Living muscle-fibres from freshly killed mice were mounted in isotonic saline / protein media and examined with a Smith interference microscope, usually with a white light-source. When the A or I bands near the edge of a fibre were observed to match the colour of the background field, their refractive indices were close to that of the mounting medium; although it is extremely probable that diffraction at the edges of the adjacent unmatched bands affected their apparent match, so that they were not exactly of this refractive index. Matched A bands were distinguished from matched I bands by examining them in plane-polarized light, by rotating the preparation through a right angle under the interference microscope to display their birefringence, and from the colour of the unmatched bands when the fringe system of the microscope was left unaltered.
In any one fibre, the refractive indices of the A-band regions were always higher than that of the I-band regions. The H bands had lower refractive indices than the A bands, and the Z bands higher than the I bands, but these were both too narrow to be matched satisfactorily by this method. The refractive indices of the solutions in which matched I bands were found ranged from 1.358 to 1.363, and those in which matched A bands were found from 1.360 to 1.366. The mean refractive index of the A and I bands was very close to 1.363, which is equivalent to a total solid content of 16% w/v.
These findings are in good general agreement with those of H. Huxley and Hanson (1957) and Bennett (1955), who measured the distribution of solid material in isolated glycerinated mammalian myofibrils; but the difference between the refractive indices of the A- and I-band regions of the living fibres appeared to be very much less. Only part of this discrepancy can be accounted for by the presence of non-fibrillar solid material, because the total amount of this is extremely unlikely to exceed 50% of the total myofibrillar fibrous protein. It therefore seems probable that, because of the diffraction from the unmatched bands, the true refractive indices of the A bands were higher than those of the solutions in which they appeared matched, and those of the I bands were correspondingly lower than those of the solutions in which they appeared matched.
The maximum error involved here (when the sarcomere interval was approximately 2.5 µ) can be quantified from independent estimations of the non-fibrillar material in whole muscle (Szent-Györgyi and others, 1955; Hanson and H. Huxley, 1957); and from this it seems highly probable that the refractive indices of the I bands were not lower than 1.350 (equivalent to a solid content of 9% w/v), and those of the A bands were not higher than 1.375 (equivalent to a solid content of 33% w/v).
