Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomography
Fig 1
Master design and aclar substrate production.
A silicon master was designed and produced in order to pattern aclar films by thermomoulding. a) The master design, including an array of 100 μm sized squares and a reference system for simple localization of cells with various imaging techniques. b) Micropatterned aclar substrates were produced from the master by thermomoulding. The master was heated to 230°C, before a piece of aclar and a glass microscopy slide was placed on top. An even pressure was applied to the assembly, before subsequently cooling down and separating the individual components. After thermomoulding, the microblocks from the master became wells in the aclar substrate. c) Illustration of how primary human macrophages could be cultured in the wells. After M. avium infection and confocal imaging, cells were fixed, stained, dehydrated and embedded in epoxy for FIB/SEM tomography. The aclar substrate was removed after resin polymerization, creating an array of protruding microblocks containing immobilized cells.