TECHNIQUES OF ADVANCED LIGHT-MICROSCOPY AND THEIR APPLICATIONS TO MORPHOLOGICAL ANALYSIS OF HUMAN EXTRAEMBRYONIC MEMBRANES

The science of light microscopy has advanced dramatically in recent ye ars through the introduction of new technology. A brief description of scanning light microscopes, laser illumination, the confocal principl e, digital imaging, and image processing reveals a number of theoretic al advantages which are particularly useful in improving epifluorescen ce microscope images. Examples of results from several studies of huma n extra-embryonic membranes conducted in our laboratory show how the a pplication of these techniques has been used to describe structures su ch as microtrabeculae and rivets for the first time, to map the micros copic distribution of a wide range of proteins, and to observe the act ivity of placental villi at the microscopic level in an environmentall y controlled microscope stage. High-sensitivity detectors have permitt ed the ''super-resolution'' detection of structures smaller than the t heoretically calculated limits of light microscope resolution. Renderi ng images in false colour is demonstrably useful in detecting subtle V ariations in fluorescence intensity at different intracellular sites a nd at different sites within tissues of fetal membranes. Processing st acks of digital images using appropriate software allows the 3-D recon struction of suitably sized extra-embryonic membrane components. These digital images created from optical sections through the tissue are o btained non-destructively, and the relationships in space of the compo nents are well preserved. (C) 1997 Wiley-Liss, Inc.