New methods for cathodoluminescence in the scanning electron microscope.

Experiments using the CL imaging mode to recognise osteoid in the polished, cut surfaces of bone biopsies embedded in PMMA led to the development of a number of new methods for contrast formation in CL images in the SEM. These involve: (1) enhancing or (2) reducing the CL signal by staining the specimen, (3) utilising the cathodoluminescence of glass microscope slides to produce images of histological sections mounted on glass so that features in the section which scatter the electron beam appear dark against a light background, and (4) enhancing the CL signal from PMMA so that features which are less penetrated by the scintillator show up dark against a bright background. Efforts to increase the efficiency of light collection resulted in the development of a new means for manufacturing reflector-cum-light guide CL detectors by wrapping aluminum foil around a wooden former. These detectors enshroud the specimen so that CL light can only escape to the photomultiplier window (or back up the final lens). A variety of such designs have proved more efficient than the conventional plastic light guides used as CL detectors. By enlarging the beam entry aperture, other SE and BSE detectors can be used simultaneously. Examples of the value of the CL mode in mineralised tissue research include the use of enhanced CL plastic embedding media to detect marrow space and of enhanced osteoid CL to detect unmineralised bone matrix; the use of tetracycline as a growth marker in pathological studies of bone and experimental studies with bone, dentine and enamel; the use of bisbenzamid to locate and count nuclei in osteoclasts, the hard tissue resorbtive cells; and the use of superficial stain absorption of auto-CL to locate stained material on tooth surfaces, with the view to monitor the efficiency of periodontal therapy.