Measurement of microtomy-induced section distortion and its correction for 3-dimensional histological reconstructions.

The presence of microtomy induced distortion in paraffin sections is a significant hindrance to the accurate alignment of sections for three-dimensional reconstructive techniques. Measurements of section distortion in various rat tissues demonstrated distortions to be present in all sections, with over 85% of such distortions being manifest as expansions when compared to the original distances between a series of eight drilled fiducial marks. Mean percentage dimensional changes in the direction of the cutting stroke and at right angles to this direction were -0.5 +/- 1.5% and 3.7 +/- 1.2% for liver, 7.6 +/- 2.4% and 9.1 +/- 1.2% for kidney, 6.6 +/- 2.3% and 10.5 +/- 1.4% for lung, and 20.3 +/- 6.6% and 8.9 +/- 5.9% for skeletal muscle. Individual sections invariably displayed measurable distortions, with only skeletal muscle showing any consistent pattern, in the form of "barrel" distortion at right angles to the cutting stroke. In addition a method of distortion correction and simultaneous image alignment is presented as a means of section alignment with full distortion correction capability. This method uses a quadratic polynomial transform in a non-linear "unwarping" algorithm, to correct for the rotational and translational misalignment as well as for microtomy and camera aspect ratio distortions. Application of this method to a sequence of 46 serial sections demonstrated an alignment accuracy to within 2.6 +/- 0.8 pixels.