Use of "reference series" to realign histological serial sections for three-dimensional reconstructions of the positions of cellular events in the developing brain.

The present study demonstrates how, predominantly by external fiducials, histological serial sections used to reconstruct patterns of individually marked cellular events in large organs or whole embryos can be realigned with the help of "reference series". Resin-embedded embryos were cut at 1 microm and consecutive sections were alternately placed on two sets of slides. For cytological diagnosis and acquisition of embryonic contours, stained sections of the first series, termed "working series", were scanned with the x 100 objective using "Huge Image", a recently established image acquisition system. For acquisition of the contours of the resin block, adjacent unstained sections of the second series, termed "reference series", were scanned with the x 5 objective. Thereafter, "hybrid sections" were created which combined vectorized embryonic contours and cellular events taken from the working series with vectorized block contours taken from the reference series. For realignment, consecutive "hybrid sections" were matched by best-fit of the block contours. Stacks of realigned "hybrid sections" were shaped like truncated pyramids and, thus, reflected repeated "trimming" of the resin block during the sectioning procedure. Among 266 "hybrid sections" at intervals of 8 microm, needed to reconstruct the brain of a 15-day-old embryo of Tupaia belangeri (Scandentia), internal fiducials were required five times for realigning a total of six adjacent truncated pyramids. Application of this method provided realistic reconstructions of the positions of apoptotic cells in the entire developing brain without the need of secondary introduction of external fiducials.