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We present a new technique for analyzing the three-dimensional structure of intact isolated islets of Langerhans. Adult rat and human islets were stained with whole-mount immunofluorescence techniques and optically sectioned with a confocal microscope. This has several advantages over traditional methods: 1) the technical difficulties in serial sectioning and handling the large numbers of sections are avoided, 2) optical sectioning by confocal microscopy gives improved resolution and strongly suppresses light from out-of-focus structures, and 3) entire islets can be rapidly imaged for the presence of positive staining. This new technique should facilitate the study of the three-dimensional structure of islets of Langerhans. Diabetes 38:808-14,1989 T he three-dimensional structure of islets of Langerhans is important in understanding the regulatory mechanisms involved in controlling the different islet cell types. Since the first immunofluorescent localization of insulin to p-cells by Lacy and Davies (1,2), glucagon to the a-cell by Baum et al. (3), somatostatin to the 5-cell by several investigators (4-7), and pancreatic polypeptide (PP) to a distinct cell type by Larsson and coworkers (8-10), fluorescence microscopy has been an important technique in investigating the structure of islets.However, a severe limitation with conventional fluorescence microscopy has been fluorescent background resulting from out-of-focus structures, which reduces image contrast and therefore limits the amount of information that can be obtained from in-focus structures. To overcome this limitation, it has been necessary to examine thin sections of

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