C. Shan Xu, PhD

Focused ion beam scanning electron microscopy (FIB-SEM) is a technique that has been used in materials science and the semiconductor industry for many decades. It uses a fine focused ion beam (FIB) as a micro sculpting tool to shape the specimen, then images the newly exposed surface with a scanning electron beam. FIB-SEM has been applied to biological imaging since 2006. Biological tissues are typically stained with heavy metals, such as osmium, which binds preferentially to the cell membranes and lipids thus enhancing the electron scattering signal at such locations. After imaging, the focused ion beam, typically comprising 30 keV gallium ions, strafes across the imaged surface and ablates a few nanometers from the top of the sample to expose a new and slightly deeper surface for subsequent imaging. Cycles of milling and imaging gradually erode away the sample while enabling the collection of a stack of consecutive 2D images, usually requiring tens of seconds to a few minutes per cycle. Such a continuous milling/imaging process generates a stack of 3D data with fine isotropic resolution (< 10 nm in x, y, and z) and excellent registration, also avoids many of the defects, such as tears and folds associated with cutting thin sections required for transmission electron microscopy. However, the conventional FIB-SEM platform comes at the cost of slower imaging speeds and lack of long-term system stability, which in turn limits the acquisition volume.

To enable FIB-SEM for large volume high resolution imaging, we redesigned the system architecture and transformed FIB-SEM from a conventional system lacking long term reliability to a robust imaging platform with 100% effective reliability: capable of years of continuous imaging without defects in the final image stack (Xu et al., eLife 2017; US patent 10,600,615; Xu et al., Neuromethods 2020).

Larger Volume

This enhanced FIB-SEM technology (eFIB-SEM) has expanded the maximum imageable volume by orders of magnitude, enabling numerous transformational discoveries in life science, many of which were major new landmarks in their fields. Particularly, the largest connectome to date has been generated (Xu et al., 2020), where the superior z resolution empowers automated tracing of neuronal processes and reduces the time-consuming human proofreading effort. The journey of enhanced FIB-SEM development and Drosophila hemibrain endeavor were featured in Nature & New York Times.

Higher Resolution

Higher resolution further improves the interpretation of otherwise ambiguous details. By trading off against imaging speed, nearly all organelles can be resolved and classified with whole cell imaging at 4 x 4 x 4 nm³ voxels. Additionally, by combining with super-resolution fluorescence imaging, the CLEM applicationsunleash the full potential of intracellular organelle identification with labeling insights. We have established an isotropic 3D reference library of whole cells and tissues (Xu et al., Nature 2021) at the finest isotropic resolution to date, with open access to all datasets in OpenOrganelle (Heinrich et al., Nature 2021). The story was featured in Nature and TheScientist.

Future Developments: invent new technology platforms beyond eFIB-SEM

Despite how much the enhanced FIB-SEM technology has contributed, the 4-nm isotropic resolution falls short of robust visualization of 3D ultrastructure of sub-10 nm features. My lab aims to transcend the SEM resolution limit to date. Such a technology does not currently exist. With success, it will bridge the fields that are currently not connected: structural biology and cell biology, in the context of probing architecture across scales from protein to organelle to cell, within its native tissue environments.

In addition, we contemplate a 3D cryo-FIB-SEM technology that can reliably image a block of vitreously frozen cells or tissues with good contrast and without the need of heavy metal staining, dehydration, and plastic embedding. This streamlined approach will offer the potential to bypass tedious EM sample preparation needed to be individually optimized for large variety of tissues from different species. Most importantly, it allows the volume EM to unveil the fine details of cells and tissues in their native states.