Biology Reference
In-Depth Information
11
ECM Macromolecules: Rotary Shadowing
and Scanning Transmission Electron Microscopy
Michael J. Sherratt, Helen K. Graham, Cay M. Kielty,
and David F. Holmes
1. Introduction
The examination of ECM macromolecules by conventional transmission
electron microscopy (TEM) or scanning transmission EM (STEM) can provide
important information on macromolecular organization and interactions.
Conventional EM preparation techniques employ contrast enhancing heavy
metal stains to visualize isolated macromolecules. Rotary shadowing (RS-TEM)
minimizes staining artefacts by coating the surface of already dehydrated mol-
ecules. Heavy metal atoms are evaporated in a vacuum from a source set at an
oblique angle relative to the substrate on which the specimen is mounted. The
gaseous metal deposits on the specimen and substrate in layers of varying thick-
ness, determined by the relief of the specimen and its substrate (
Fig. 1
). The
image resolution of shadowed biological specimens is limited by specimen
preparation techniques, which rely on dehydration, and by the granularity of
the shadowed metal.
STEM is a well-established technique that is capable of providing quantita-
tive mass distribution data on unstained and unshadowed macromolecular
assemblies
(1)
. The techniques of mass measurement and mapping were origi-
nally developed on dedicated field-emission STEM instruments in which an
annular dark-field detector (ADF) provided efficient collection of elastically
scattered electrons from unstained biological samples
(2)
. The freedom from
staining or shadowing allows the investigation of subtle changes in absolute
mass and mass distribution within a system following experimental interven-
tion. The assembly and growth mechanisms of collagen fibrils formed in vivo
and in vitro have been investigated by STEM (
Fig. 2
)
(3)
. Fibrillin microfibrils
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