FIB-SEM-based analysis of Borrelia intracellular processing by human macrophages.

Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic disorder affecting primarily skin, joints and nervous system. Successful internalization and intracellular processing of borreliae by immune cells, like macrophages, is decisive for the outcome of a respective infection. Here, we use, for the first time, focused ion beam scanning electron microscopy tomography (FIB-SEM tomography) to visualize the interaction of borreliae with primary human macrophages with high resolution. We report that interaction between macrophages and the elongated and highly motile borreliae can lead to formation of membrane tunnels that extend deeper into the host cytoplasm than the actual phagosome, most probably as a result of partial extrication of captured borreliae. We also show that membrane tubulation at borreliae-containing phagosomes, a process suggested earlier as a mechanism leading to phagosome compaction but hard to visualize in live-cell imaging, is apparently a frequent phenomenon. Finally, we demonstrate that the endoplasmic reticulum (ER) forms multiple STIM1-positive contact sites with both membrane tunnels and phagosome tubulations, confirming the important role of the ER during uptake and intracellular processing of borreliae.

Label-free 3D-CLEM Using Endogenous Tissue Landmarks.

Emerging 3D correlative light and electron microscopy approaches enable studying neuronal structure-function relations at unprecedented depth and precision. However, established protocols for the correlation of light and electron micrographs rely on the introduction of artificial fiducial markers, such as polymer beads or near-infrared brandings, which might obscure or even damage the structure under investigation. Here, we report a general applicable "flat embedding" preparation, enabling high-precision overlay of light and scanning electron micrographs, using exclusively endogenous landmarks in the brain: blood vessels, nuclei, and myelinated axons. Furthermore, we demonstrate feasibility of the workflow by combining in vivo 2-photon microscopy and focused ion beam scanning electron microscopy to dissect the role of astrocytic coverage in the persistence of dendritic spines.