Multi-resolution correlative focused ion beam scanning electron microscopy: Applications to cell biology

Abstract

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.

Calcium signaling at ER membrane contact sites

“…Serial sectioning circumvents this problem but is impractical for MCS quantitation. 3D-EM techniques have recently been developed that progressively remove an ultrathin layer of the specimen with a focused ion beam [38] or by automated serial sectioning with an ultramicrotome. The newly exposed blockface is imaged by scanning-EM (SEM) between each section and a 3D reconstruction can be made with nanometer resolution (Fig.…”

Correlative Light Microscopy and Electron Tomography to Study Von Willebrand Factor Exocytosis from Vascular Endothelial Cells

“…Correlating multiple regions imaged by confocal microscopy with 3D volume SEM can be performed easily, vastly increasing the imaging throughput relative to other CLEM methods. Although it is more challenging to visualize small and delicate structures such as actin, FIB-SEM imaging also provides a good alternative for electron tomography as it can generate large 3D stacks with slices up to 3 nm in thickness (Narayan et al, 2013). For this, grid-engraved MatTek dishes could provide a good navigation tool for both LM and SEM.…”

Recent advances in focused ion beam technology and applications

“…117 Narayan et al recently presented 3D correlative FIB analysis of HIV infected cells where they applied a multi-resolution imaging strategy to capture relevant details (HIV cores) at a very high resolution (3 nm × 3 nm × 3 nm voxel), while contextual information (cellular structures) was captured at lower resolutions. 118 Effective use of correlative techniques has enabled precise localization of VOIs, as demonstrated by Narayan et al 118 and Maco et al 119 Another challenging aspect of 3D analysis is striking the optimal balance between mutually confl icting experimental conditions needed for milling, imaging, and spectral analysis without introducing artifacts or damage. 3D analysis is increasingly being applied to biological and "soft"' polymeric materials.…”

“…This method is obviously not very scalable. One possible approach is multi-resolution analysis similar to that presented by Narayan et al 118 In this case, a priori knowledge of the HIV core locations obtained from fl uorescence imaging was used to specify areas of high resolution imaging. However, borrowing from the remote sensing and machine vision communities, 134 -136 some form of active or adaptive multi-resolution image acquisition strategy could also be devised, enabling real-time determination of required imaging conditions based on the information content of the most recent image frames.…”