Islet MorphometryIslet morphometry was analyzed with Volocity version 6.1.1 (PerkinElmer) (12,19). Axio Imager (Zeiss) with X-Y stage and Orca-ER digital camera (Hamamatsu) was used to acquire thousands of islet images, with tens of thousands of nuclei analyzed per sample (Supplementary Table 4). All visible islets within one pancreatic section per individual were imaged for insulin with long exposure imaging (≥20× shutter time of standard exposure). Acinar and ductal tissue images were captured as negative controls for insulin staining.

Blinded Study 1: Classifying Insulinlow IsletsVirtually all islets in a pancreatic section were identified by DAPI and imaged. Negative control images (nonislet containing) were inserted into image stacks for subsequent blinded classification. For classification of insulinlow cells, T1D islets were separated into those exhibiting strong, moderate, or no insulinlow (described in detail below). Two examiners blinded to disease duration or phenotype independently classified and quantified insulinlow phenotypes in T1D islets. Islets were defined as containing five or more islet endocrine cells.

Blinded Study 2: Calculating Insulinlow Islet AbundanceMost, if not all, T1D islets were imaged for synaptophysin, insulin, and Nkx6.1 for 10 control and 15 T1D pancreatic sections. Control islets were imaged with standard exposure; T1D islets were imaged with long exposure for insulin only. Blinded investigators quantified insulinlow islets as percentage of total synaptophysin-positive islets.