Biology Reference
In-Depth Information
3. Sodium phosphate provides excellent buffering for fixatives at 0.1
M
and is
often used in place of cacodylate. Each buffer system has certain advantages
and limitations. Cacodylate is an organic arsenic compound, dimethylarsenate,
which is poisonous. Whereas it may provide additional TEM contrast in biological
samples, it is a relatively weak buffer at pH 7.2. Alternatively, phosphate requires
fewer safety precautions. However, our laboratory has observed precipitants in
some biological samples, such as avian erythrocytes, that were attributed to the
phosphate buffer.
4. Epoxy resins emit noxious and potentially carcinogenic fumes. Therefore, curing
ovens should be located within a fume hood or designed to be vented away from
occupied spaces as with vacuum ovens.
5. Removal of Thermanox® cover slips can be facilitated by immersing the cover
slip into liquid nitrogen for about 5 s. Prolonged exposure of the resin to liquid
nitrogen should be avoided, as cracks can develop within the block. Similarly,
silicon chips are removed cleanly after pressing the chip onto dry ice for
10-15 s.
6. The quality of sections are affected by many variables including personal ability,
type of microtome, type of knife, flaws in the knife, cutting speed, sample
and block integrity, ambient temperature, air circulation, humidity, and others.
Section thickness settings on ultramicrotomes are approximate values. Typically,
sections are cut between 50-70 nm in thickness. At this thickness, the sections
appear silver to light-gold in color and provide good characteristics of stability,
contrast, and resolution under typical biological imaging conditions of 80-100 kV.
Furthermore, compression artifact during sectioning can be minimized by
orienting the block face such that parallel edges of the block face are aligned
parallel with the knife blade, and that the wider edge advances across the knife
first.
7. In some cases, the SEM substrate can be a TEM grid. More typically, ideal
substrates are flat or relatively featureless or both. Pre-cleaned glass or plastic
cover slips are commonly used for cell layers and cells that can adhere. Use
of pre-cut silicon chips as a substrate is gaining popularity, as these are very
flat and featureless and sufficiently conductive to minimize charge buildup in
samples. In many cases, cells and bacteria adhere to cover slips and chips without
further treatment. If necessary, the substrates can be coated with polypeptides
or other agents such as collagen, fibronectin, serum, or polylysine to promote
adherence.
8. In our laboratory, backscatter detection of quantum dots has been achieved
at accelerating voltages ranging from 0.5 to 10 kV. However, during testing,
backscatter contrast decreased steadily as the accelerating voltage was increased
beyond 2 kV, presumably due to reduced scattering by the nanocrystal or increased
scattering by the substrate or both. If available on the SEM, mixing signals from
the secondary and backscattered electron detectors, or adjusting for detection
of optimum electron energy levels may enhance image quality with respect to
compositional contrast and sample topography.
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