For comparison with chemically fixed osteoclasts, we prepared chick
osteoclasts by quick freezing followed
by freeze-substitution. In spite of technical difficulties this demonstrated
that
osteoclasts can be satisfactorily
frozen in situ by the metal contact method. Ultrastructural differences
were
revealed between conventional
fixation and quick freezing. Compared with conventional fixation, the quick
freezing method appeared to
improve preservation: (1) a discrete trilaminar plasma membrane and other
intracellular membranes showed
a smooth profile without undulation or rupture; (2) cytoskeletal components
appeared to be clearer,
straighter, and more numerous; (3) the interior of the ruffled finger contained
interconnected lattice
structures whereas highly organised microfilaments were seen in the clear
zone;
(4) well developed
tubulovesicular structures (TVSs) that branched or anastomosed with each
other
were revealed in the
cytoplasm; (5) the contents of intracellular membrane systems including
the
nuclear envelope, endoplasmic
reticulum, and Golgi complex were stained to a various extent; (6) vesicles
and
vacuoles were much smaller,
round and well-defined with electron-dense contents; (7) crystalline structures
were seen at the extracellular
channels of the ruffled border, in the lumen of TVSs, and in vesicles;
(8) in
some instances mitochondrial
granules were visible; (9) within the resorptive lacuna, osteoclasts adhered
to
the degraded bone matrix without any intervening empty space.