster scan for pencil beam imaging. The pencil photon beam scanning mode acquired simultaneously KES and FSI measurements with an ionization chamber measuring the KES data and a multielement germanium detector measuring the FSI data. A description of the system is given as well as preliminary results using an iodine test object.