Imaging FAQ Sheet

How does imaging work?
The tissue are deeply stained with heavy metal stains (osmium, uranium, lead) and embedded in plastic (Epon in our lab).  Once the resin hardens, it is trimmed to sample regions of about 0.5 x 0.5 mm. They are imaged in an SEM fitted with an in-chamber ultramicrotome. Unlike traditional biological SEM images, these systems use backscattered electrons to create an image that looks like any conventional transmission EM.  The surface is then cut away by the diamond knife of the ultramicrotome, and the fresh surface is then available for imaging. Cycles of cutting and imaging result in stacks of images, like those from confocal microscopy.

How long does imaging take?
Standard samples takes about 24h to run including set up, though many take 48 hrs and others can be combined to run in less time.  It depends on how many slices (images) you need and how long collecting each one takes.

The sample is completely opaque – how you I know where to image?
Usually people sketch or image the material by LM or eye prior to staining.  Attention is paid to possible fiducials (landmarks), which can be cut into the tissue before staining (eg nick on one corner).  But there are lots of other ways to keep track of your sample orientation.

Should I learn to do my own imaging?
If you are genuinely interested in the technology, and have done confocal microscopy or EM previously, then you should find SBF-SEM remarkably fun to do.  If you hate computers and don’t care much about imaging, then we recommend using core facilities or collaborators, who can save you a lot of anguish.

How big an area is imaged?
Depends on the research question, and is constrained by your imaging time budget.
Typical samples are about 60-100 µm at about 7nm/pixel resolution, and 75nm cuts to produce a stack of 300-500 slices covering up to 40um deep.  However, you can survey the entire 0.5mm block-face but you would only get one or two slices per hour, and end up with hundreds or thousands of ROIs.

Can I image multiple areas? Letterbox formats? Different mags?
All of the above.

What kind of images do I get?
Images can be 2Kx2K pixels up to about 20K x 16K pixels (~200gB each image).  Images come with proprietary naming and some formats: Gatan images have their own DM format, while Thermofisher uses Tiff.  Images saved as DM3 images from a Gatan system can be opened in ImageJ using the BioFormats reader.   We recommend having a batch process to convert them to standard tiff files and scaled versions etc that you can readily make use of.  We also advise that you start off with a standard way of naming folders (notes to be added as a link here) and stick to it