Three-dimensional reconstruction from serial sections. IV. The reassembly problem.

In many fields of biology and medicine there is a pressing need for quantitative descriptions of biological structures at a resolution of micrometers. This need is currently met best by three-dimensional reconstruction from serial sections. The preliminary steps in three-dimensional reconstruction include fixation, embedding in plastic, introduction of fiducials, serial sectioning, and staining. At the light microscope level, with which we are chiefly concerned, one will usually want to do photomicrography (or videomicrography) of adjacent fields within individual tissue sections. The resultant images are projected onto a digitizer pad and the contours of interest manually digitized. From the digitized coordinates generated thereby, one wishes to generate a likeness of the original object, using computer graphic displays, and to then do interactive morphometrics. The problem of combining the digitized coordinates so as to produce a numerically faithful representation of the original object (i.e., the reassembly problem) is, as a practical matter, nontrivial. A technical description of the reassembly problem is presented. The main factors entering into a solution of the problem are discussed and a mathematical statement of the solution is given.

Three-dimensional reconstruction from serial sections III. AUTOSCAN, a software package in FORTRAN for semiautomated photomicrography.

A FORTRAN program, called AUTOSCAN, is described. This program permits the collection of photomicrographic data from serial sections to be semiautomated. In essence the user defines a box around a microscopic field of interest. Then the program drives the stage incrementally in the x and y directions, taking photographs of contiguous subfields. The box is defined by the use of a joystick and the "return" key. That is, movements of a joystick cause the stage to translate in the x and y directions. When a corner of the object is reached, as defined by cross-hairs in the microscope eyepiece, the user hits the return key. Repetition of this process at each corner defines a "box" within which photographs are to be taken. AUTOSCAN then calculates the step size and the number of frames to be taken from the user-defined values for the magnification. The actual movements of the stage in the x and y directions and the photography are fully automated. Each frame of film has the x and y coordinates of the center of the subfield being photographed imprinted in one corner, along with other relevant data. The x and y coordinates permit the resultant information to be assembled correctly into a two-dimensional montage.