Functional Imaging of Intact Pancreatic Islets by Inverted Selective Plane Illumination Microscopy

Abstract

The islet of Langerhans is a complex aggregate of cells ∼100 μm in diameter, which plays a central role in glucose homeostasis through the regulated secretion of glycolytic hormones. However, the mechanisms underlying these secretions are not yet fully understood. The behaviors of individual islet cells completely change when they are dislocated from the islet environment, so it is essential to choose experimental approaches that permit investigations in the whole islet. Fluorescence microscopy is well-suited to functional imaging in biological samples, thanks to its specificity and non-invasive quality. Selective Plane Illumination Microscopy (SPIM) has been proven to be a useful technique for imaging thick samples. It provides intrinsic optical sectioning by specifically illuminating the sample with a thin sheet of light, yielding the imaging speed of a conventional widefield microscope coupled with less photobleaching compared to confocal imaging techniques. We have developed a version of the inverted SPIM (iSPIM) [Wu et al., 2011. PNAS 108:17708-13] on a widefield inverted microscope, to study the functional behavior of intact mice islets of Langerhans. By scanning the light sheet through the static sample, it is possible to obtain a volume reconstruction of the whole islet in less than 3 seconds, enabling the study of fast dynamics within the islet. Moreover, the resolution provided by a 0.8 NA objective allows for definitive measurements of individual cell's functional details within the islet milieu. Together, the results demonstrate the multi-purpose capability of this technique.