Ultrastructural analysis of discrete neurobiological structures by volume scanning electron microscopy (SEM) often constitutes a "needle-in-the-haystack" problem and therefore relies on sophisticated search strategies. The appropriate SEM approach for a given relocation task not only depends on the desired final image quality but also on the complexity and required accuracy of the screening process. Block-face SEM techniques like Focused Ion Beam or serial block-face SEM are "one-shot" imaging runs by nature and, thus, require precise relocation prior to acquisition. In contrast, "multi-shot" approaches conserve the sectioned tissue through the collection of serial sections onto solid support and allow reimaging. These tissue libraries generated by Array Tomography or Automated Tape Collecting Ultramicrotomy can be screened at low resolution to target high resolution SEM. This is particularly useful if a structure of interest is rare or has been predetermined by correlated light microscopy, which can assign molecular, dynamic and functional information to an ultrastructure. As such approaches require bridging mm to nm scales, they rely on tissue trimming at different stages of sample processing. Relocation is facilitated by endogenous or exogenous landmarks that are visible by several imaging modalities, combined with appropriate registration strategies that allow overlaying images of various sources. Here, we discuss the opportunities of using multi-shot serial sectioning SEM approaches, as well as suitable trimming and registration techniques, to slim down the high-resolution imaging volume to the actual structure of interest and hence facilitate ambitious targeted volume SEM projects.      «

Ultrastructural analysis of discrete neurobiological structures by volume scanning electron microscopy (SEM) often constitutes a "needle-in-the-haystack" problem and therefore relies on sophisticated search strategies. The appropriate SEM approach for a given relocation task not only depends on the desired final image quality but also on the complexity and required accuracy of the screening process. Block-face SEM techniques like Focused Ion Beam or serial block-face SEM are "one-shot" imaging r...     »