Rapid Publication Three-Dimensional Imaging of Intact Isolated Islets of Langerhans With Confocal Microscopy T. CLARK BRELJE, DAVID W. SCHARP, AND ROBERT L. SORENSON 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 Langer- hans 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 co- workers (8-10), fluorescence microscopy has been an im- portant technique in investigating the structure of islets. However, a severe limitation with conventional fluores- cence microscopy has been fluorescent background re- sulting 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 From the Department of Cell Biology and Neuroanatomy, University of Min- nesota Medical School, Minneapolis, Minnesota; and the Department of Sur- gery, Washington University School of Medicine, St. Louis, Missouri. Address correspondence and reprint requests to Dr. Robert L. Sorenson, Department of Cell Biology and Neuroanatomy, University of Minnesota, 321 Church Street, SE, Minneapolis, MN 55455. Received for publication 30 January 1989 and accepted in revised form 27 February 1989. islets (11-14). Physical sectioning is not only time consuming but, more important, introduces technical difficulties and problems in interpreting three-dimensional structure be- cause of the surface destruction that occurs during the sec- tioning process. We present a new method for examining the three-di- mensional structure of islets without the need for physical sectioning. Instead, islets are stained with whole-mount immunofluorescence techniques and imaged with a confocal microscope. Confocal microscopy dramatically reduces interference from out-of-focus structures, allowing optical sectioning of a specimen by adjusting the plane of focus (15-17). This removes one of the most severe limitations of conventional microscopy, the necessity to examine thin sec- tions. Furthermore, a theoretical improvement in resolution in the plane of focus by a factor of 1.4 is obtained (16). This article presents preliminary observations from rat and human islets examined to assess the utility of confocal imaging in studying the three-dimensional structure of islets. MATERIALS AND METHODS Primary antiserums. Guinea pig anti-porcine insulin, rabbit anti-glucagon, and rabbit anti-somatostatin antiserums were previously prepared and have been extensively character- ized. Rabbit anti-bovine pancreatic polypeptide (PP) anti- serum was obtained from Lilly (Indianapolis, IN). Mouse monoclonal antibodies against somatostatin (SOM-018), glu- cagon (GLU-001), and human proinsulin (HPI-005) were ob- tained from Novo (Danbury, CT). Isolation of rat islets. Islets were isolated from 200- to 250- g Sprague-Dawley rats by pancreatic distension with a col- lagenase solution followed by stationary in vitro digestion (18). Islets were purified on a discontinuous dextran gradient of 27, 23, and 11 % solutions of 60,000- to 90,000-/W r dextran (50 x g for 5 min, then 400 x g for 5 min). Isolation of human islets. Islets were isolated from pan- creases removed from life-supported cadavers in St. Louis by the intraductal collagenase-distension and automated screen-isolation method (19). Purified islets were cultured 808 DIABETES, VOL. 38, JUNE 1989