It is shown how serial block-face electron microscopy (SBEM) of insulin-secreting β-cells in wild-type mouse pancreatic islets of Langerhans can be used to determine maturation times of secretory granules. Although SBEM captures the β-cell structure at a snapshot in time, the observed ultrastructure can be considered representative of a dynamic equilibrium state of the cells since the pancreatic islets are maintained in culture in approximate homeostasis. It was found that 7.2 ± 1.2% (±st. dev.) of the β-cell volume is composed of secretory granule dense-cores exhibiting angular shapes surrounded by wide (typically ≳100 nm) electron-lucent halos. These organelles are identified as mature granules that store insulin for regulated release through the plasma membrane, with a release time of 96 ± 12 h, as previously obtained from pulsed ³⁵S-radiolabeling of cysteine and methionine. Analysis of β-cell 3D volumes reveals a subpopulation of secretory organelles without electron-lucent halos, identified as immature secretory granules. Another subpopulation of secretory granules is found with thin (typically ≲30 nm) electron-lucent halos, which are attributed to immature granules that are transforming from proinsulin to insulin by action of prohormone convertases. From the volume ratio of proinsulin in the immature granules to insulin in the mature granules, we estimate that the newly formed immature granules remain in morphologically-defined immature states for an average time of 135 ± 14 min, and the immature transforming granules for an average time of 130 ± 17 min.

显示了如何使用朗格汉斯野生型小鼠胰岛中胰岛素分泌 β 细胞的连续块面电子显微镜 (SBEM) 来确定分泌颗粒的成熟时间。尽管 SBEM 及时捕获了 β 细胞结构，但观察到的超微结构可以被认为是细胞动态平衡状态的代表，因为胰岛在培养中保持近似稳态。发现 7.2 ± 1.2% (±st. dev.) 的 β 细胞体积由分泌颗粒致密核心组成，呈角形，周围环绕着宽（通常≳ 100 nm）电子透明晕。这些细胞器被鉴定为成熟的颗粒，可储存胰岛素以通过质膜调节释放，释放时间为 96 ± 12 小时，如先前从脉冲³⁵半胱氨酸和甲硫氨酸的 S-放射性标记。对 β 细胞 3D 体积的分析揭示了一个没有电子透明晕的分泌细胞器亚群，被确定为未成熟的分泌颗粒。分泌颗粒的另一个亚群被发现具有薄（通常 ≲ 30 nm）电子透明晕，这是由于未成熟颗粒在激素原转化酶的作用下从胰岛素原转化为胰岛素。根据未成熟颗粒中胰岛素原与成熟颗粒中胰岛素的体积比，我们估计新形成的未成熟颗粒保持形态定义的未成熟状态平均时间为 135±14 分钟，而未成熟转化颗粒的平均时间为130 ± 17 分钟的时间。