Techniques of advanced light microscopy and their applications to morphological analysis of human extra-embryonic...

The science of light microscopy has advanced dramatically in recent years through the introduction of new technology. A brief description of scanning light microscopes, laser illumination, the confocal principle, digital imaging, and image processing reveals a number of theoretical advantages which are particularly useful in improving epifluorescence microscope images. Examples of results from several studies of human extra-embryonic membranes conducted in our laboratory show how the application of these techniques has been used to describe structures such as microtrabeculae and rivets for the first time, to map the microscopic distribution of a wide range of proteins, and to observe the activity of placental villi at the microscopic level in an environmentally controlled microscope stage. High-sensitivity detectors have permitted the "super-resolution" detection of structures smaller than the theoretically calculated limits of light microscope resolution. Rendering images in false colour is demonstrably useful in detecting subtle variations in fluorescence intensity at different intracellular sites and at different sites within tissues of fetal membranes. Processing stacks of digital images using appropriate software allows the 3-D reconstruction of suitably sized extra-embryonic membrane components. These digital images created from optical sections through the tissue are obtained non-destructively, and the relationships in space of the components are well preserved.