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Microtubules play a major role in intracellular trafficking of vesicles in endocrine cells. Detailed knowledge of microtubule organization and their relation to other cell constituents is crucial for understanding cell function. However, their role in insulin transport and secretion is under debate. Here, we use FIB-SEM to image islet β cells in their entirety with unprecedented resolution. We reconstruct mitochondria, Golgi apparati, centrioles, insulin secretory granules, and microtubules of seven β cells, and generate a comprehensive spatial map of microtubule–organelle interactions. We find that microtubules form nonradial networks that are predominantly not connected to either centrioles or endomembranes. Microtubule number and length, but not microtubule polymer density, vary with glucose stimulation. Furthermore, insulin secretory granules are enriched near the plasma membrane, where they associate with microtubules. In summary, we provide the first 3D reconstructions of complete microtubule networks in primary mammalian cells together with evidence regarding their importance for insulin secretory granule positioning and thus their supportive role in insulin secretion.

Diarrhea is the leading cause of mortality in beef and dairy calves during the first week of life and results in substantial financial loss [1]. Diarrhea is a multifactorial disease and can be infectious or non-infectious. However, in the majority of calves, infectious organisms, especially Cryptosporidium parvum, rotavirus, coronavirus, and E. coli, are the primary cause [2]. The aim of this study was to generate a decision tree, based on prevalence, diagnostic testing and treatment and to estimate associated costs or risk. For each of the four main pathogens, two principal approaches are outlined and compared. The first approach relies on a detailed diagnostic workup and allows for specific etiological treatment. The second approach relies on the trial-and-error method, which involves the use of a first-choice antibiotic, followed by a second- and third-choice antibiotic if the previous ones failed to resolve the disease. In Switzerland, the prevalence of diarrheic calves infected with E. coli is approximately 1% suggesting that the use of antimicrobials for the treatment of scouring calves, in the absence of a diagnostic workup, is not always justified. However, for all four major pathogens, the trial-and-error method affords cheaper treatment compared with treatment based on an etiological diagnosis. This creates a quandary in view of the current worldwide efforts to reduce the use of antibiotics in animal agriculture.

Correlative microscopy combines the versatility of the light microscope with the excellent spatial resolution of the electron microscope. Here, we describe fast and simple methods for correlative immunofluorescence and immunogold labeling on the very same ultrathin section. The protocols are demonstrated on sections of tissue samples embedded in the methacrylate Lowicryl K4M. Ultrathin sections are mounted on electron microscopy (EM) grids and stained simultaneously with fluorescent and gold markers. For the detection of primary antibodies, we applied either protein A gold or immunoglobulin G (IgG) gold in combination with secondary antibodies coupled to Alexa488 or Alexa555. Alternatively, the correlative marker FluoroNanogold was used, followed by silver enhancement. The samples have to be analyzed first at the light microscope and then in the transmission electron microscope (TEM), because the fluorescence is bleached by the electron beam. Labeled structures selected at the fluorescence microscope can be identified in the TEM and analyzed at high resolution. This way, fluorescent signals can be directly correlated to the corresponding subcellular structures in the area of interest.