Snapshot Hyperspectral Light-Sheet Imaging allows 5D Investigation of Pancreatic Islets

Abstract

The islet of Langerhans is a micro-organ which contributes to blood glucose homeostasis through the regulated secretion of glucagon (from α-cells) and insulin (from β-cells). Cell-cell interactions allow for specific dynamics and responses to glucose that are not seen in equivalent populations of isolated cells. For this reason, investigations of cellular signaling within the intact islet is crucial to a better understanding of diseases as type 1 and type 2 diabetes. Fluorescent markers are powerful non-invasive tools to follow dynamics within cells, thanks to their specificity and reliability for long term experiments. However, the molecular processes of hormones’ secretion are fast, and scanning microscopy techniques often lack of temporal resolution to follow these dynamics. Light sheet microscopy, with its intrinsic optical sectioning, permits the acquisition of fast, three-dimensional data sets from living samples, with low photobleaching. However, it is also needed to visualize multiple probes simultaneously, which increases the complexity of the system. Snapshot hyperspectral imaging overcomes these limitations, allowing real-time acquisition of (x,y,λ) voxels containing all the spatial and spectral information for each pixel in the image. We demonstrate the combination of a snapshot hyperspectral system, the Image Mapper Spectrometer (IMS), with an inverted Selective Plane Illumination Microscope (iSPIM). This novel snapshot hyperspectral light-sheet architecture provides three dimensional, high (spatial and temporal) resolution images of multiple fluorophores with a single excitation wavelength. This approach allows distinguishing α- from β-cells in an intact islet, while simultaneously acquiring information on the calcium activity and other cellular dynamics including changes in intracellular Zn2+.