A two-step alignment method for 3D computer-aided reconstruction based on fiducial markers and applied to mouse embryonic hearts.

An accurate three dimensional computer reconstruction of microscopic biological objects or distribution of molecules identified on serial sections must solve two major problems: 1) the alignment of sections using adequate extrinsic references (fiducial markers); 2) the impossibility of observing these references and the cellular or molecular structures in the microscope at the same magnification. To provide extrinsic references for objects embedded in soft media, we have modified and simplified the charcoal-paraffin method described by Langemeijer and Simons (1973). It consists of drilling three or four small holes into the paraffin block, sealing this block at the extremity of a glass holder and, from the other extremity of the holder attached to a rubber hose, aspirating a liquefied mixture of charcoal-paraffin to fill these cylindrical holes. An alignment procedure was developed using serial sections of mouse embryonic hearts with bromodeoxyuridine-labelled DNA synthesizing cells. From each fourth section, two sets of contours have been drawn and digitized: 1) at low magnification (about 40x), embryo body wall, heart, neural tube and extrinsic reference marks (black dots); 2) at higher magnification (240-300x): heart contours alone (without extrinsic references, but with individual labelled cells). Different operations of the computer-aided alignment, as well as checking of results by inverse alignment, are described in detail. This two-step alignment method offers a practical, efficient compromise between: a) purely subjective alignment based only on tissular landmarks interpreted by the operator; b) ideal perfect alignment based not only on adequate references, but on computerized correction of section deformation, as well.