Digital micrograph

The resin-embedded, gold-coated blocks were imaged using a Gatan 3View serial block-face imaging system mounted in the specimen chamber of a Zeiss SIGMA-VP SEM. A total of five blocks (one islet in each), taken from three wild-type mice, were imaged to collect the data used in this study. The microscope was operated at high vacuum, with accelerating voltage ranging from 1.3-1.5 kV, using a 30 μm condenser aperture. Images were collected at an intermediate magnification, with a pixel size of 11.1 nm and a dwell time of 2.0 μs/pixel. The size of each image collected was 4,000 x 4,000 pixels in the x-y plane. The thickness of each slice in z-direction was 50 nm. Data sets were collected as stacks of 500 images, resulting in data volumes of 4.93 x 10⁴ μm³. The 3D image stacks were aligned using Digital Micrograph software (Gatan Inc., USA). Visualization and analysis of the data were performed using Digital Micrograph and Amira (FEI Inc., U.S.A.), as well as the NIH ImageJ software (Schneider et al., 2012 ).

Field emission TEM images of the ReApoBds were obtained using FEI-Tecnai G2 F20 X-TWIN that was operated at an acceleration voltage of 200 kV. Images were obtained using an ORIUS CCD camera through Digital Micrograph, and energy dispersive X-ray spectra (EDS) were recorded through FEI-TIA interface. For sample preparation, 10 μL of ReApoBds suspension were drop casted on glow discharged copper grids with pure carbon support film, incubated for 10–15 min, and then washed with ultrapure water. Finally, the sample grids were negatively stained with 1% uranyl acetate solution, and excess solution was wicked away with absorbent paper before imaging.