Backscattered electron imaging of lectin binding sites in tissues following freeze-fracture cytochemistry

Recently, cytochemical techniques have been applied for localizing membrane components; however, transmission electron microscopy only provides two-dimensional information about their distribution. Scanning electron microscopy, on the other hand, offers the possibility of examining the three-dimensional architecture of biological samples. The fracture-label cytochemical technique was combined with the backscattered electron imaging (BEI) mode of the scanning electron microscope to visualize the in vivo distribution of lectin binding sites on freeze-fractured biological membranes in tissues and cells. Pancreatic and testicular tissues, fixed with glutaraldehyde, were freeze-fractured and labeled with Helix pomatia lectin-gold or Ricinus communis I-gold complexes. The labeled specimens were then critical-point dried and replicated with platinum-carbon for routine transmission electron microscopy or with carbon alone for BEI. Lectin-gold labeling of fractured plasma and intracellular membranes observed with BEI showed a labeling pattern similar to that seen by the replica method. However, BEI-fracture-label provided additional information about the distribution of the labeling with respect to three-dimensional organization of tissues and cells. Large sample areas could be examined, making this technique particularly useful as a survey method for specimens that are either differentially labeled or composed of heterogenous cell populations.