Ultrastructural Studies of Cell—Collagen Interactions

To analyze cell morphology, the organization of the substratum, and physical relationship of cells to the substratum, it is necessary to use ultrastructural methods. This chaper describes the techniques for using scanning and transmission electron microscopy (SEM and TEM) to study cell interactions with dried and hydrated collagen gels. Samples for SEM analysis are fixed with glutaraldehyde in sodium phosphate. Cells are then washed in phosphate buffer containing sucrose and stored in phosphate sucrose overnight. The specimens are then dehydrated through a graded series of ethanol. The dehydrated samples are critically point dried with liquid CO₂. Finally, the samples are placed in an argon atmosphere in a vacuum evaporator and coated with metal from a 60% gold/ 40% palladium target. When viewing the coated specimens with the scanning electron microscope, accelerating voltages ranging from 10 to 25 kV are used depending upon the stability of the samples and the magnification desired. Samples for TEM analysis are fixed and washed similarly to samples for SEM except that 2% glutaraldehyde/l% tannic acid is used instead of 3% glutaraldehyde to enhance the contrast of membranes. An alternative method for preparing samples is useful when it is necessary to view a large number of cells in each section.