Figure 1.

Molecular diffusion into islets—imaging and method development. (a) Lateral optical cross-sections of a pancreatic islet on top of a coverslip (unflattened) imaged by a confocal microscope (FDA/PI/Hoechst 33342 staining for 15 min at room temperature); each slice is labelled with distance in μm from the top of the islet. (b) Novel method to study diffusion: isolated pancreatic islets were incubated with Hoechst 33342 nuclear stain under different conditions, washed and immediately embedded into Optimal Cutting Temperature compound (O.C.T.) to stop the diffusion of the dye further into the islet. Frozen sections were cut on a cryostat and coverslip was added using a dry mounting method (double-sided tape) to prevent any exposure to moisture. Each slide was imaged immediately after being thawed to prevent further diffusion of the dye into the islet. (c) Confocal images of the sectioned islets were analysed using ImageJ plugin Concentric Circles. Parameters of the plugin were set to align with the outer edge of the imaged islet and to draw circles 5 pixels (corresponding to 1.89 μm) apart. Average intensity of each circle was measured, and results were plotted to show a staining profile. (d) Average staining profiles of pancreatic islets incubated with Hoechst 33342 at room temperature for 15 min, 6 h or 12 h (n = 3–5 islets). To get an average staining profile for a certain staining condition, profiles from multiple islets were aligned at the outer edge (0 μm). (e) Linear relationship between time and square of distance shown for four different fluorescent intensities from plot shown in (c). Figure (b) was created using Inkscape (version 0.91, http://www.inkscape.org/), figures (d) and (e) using GraphPad Prism (version 8.4.1, https://www.graphpad.com). RFU relative fluorescence intensity.