Efficient correlative imaging of small targets within large fields is a
central problem in cell biology. Here, we demonstrate a series of technical
advances in focused ion beam scanning electron microscopy (FIB-SEM) to address
this issue. We increase the speed, robustness and automation of the process, and
achieve consistent z slice thickness of ~3 nm. We introduce “keyframe
imaging” to simultaneously image large fields of view and obtain
high-resolution 3D images of targeted sub-volumes. We apply these advances to
image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently
labeled cell membranes, proteins and HIV cores, we first produce a
“target map” of an HIV infected cell by fluorescence microscopy.
We then generate a correlated 3D EM volume of the entire cell as well as
high-resolution 3D images of individual HIV cores, achieving correlative imaging
across a volume scale of 109 in a single automated experimental
run.
A key feature in the understanding of the mechanisms of integration versus rejection of implanted materials is a deepened understanding of the elemental and molecular compositions of the interface zone between the surface of the synthetic man-made material and the biological components of tissue. Intact interfaces between metallic implants and tissues have not been able to image and analyse on the ultrastructural level with the common transmission electron microscopy (TEM) sample preparation techniques. By using focused ion beam microscopy for site-specific preparation of TEM samples, intact interfaces between metal implants and calcified tissue were imaged for the first time. The interface's elemental and crystallographic compositions were determined using energy dispersive X-ray mapping and electron diffraction. The developed technique fulfills a long-sought-for demand to correlate the surface properties of implanted metal prostheses with the fine structure and composition of preserved interfaces with tissues.
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