Over the last few years we have developed a new generation of dual beam electron microscopes capable of site-specific imaging of the interior of cellular and tissue specimens at spatial resolutions over an order of magnitude better than those currently achieved with optical microscopy. The principle of imaging is based on using a focused ion beam to create a cut at a designated site in the specimen, followed by viewing the newly generated surface with a scanning electron beam. Iteration of these two steps several times thus results in the generation of a series of surface maps of the specimen at regularly spaced intervals, which can be converted into a three-dimensional map of the specimen. We have explored the potential of this sequential slice-and-view strategy for site-specific 3D imaging of a variety of eukaryotic cells, and demonstrated that an all-electronic lift-out of plastic-embedded cell and tissue specimens is possible, thus potentially eliminating the need for manual microtome operation for generating thin sections. Our results demonstrate that this technology, which we term Ion Abrasion Scanning Electron Microscopy, is a powerful tool for cellular and sub-cellular imaging in 3D for biomedical and clinical applications. One recent application of this developing technology has been directed towards understanding disease mechanisms in methylmalonic acidemia (MMA), an autosomal recessive inborn error of metabolism caused by an inability of the 5-prime-deoxycobalamin-dependent enzyme methylmalonyl-CoA mutase (MUT) to convert methylmalonyl-CoA to succinyl-CoA. Patients suffer frequent metabolic decompensation, which causes an accumulation of methylmalonic acid in blood and tissues. Multiple organs in the patients are compromised including the liver, kidney, pancreas and basal ganglia. Patients have a shortened lifespan and require liver or kidney transplantation for survival. Previous studies using tissues from Mut knockout mice and the native liver from a patient with methylmalonic acidemia have demonstrated that morphological changes are present in mitochondria in the hepatocytes, proximal tubular renal epithelium and the pancreas, but not in other high energy tissues, such as the heart or skeletal muscle. We have used IASEM to carry out a quantitative analysis of comparative mitochondrial morphologies obtained from the liver of 4 day old, healthy, control and 4 day old, diseased, Mut knockout mice. Under these conditions, hepatocytes in the diseased mice are captured at a stage when structural changes to the mitochondria begin to appear, but before completion of conversion to megamitochondria. The technical focus of this work was to develop and evaluate predictive, quantitative tools to analyze 3D volumetric data in IASEM as they relate to describing subcellular changes in diseased cells and tissues. Using IASEM, we show that mitochondria are more convoluted and have a broader distribution of sizes in the mutant tissue. Compared to normal cells, mitochondria from mutant cells have a larger surface-area-to-volume ratio, which can be attributed to their convoluted shape and not to their elongation or reduced volume. The 3D imaging approach and image analysis described here provides a paradigm for how IASEM imaging could be useful as a diagnostic tool for the evaluation of disease progression in aberrant cells, and as a tool for early stage disease prediction. We have recently carried out several methodological advances that extend the current capabilities of IASEM by describing a detailed approach for carrying out correlative live confocal microscopy and IASEM on the same cells. We have demonstrated that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010278-14
Application #
8348961
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
14
Fiscal Year
2011
Total Cost
$611,814
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Glancy, Brian; Hartnell, Lisa M; Combs, Christian A et al. (2017) Power Grid Protection of the Muscle Mitochondrial Reticulum. Cell Rep 19:487-496
Merk, Alan; Bartesaghi, Alberto; Banerjee, Soojay et al. (2016) Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery. Cell 165:1698-1707
Subramaniam, Sriram; Earl, Lesley A; Falconieri, Veronica et al. (2016) Resolution advances in cryo-EM enable application to drug discovery. Curr Opin Struct Biol 41:194-202
Matthies, Doreen; Dalmas, Olivier; Borgnia, Mario J et al. (2016) Cryo-EM Structures of the Magnesium Channel CorA Reveal Symmetry Break upon Gating. Cell 164:747-56
Glancy, Brian; Hartnell, Lisa M; Malide, Daniela et al. (2015) Mitochondrial reticulum for cellular energy distribution in muscle. Nature 523:617-20
Narayan, Kedar; Subramaniam, Sriram (2015) Focused ion beams in biology. Nat Methods 12:1021-31
Narayan, Kedar; Danielson, Cindy M; Lagarec, Ken et al. (2014) Multi-resolution correlative focused ion beam scanning electron microscopy: applications to cell biology. J Struct Biol 185:278-84
Meyerson, Joel R; Kumar, Janesh; Chittori, Sagar et al. (2014) Structural mechanism of glutamate receptor activation and desensitization. Nature 514:328-34
Schauder, David M; Kuybeda, Oleg; Zhang, Jinjin et al. (2013) Glutamate receptor desensitization is mediated by changes in quaternary structure of the ligand binding domain. Proc Natl Acad Sci U S A 110:5921-6
Wu, Xiongwu; Subramaniam, Sriram; Case, David A et al. (2013) Targeted conformational search with map-restrained self-guided Langevin dynamics: application to flexible fitting into electron microscopic density maps. J Struct Biol 183:429-440

Showing the most recent 10 out of 40 publications