Serial Block-Face SEM: Automated Sectioning & Image Capture
With the Gatan 3View system you can image your samples in 3D with a remarkably fine depth resolution on an FE-SEM (Field Emission Scanning Electron Microscope).
Using serial block-face scanning electron microscopy (SBFSEM) technology, you can acquire the 3D ultrastructure by automatically sequentially imaging a freshly cut, resin embedded block-face. Unlike the light microscopy 3D techniques, the spatial resolution in the Z direction is comparable to the X,Y resolution and does not degrade with depth.
- Minimal errors and distortion compared to focus ion beam imaging or traditional serial section imaging.
- Collect large format images (32k x 24k)
- Precise stage motion – image multiple regions without losing data.
- 15 nm Z section thickness without the need to unblur multi-kV images.
- Fast imaging at low kV without compromising image quality.
Biological samples should be prepared in a similar way to a microtome protocol optimised for TEM. The sample is then fixed, stained and embedded. This method ensures that the sample’s integrity will be maintained for hours while it’s being imaged.
Set-up and operation is simple. First remove the standard stage from your FE-SEM by venting the chamber. This is then replaced by a 3View specific to that microscope. This ensures stability over long sample runs.
You’re then ready to customise your experiment:
- Cut step size can be 5 – 200 nm to isolate and collect distinct sample features.
- Diamond knife cutting speed can be 0.05 – 5.0 mm/s to prevent artefacts and accommodate different sample types.
- Number of images collected can be hundreds or thousands in a single run. This depends on how much material you want to section through. The maximum sample depth is 600 μm (Z).
Images are acquired from the block-face, rather than the sections. They are captured using a Gatan backscatter detector specifically optimized for high signal collection at low accelerating voltage.
Sample charging is unavoidable, because the sample is embedded in a non-conductive resin or epoxy. The best way to deal with this is with a variable pressure SEM and low accelerating voltage.
Image collection can also be customised:
- Choose an image to collect after each cut
- One or several images per cut
- Capture images at various magnifications
- Create a montage from a series of images