Collection and handling of ultrathin serial sections for 3-dimensional reconstruction

Serial sectioning for 3-D reconstruction requires a highly skilled and experienced individual to collect ribbons of ultrathin sections on formvar-coated grids, and to handle the grids after section collection. A simple method is described for placing ribbons in an orderly serial fashion on formvar-coated grids, by a microtomist with average experience. Prior to sectioning, a wax ledge is prepared on the sloping edge of a glass knife in order to support a formvar-coated grid held in a horizontal slot cut in the wax. After a ribbon is formed, the water in the trough is slowly withdrawn to allow the ribbon to settle on the grid. The grids are then placed in an easy-to-make plastic chamber so that the formvar does not get ruptured during drying. The chamber can also be used for staining and storage of grids thereafter. Approximately 4000 sections from mudpuppy retinal cells have been successfully collected using this method. Computer 3-D reconstruction of the individual cells has been done.