Cryoultramicrotomy of muscle: improved preservation and resolution of muscle ultrastructure using negatively stained ultrathin cryosections.
Ultrathin sections of rapidly frozen, briefly pre-treated muscle tissue are cut and thereafter are thawed and contrasted using a negative staining technique. The method has provided micrographs in which the in-vivo order in the muscle fibres has been preserved well enough to enable both a more complete interpretation of X-ray diffraction evidence from muscle, and also a gain of new ultrastructural information on aspects of myofibril and myofilament architecture in different types of fibre. Examples here are taken from chicken, rabbit and fish muscles and show both the M-band and the bridge region of the A-band in great detail. To enhance the detail in the original images, one-dimensional (1-D) and 2-D averaging techniques (lateral smearing and step averaging, respectively) are used. Although there is major shrinkage in section thickness to about one-third of its original value, demonstrated here for the first time is the fact that the characteristic A-band lattice planes are preserved in these sections in 3-D. This confirms the usefulness of cryosections not just for 1-D and 2-D image processing, but also for 3-D reconstruction. Thus, in combination with techniques of image processing, cryoultramicrotomy can give the muscle morphologist the detailed data that are needed to match the molecular biologists, biochemists and immunologists in the interpretation of their data about physiological and pathophysiological events in muscle fibres at the macromolecular level.
Sjöström
M
M
Department of Social Medicine, University of Umea, Sweden.
Squire
J M
JM
Luther
P
P
Morris
E
E
Edman
A C
AC
D000818:Animals; D002645:Chickens; D016616:Cryoultramicrotomy; D003720:Densitometry; D006054:Goldfish; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D009132:Muscles / Q000648:ultrastructure*; D016613:Plastic Embedding; D011817:Rabbits; D012518:Sarcomeres / Q000648:ultrastructure; D013194:Staining and Labeling; D014961:X-Ray Diffraction