It was immediately evident from our studies that the x-ray
reflections arising from the regularly packed collagen fibrils in the
6-month-old lumican-null corneas were substantially more diffuse than
the corresponding reflections from normal murine corneas of the same
age. This was true of tissues that had been stored frozen and of those
that had been chemically fixed. However, because fixation itself
diminished the sharpness of the diffraction patterns, the frozen data
were the more useful for structural measurements, and it is this that
is shown in
Figures 1 2 and 3 . (Chemical fixation for electron
microscopy is known to cause some shrinkage in corneal
tissue.
30 It now appears that it also induces some
alteration in the arrangement of the collagen fibrils, something that
we intend to investigate further). The diffuseness of the
interfibrillar x-ray reflections from the
lum tm1sc/
lum tm1sc corneas was confirmed by the fact that the ratio of the height of each
interfibrillar reflection to its width at half height on each intensity
scan was less than half the ratio in the
lum +/
lum + corneas
(Table 1) . As is outlined in more detail
elsewhere,
23 a perfect lattice of scattering centers (in
this case, collagen fibrils) gives rise to true diffraction maxima,
similar to those from a crystal, that appear as very sharp peaks on an
x-ray intensity scan. Totally amorphous structures, on the other hand,
give rise only to diffuse x-ray scatter. Structures part way between
these two extremes form interference maxima whose sharpness is a
function of the level of local order in the tissue. The sharper the
peak, the narrower the range of nearest-neighbor distances between the
scatterers and vice versa. Thus, on average, throughout the whole
thickness of the tissue, the configuration of collagen fibrils in the
6-month-old lumican-null corneas was demonstrably less well ordered
than the array of collagen fibrils in wild-type corneas of the same
age. Moreover, because the height-to-width at half-height ratio of the
interfibrillar reflection from 2-month-old mutant corneas was also
noticeably reduced
(Table 2) , it seems unlikely that a proper fibrillar
arrangement is attained in the weeks after eye opening (i.e., by 2
months of age) and subsequently lost. Rather, collagen in the corneas
of lumican-null mice appears to be in a state of disarray from early in
life. This meshes with the finding by electron microscopy of a
disorganized lamellar architecture in the corneas of lumican-deficient
mice of ages similar to those studied here.
17 It should be
noted, however, that previous electron microscopic work
17 has documented different amounts of disorder depending on stromal
depth, of which more later.