Section Mounting
Specimen Grids
•3 mm support for TEM specimens. (a few are 2.6mm)
•Different materials..usually copper...also nickel, gold, aluminum,
platinum, stainless steel, beryllium, carbon, nylon.
•Most are manufactured individually by electroplating; some are
punched from screen stock; a few are woven.
•Also differ by mesh size (bars per inch) 0 - 1000m
•The smaller the mesh size, the greater the support (section
drifting, splitting), but the less open area for viewing.
Section Mounting
•A 200m grid has 60% open
area; a 400m grid only 40%
•Thin-bar grids...more fragile,
more expensive.
•Ultrathin sections can be
supported on a bare grid of no
greater than 200m.
•Commonly used TEM grid
types:
Picking up sections
Slot grids
Mesh grids
Slot grids: typically come up from
bottom.
Mesh grids: either from under or
top (naked). If coated, from under.
Collecting on slot grids
Sections floating
on water
Dried on bridge, then
punched out for viewing
Section Mounting
An ultrathin section on a 50m support filmed grid at 200X mag.
Support Films
Formvar, Carbon, Collodion
-Used when sections or samples are smaller than
support of grid.
-100 mesh or less, slot grids
-Fragile or very thin sections
Avoid when possible because:
Usually has holes or uneven thickness
Added thickness affects clarity and contrast
Formvar Coating
Formvar coated grids
Holey formvar
Formvar and carbon
Contrast
Light Microscopy
•Contrast achieved by:
•Use of special optics and filters which impart
selective colors or brightness to areas differing in
thickness or composition. E.g. - phase contrast,
D.I.C. optics.
Phase contrast
Contrast achieved by:
•Selective staining
•Chromatic stains selectively bind to specific components in
the specimen. E.g. - Hematoxylin/Eosin
Contrast
Transmission Electron Microscopy:
•Contrast is produced by the adsorption of heavy metals
to specimen macromolecules.
•The ability of an atom to absorb electrons is directly
related to its mass.
•Since biological specimens are composed mostly of low
atomic # elements (C,O,H,N), they lack endogenous
contrast....thus contrast is induced by "staining" with
heavy metals.
•Microscopists refer to the measure of a specimen's
ability to absorb electrons as its electron density (vs
electron “transparency”).
Contrast
Transmission Electron Microscopy:
•Heavy metals commonly used for contrasting in TEM: uranium,
lead, osmium, ruthenium, molybdenum, gold, silver.
•It is the differential adsorption of various heavy metals to tissue
components that produces the electron image of biological thinsectioned materials.
•The image may be composed of areas ranging from completely
black to completely white with all ranges of grey in between.
•Images with mostly pure blacks and whites are "contrasty"
images, while those containing mainly greys are "flat” images.
Post-Staining
•Typically always used, even if en bloc staining (ie uranyl acetate)
has been done.
•Uranyl acetate - 0.5 - 2% aqueous. Also can use saturated
ethanolic or methanolic UA
•Lead citrate - several formulations (Venable and Coggeshell or
Reynolds). Common is using lead nitrate chelated with sodium
citrate.
•Adequate rinsing between and after staining is essential to
prevent post-stain contamination.
Particular care must be used to exclude CO2 to
inhibit lead carbonate formation - black
cannonballs.
Staining with UA
Lead staining
Typical protocol:
- 30 minutes UA
- Wash with water
- 5 minutes lead citrate
- Wash once in 0.02M NaOH
- Wash well with water.
- Dry by wicking with filter paper
Negative Staining
Positive staining - forms a complex with specimen
Negative - stain and specimen do not interact and specimen
remains electron transparent
Advantages:
1) Improved resolution
2) Speed
3) Unique information
4) Simplicity
Disadvantages:
1) Repeatability
2) Limited surface topography
3) Toxicity
Choice of stain:
1) High density to provide high contrast
2) High solubility and minimal reaction to sample
3) High melting and boiling point (beam stable)
4) Precipitant formed is extremely fined grained
Stains commonly used:
Phosphotungstate, sodium tungstate, uranyl acetate
and uranyl nitrate
Brief procedure:
Small grid and support film (formvar,
paraloidin. Sometimes carbon added.
Thin suspension of sample and excess
removed.
Dry then add negative stain and remove
Factors affecting staining:
concentration of stain
pH of stain
time
- Dry and view.
Negatively stained Ad2 (K. Boucke)
Bacteria with flagella
SARS inducing virus
(coronavirus)
Negative stain of purified RhMV virus labelled with antiRhMV and detected with anti- rabbit conjugated to 10 nm
gold. Bar = 100 nm.
Photograph provided by Fred Gildow Lab, Department of
Plant Pathology, Penn State.