Imaging Parameters FAQ

In case you are getting started alone, or through a remote collaborator or organization, this may help.

What things affect imaging the most?
Staining density and nature of the cells/tissue (membranous organelle content) make the greatest differences when imaging. Weak staining requires longer scans and creates charging, and sparse cellular material promotes charging and consequent image drift.  Staining can be fixed by longer incubations, but sparse tissue needs to be handled by imaging conditions.  After that, a combination of dwell time, pixel size, number of pixels in the field, and cut thickness each has an effect and need to be managed.

What is the highest resolution I can get?
That is a common question, and people argue about it a lot.  The highest resolution of the SEM instrument using a standard gold sample, on a well aligned system in a room with minimal vibration, noise and electromagnetic radiation, is probably around 1nm or better with the optimal kV setting and aperture size, using a small field and high magnification.

What is the highest resolution using plastic-embedded biological samples?
That is a better question.  If you have a well-stained sample with dense membranous material, the volume in which the beam electrons interact with that material at each scan location (interaction volume) is thought to be around 4-5 nm and maybe 20-30nm in (z) direction. Explained in more detail below.

What are point size, interaction volume, and resolution?
A scanning EM images the sample one “point” at a time. That point extends beneath the sample surface and is called the “interaction volume”. It is a teardrop-shaped “point”. Backscattered electrons come from the mid-region. Secondary (surface) electrons come from the apex, and x-rays come from the wide base. For biological imaging, the usual kV range is between about 1.0 and 3.0kV.  At around 2kV and the smallest interaction volume is ~4 nm in (x,y) diameter and ~30 nm deep.   This value represents the best that the resolution can be in that image.  More or less*.

Why state magnification in nm/pixel?  What is the magnification?
Two things primarily determine the size of something in an image.  One is the magnification in the SEM column.  The other is the the pixel density in the imaged area.  Nanometers/pixel expresses both of these things.  For example, in one of our systems, at x2.974K magnification, if an of area 80µm x 80µm is captured as an image of 6k x 6k pixels, the pixel size is 13.3nm/pixel.  If it is imaged at same magnification (x2.974K) but with 16K x 16K pixels, the image will have 5 nm pixels.  More or less*.

*More or less?
In real situations, the theoretical/stated values for point size etc do not take beam drift and sample motion and charging effects and surface damage into account.  So calculated resolution more aptly represents the best that the resolution could be.

What do people really mean by the term resolution?
Resolution is generally defined as how far apart two thing can be and still be imaged as having a space between them.  It depends purely on what you can see in a given image, and not theoretical values the instrument may indicate.

Can slower scanning speed provide better images?
Yes – Normally slower scans produce better signal-to-noise ratios and clearer images. The price though is that they take longer. Slow scans also potentially permit more surface damage to the block, which may require that thicker slices be cut to compensate. Paradoxically, slow scans sometimes exhibit less charging artifact than fast scans in difficult material.

Why is it that small changes in pixel size (nm/pixel) can help cutting and charging?

Because images represent areas, there is a square relationship between pixel size and scan time and area.  Changing from 5nm/pixel and 6nm/pixel reduces the area (in µm2) scanned by about 30% but doesn’t change resolution all that much. So highest mag is not usually best.

How thick should I cut?
Generally between about 40nm and 120nm, with 60nm usual. If the cuts are too thin for the amount of beam damage, then the knife skips over the surface and no cut occurs.  Obviously it depends on what you are trying to resolve or follow in 3D.

What effect does kV have on resolution?
The kV is the beam energy and with backscattered electron imaging, it can be thought to determine how deep and wide the electrons penetrate.  kV also determines how much charging occurs – at higher kV more electrons trapped in the sample. It also determines how much damage occurs and how deep both charging and damage extend.

What is charging and what does it look like?
Charging looks like a cloud of blackness (on black on white background images). It can also promote sample drift which introduces zig-zags between lines in the scan.

What does beam damage look like?
A scuffed appearance to the tissue and sometimes cracking of dense regions might be present. It also impedes cutting and so some slice skipping is usually the first problem that you see: playing back the first few slices shows if they are cutting, part cutting, or skipping alternate sections.

Why are there flecks of debris on my sample?
Sample debris from the knife may be attracted to charged areas of the block (e.g. vessels and nuclei) that are sparsely stained.  On one instrument, these can be removed as part of the sample run settings, but at the cost of reducing image acquisition rate.

Can I use”low vacuum” imaging to reduce charging?
Yes you can – by introducing water vapor or N2 into the chamber, the charging effects can be dissipated. Usually there is a penalty for that, which is reduced resolution and slower scans.  How much that matters depends on the project.