Imaging cellular assemblies with three-dimensional electron microscopy

Division Of Basic Sciences - Nci

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Abstract

Focused ion beam scanning electron microscopy (FIB-SEM), also referred to as ion abrasion scanning electron microscopy (IA-SEM), is a technology that we have been developing in the lab to image cells and tissues in 3D at high resolution. Imaging cells and tissues by FIB-SEM at high resolution offers many exciting possibilities for biological research; however, at high resolution, this technology produces enormous amounts of data, and is extremely slow. Moreover, one of the most promising aspects of this technology is the ability to quantitatively analyze ultrastructural morphology. Thus in addition to using FIB-SEM to study 3D architecture in cells and tissues, we have also been developing imaging methods and techniques that align the technology with the goal of automated, quantitative analysis of 3D structure at electron microscopy resolutions. While developing these techniques, we have also applied the FIB-SEM technology to new biological problems. One exciting area of FIB-SEM analysis has been a collaboration with the Balaban laboratory in NHLBI to visualize the 3D mitochondrial network in muscle cells. During peak contraction, the energy requirements for skeletal muscle can be intense, requiring significant transfer of energy from the area immediately surrounding blood vessels to the interior of the muscle fiber. It has been hypothesized that this energy transfer occurs via transfer of metabolites such as ATP; however, the distances involved indicate that this would require facilitated diffusion, which genetic evidence suggests is not required in muscles except during peak performance. In a 3D study of the mitochondria in skeletal muscle we published recently in the journal Nature, we showed that the mitochondrial reticulum network extends directly and contiguously from the area surrounding the blood vessel deep into the muscle fiber, enabling direct energy transfer via electrical conduction within the mitochondria. While facilitated metabolite diffusion is likely important for peak performance, the electrical conduction through the mitochondrial reticulum, as demonstrated in this study, may provide the majority of skeletal muscle energy requirements. This year, we have begun extending our skeletal muscle study with a similar investigation of mitochondrial networks in cardiac muscle, with the expectation that the comparison will provide interesting new information on mechanisms of electrical conduction. Building upon these advances, we have also initiated a new study, in collaboration with Dr. Luigi Ferrucci at the National Institute of Aging, aimed at understanding the differences in skeletal muscle morphology in humans during aging. While this investigation is still in its early stages, we anticipate that the use of FIB-SEM technology to explore differences in mitochondrial density, network formation, and connectivity at different stages during the aging process will provide new insights.