Nuclear microscopy of normal and necrotic skeletal muscle fibers: an intracellular elemental microanalysis.

The in situ total elemental composition and elemental concentrations present in mouse soleus (type I) and gastrocnemius (type IIA) muscle fibers were analyzed by using nuclear microscopy (NM). Elemental changes in necrotic fibers, induced by intramuscular injection with snake venom (Pseudechis australis), were also studied 3 h post-injection. Nuclear microscopy is a new method based on nuclear technology that utilizes the interaction between a million-electron-volt nuclear particle beam and the muscle sample (in the case of the present study). Elemental analysis was done at the parts per million (ppm) level of sensitivity on unfixed, rapidly frozen and unstained single fast- and slow-twitch muscle fibers with imaging capabilities of micron spatial resolution and in multi-elemental mode. In total, 12 different intracellular elements were mapped, co-localized and analyzed in single normal and necrotic skeletal muscle fibers from mice. Elements such as potassium, sulfur, phosphorus, chlorine and sodium were found in concentrations from 1000 to 18,000 ppm. Unlike conventional electron-probe X-ray microanalysis, NM also detected and analyzed the trace elements such as magnesium, calcium, iron and zinc that were found in concentrations of 50 to 1000 ppm. Other elements--copper, manganese and rubidium--were also detected in concentrations of less than 50 ppm. The trace elements calcium, iron and zinc were more abundant in the soleus than the gastrocnemius (the level of iron was statistically significant). Calcium, sodium and chlorine were significantly elevated in venom-induced necrotic soleus muscle fibers.

The onset of atherosclerotic lesion formation in hypercholesterolemic rabbits is delayed by iron depletion.

The theory that iron may play a significant role in atherogenesis by promoting the formation of free radicals is controversial. Previous results using the new technique of nuclear microscopy showed a seven-fold increase in iron concentrations within newly formed atherosclerotic lesions in hypercholesterolemic rabbits compared to healthy artery tissue. In a follow-up time sequence study described here, we show that iron accumulation occurs at the onset of lesion formation. In addition, weekly bleeding decreases the iron uptake into the artery wall and delays the onset of atherogenesis. These results provide direct evidence for a key role of iron in initiating atherogenesis.

A nuclear microscopic study of elemental changes in the rat hippocampus after kainate-induced neuronal injury.

The effect of intracerebroventricular kainate injection on the elemental composition of the hippocampus was studied in adult Wistar rats, at 1 day and 1, 2, 3, and 4 weeks postinjection, using a nuclear microscope. An increase in calcium concentration was observed on the injected side from 1 day postinjection. The increase peaked at 3 weeks postinjection, reaching a concentration of 18 times normal. Large numbers of glial cells but no neurons were observed in the lesioned CA fields at this time, suggesting that an increased calcium level was present in glial cells. This was confirmed by high-resolution elemental maps of the lesioned areas, which showed very high intracellular calcium concentrations in almost all glial cells. It is possible that the high intracellular calcium level could activate calcium-dependent enzymes, including calpain II and cytosolic phospholipase A2, shown to be expressed in reactive glial cells after kainate injections. In addition to calcium, an increase in iron content was also observed at the periphery of the glial scar at 4 weeks postinjection. Because free iron could catalyze the formation of free radicals, the late increase in iron content may be related to oxygen radical formation during neurodegeneration.

Nuclear microscopy in the life sciences at the National University of Singapore. A review.

The nuclear microscope is now gaining popularity in the field of life sciences. In particular, the combination of proton-induced X-ray emission to measure the elemental concentrations of inorganic elements, Rutherford backscattering spectrometry to characterize the organic matrix, and scanning transmission ion microscopy to provide information on the density and structure of the sample represents a powerful set of techniques that can be applied simultaneously to the specimen under investigation. These techniques are extremely useful for measuring any imbalances in trace elements in localized regions of biological tissue and, as such, can provide unique information on many diseases. In this article, we describe the nuclear microscope and its related ion-beam techniques, and we review the biomedical work carried out using the nuclear microscope in the National University of Singapore.

Absence of aluminium in neurofibrillary tangles in Alzheimer's disease.

Using the new technique of nuclear microscopy, aluminium is not detected in pyramidal neurons in brain tissue from Alzheimer's disease (AD) patients. The analytical technique of nuclear microscopy can simultaneously image and analyse features in unstained and untreated tissue sections. In tissue which had been previously subjected to conventional procedures such as fixation and osmication, aluminium was observed in both neurons and surrounding tissue. This result shows that the analysis of tissue prepared using conventional chemical techniques may produce contamination or elemental redistribution, and supports our previous investigations which implied that aluminium is not involved in the aetiology of AD. In addition, significant increases in iron, phosphorus and sulphur concentrations were noted between neurons from Alzheimer tissue and neurons from age-matched controls, and between the supporting Alzheimer tissue and supporting control tissue, implying an overall increase in these elements. No significant increase in calcium was observed between neurons from Alzheimer tissue and neurons from age-matched controls.

A review of nuclear microscopy and applications in medicine.

Nuclear Microscopy, the extraction of analytical information from microscopic regions of a sample using a scanning focused high energy ion beam, has been increasing in popularity recently, despite its technical complexity. The three ion beam related techniques Particle Induced X-ray Emission (PIXE), Rutherford Backscattering Spectrometry (RBS) and Scanning Transmission Ion Microscopy (STIM) can be carried out simultaneously at sub-micron spatial resolutions, and provide structural and quantitative elemental analysis down to the parts per million levels of analytical sensitivity. These techniques are extremely useful for measuring any imbalances in trace elements, including metal ions, in localised regions of biological tissue, and as such can provide unique information on many diseases. In this paper we briefly describe the nuclear microscope and its related ion beam techniques, and briefly review recent work carried out using the nuclear microscope into the degenerative diseases Alzheimer's disease, Parkinson's disease and atherosclerosis.