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Unique System Features

  • 4-16nm voxel size achievable over larger volumes than is possible with commercial systems.

  • Acquire a full cell volume in ca. 2 weeks, depending on sample size and resolution.

  • Automated focus and astigmatism correction maximizes image quality throughout the sample volume.

  • Both secondary and backscatter electron detection available.  

  • Increased imaging speed compared to commercial systems.

  • Altered FIB milling angle for better image quality.

  • Novel FIB control maintains consistent sample milling throughout an imaging experiment.

  • Environmental and system reliability monitoring and control for improved long-term imaging.

  • "Gentle" system shut-down and "smart" start-up guard against accidental data loss.

System Specifications

  • The AIC FIB-SEM system is based on a custom 90° mounting of a Zeiss Capella focused ion beam column mounted horizontally onto a Zeiss Merlin scanning electron microscope

  • SEM column: 0.2-4nA beam current, 0.7-1.5kV landing energy. 

  • Imaging readout speed: 200kHz - 10MHz

  • FIB column: Gallium ion source, 15nA/30nA, 30kV

  • Both backscatter and secondary electron detection is available via In-lens and ESB detectors

  • Typical resolution range: 4-16nm

Limitations and Considerations

  • FIB-SEM is not ideal for experiments requiring large sample numbers.  Typically, a full cell volume can be collected in several days to two weeks, depending on the resolution required, and the sample preparation quality.

  • Samples must be stained "en bloc", therefore good stain penetration into cells/tissues is essential.

  • Molecular-specific contrast is limited compared to fluorescence imaging.  Therefore, digital segmentation algorithms are generally necessary to isolate structures of interest.

  • Samples should be pre-imaged in a transmission electron microscope (TEM) prior to FIB-SEM to verify good staining quality.

  • Multiple rounds of optical and/or X-ray imaging are often needed to identify and correctly orient the cell(s) of interest prior to FIB-SEM.

Instrument Summary

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) was originally developed as a materials characterization method that was more recently adapted to imaging biological samples.  It operates via alternating "milling" and "imaging" cycles.  The surface of a sample is imaged using SEM, followed by exposure to a focused ion beam (FIB), consisting of high energy gallium ions.   The FIB grazes the top of the sample and ablates a small layer from the surface.  Another SEM image is then acquired of the newly exposed surface, and the process repeats, often for many thousands of imaging/milling cycles.   In this way, a three-dimensional image is constructed of a sample, as shown below.

An advantage of FIB-SIM lies in its ability to attain isotropic resolution over a relatively large volume - that is, the resolution is equal in both the lateral (xy) and axial (z) directions, over sample sizes of tens to hundreds of microns.  This is in contrast to other 3D electron microscopy methods such as serial sectioning TEM, block face SEM, or tomomgraphic techniques. 

However, commercially available FIB-SEM systems suffer from practical limitations that result in only a small portion of the sample volume being imaged without errors.  Sample and/or instrument drift, FIB milling inconsistency, as well as both planned and unplanned maintenance and shutdowns (such as needed for gallium source replacement) often conspire to limit the amount of error-free data that can be acquired.

The enhanced FIB-SEM system offered at the Advanced Imaging Center has been engineered by to overcome these limitations via a number of improvements.  First, it features environmental and system monitoring and control mechanisms that can (1) alert the user if certain conditions (such as temperature or column pressure) deviate from operational bounds and (2) gently, but swiftly, pause the system until such time conditions return to normal.  Second, a sophisticated feedback control mechanism (shown in the figure below) has been developed for tightly controlling the FIB milling rate.  This is critical to maintain consistent z-spacing between successive SEM images and to ensure smooth re-engagement after any system interruptions.  Third, an automated focus adjustment and astigmatism correction routine has been built into the system to account for both sample and instrument drift over time.  This optimizes image quality throughout the volume acquisition.

The critical factor that users should consider is the time required to image a specified volume at a given resolution.  The graph below indicates the volumes and resolutions where FIB-SEM has traditionally been most used (dark green), and where Janelia's improved FIB-SEM system can extend current capabilities (light green).  As a guide, the table at bottom indicates estimated volume sizes that can be acquired at the specified resolutions over a 2-week imaging period.

Suggested Reading

  1. Xu, C. S., Hayworth, K. J., Lu, Z., Grob, P., Hassan, A. M., Garcia-Cerdan, J. G., ... & Hess, H. F. (2017). Enhanced FIB-SEM systems for large-volume 3D imaging. Elife, 6, e25916.

  2. Wu, Y., Whiteus, C., Xu, C. S., Hayworth, K. J., Weinberg, R. J., Hess, H. F., & De Camilli, P. (2017). Contacts between the endoplasmic reticulum and other membranes in neurons. Proceedings of the National Academy of Sciences, 114(24), E4859-E4867.

  3. Xu, C., & Hess, H. (2011). A closer look at the brain in 3D using FIB-SEM. Microscopy and Microanalysis, 17(S2), 664-665.

  4. Kopek, B. G., Shtengel, G., Xu, C. S., Clayton, D. A., & Hess, H. F. (2012). Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes. Proceedings of the National Academy of Sciences, 109(16), 6136-6141.

  5. Nixon-Abell, J., Obara, C. J., Weigel, A. V., Li, D., Legant, W. R., Xu, C. S., ... & Blackstone, C. (2016). Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER. Science, 354(6311), aaf3928.

  6. Takemura, S. Y., Xu, C. S., Lu, Z., Rivlin, P. K., Parag, T., Olbris, D. J., ... & Weaver, C. (2015). Synaptic circuits and their variations within different columns in the visual system of Drosophila. Proceedings of the National Academy of Sciences, 112(44), 13711-13716.

  7. Shinomiya, K., Huang, G., Lu, Z., Parag, T., Xu, C. S., Aniceto, R., ... & Ogundeyi, O. (2019). Comparisons between the ON-and OFF-edge motion pathways in the Drosophila brain. Elife, 8, e40025.