Brucella abortus is a Gram-negative intracellular pathogen that causes a wide spread zoonotic disease known as Malta fever. During infection, Brucellae enter host cells and reside in membranous compartments called Brucella-containing vacuoles (BCVs). With time, the BCVs acquire endoplasmic reticulum (ER)-like characteristics and become a suitable environment for the replication of bacteria. After several rounds of division, the host cytoplasm becomes completely occupied by growing bacteria. From there, Brucellae manage to escape the host cells and disseminate across the body.
The goal of this project is to obtain a detailed description of the life cycle of Brucella abortus in host cells using a combination of light and electron microscopy (LM/EM).
A correlative technique that combines confocal imaging by LM with focused ion beam/scanning electron microcopy (FIB/SEM) is being employed to obtain the required information. Fluorescent LM allows the localization of interesting events within cells infected with Brucella, such as interaction with specific host markers. Later, these events can be imaged at high resolution by electron microscopy (EM).
FIB/SEM is used to generate 3-dimentional (3-D) volumes of large samples at intermediate resolution (up to 30-50nm). The block face of an embedded sample is imaged using backscattered electron (BSE) detectors in the SEM. To obtain 3-D data, the block face needs to be repeatedly imaged, with the top slice removed between image acquisitions using the FIB.