One key area of interest for the lab has been to close the 3D imaging gap, finding ways to image whole cells and tissues at high resolution. Focused ion beam scanning electron microscopy (FIB-SEM, or otherwise known as ion abrasion scanning electron microscopy, IA-SEM) uses a scanning electron beam to image the face of a fixed, resin-embedded sample, and an ion beam to remove “slices” of the sample, resulting in a sequential stack of high resolution images
Early work from the lab using FIB-SEM included imaging of a number of samples, including melanoma cells and the marine diatom Thalassiosira pseudonana (a biomineralizing organism), showing that this technology can visualize both “soft” (organic) and “hard” (siliceous) components, as well as the 3D architecture of intracellular organelles. More recently we have expanded the capability of this technology, introducing techniques such as keyframe imaging, which allows for selective high resolution imaging of small volumes, with lower resolution imaging of much larger volumes, as well correlative light microscopy with FIB-SEM.
We have used this technology to study the architecture of HIV-1 virological synapses, including those of macrophages, immature and mature dendritic cells, T cells, and astrocytes. A major feature of these synapses is the highly complex three-dimensional interactions between the membranes of neighboring cells. For example, within the synapses formed between CD4+ T cells and HIV-pulsed dendritic cells, filopodial extensions emanating from CD4+ T cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic cell. Synapses between infected and uninfected CD4+ T cells and between infected T cells and astrocytes have suggested that HIV likely hijacks key cellular interactions between immune cells to facilitate the transfer of virions along filopodia or membrane-enclosed extensions from both the infected and uninfected cells. The complexity of these interactions, which can shield virions from immune surveillance and facilitate virus transfer, highlight novel aspects of cell-cell HIV transmission, and suggest new approaches for limiting the spread of HIV/AIDS. A video describing the mechanisms of HIV viral transmission and entry can be seen here.
Because the FIB-SEM the technique can reveal the 3D structures of intracellular objects to high resolution, it is particularly well-suited for studying the architecture of mitochondrial networks. A recent area of interest for us has been applying the FIB-SEM technique to skeletal muscle tissue, revealing a highly organized network of mitochondria extending from the perivascular region deep into the muscle, enabling rapid transfer of energy.
In several collaborative projects, we are continuing to explore the role of architecture in mitochondrial function, and how mitochondrial structure affects tissue function in health and disease.