Three-dimensional visualization of human Langerhans' cells using confocal scanning laser microscopy.

Most techniques used to visualize cells in tissues are accompanied by some distortion of the tissue due to fixation and sectioning. We here present a technique involving "optical sectioning" and three-dimensional reconstruction of fluorescence-labelled Langerhans' cells in human skin that avoids such problems. The instrument used to study the specimens was a PHOIBOS Confocal Scanning Laser Microscope (CSLM) built around a Zeiss Universal microscope. A software package for three-dimensional reconstructions of confocal sections was employed for calculations in a Hewlett-Packard HP9000-350 minicomputer running UNIX. Normal human skin obtained at plastic surgery or skin biopsy specimens from allergic, irritant, or control patch test reactions was studied. Epidermis separated from dermis by incubation in thermolysin or thick (25 microns) cryostat sections were incubated with fluorescein-isothiocyanate labelled mouse monoclonal antibodies directed against HLA-DR. To reduce the fading of immunofluorescence during microscopy, the specimens were mounted with glycerol in PBS containing p-phenylenediamine. The results indicate that CSLM is a powerful tool for investigations on Langerhans' cells in different conditions of the skin allowing a three-dimensional view of cells in unfixed or acetone-fixed preparations at the light microscope level.