Hyperspectral imaging approaches to characterize the distribution and expression of FRET in tissues isolated from a novel transgenic rat expressing a FRET-based probe

Abstract

Fӧrster resonance energy transfer (FRET) based cAMP sensors have become a standard approach to visualize cAMP signals in cells. However, FRET sensors inherently offer low signal-to-noise ratios, making cAMP measurements challenging, especially in 3 spatial dimensions. In previous studies, we used 4-dimensional (x, y, z, and λ) hyperspectral imaging and analysis approaches to measure agonist – induced distribution of cAMP signals in cultured pulmonary microvascular endothelial cells. In many cell types including PMVECs, transient transfection of FRET sensors results in a low transfection efficiency (1-5%), which limits the possibilities of studying spatial distribution of FRET signals in many cells simultaneously. We developed a transgenic rat model that expresses the H187 FRET sensor. In current studies, we utilize hyperspectral imaging and analysis approaches to characterize the distribution of FRET signals in different tissues and vessels harvested from transgenic rats. Hyperspectral z-stacks of tissues and vessels were acquired using a Nikon A1R confocal microscope equipped with 20X multi-immersion objective and a 32 channel PMT detector. Samples were excited using 405 nm and 561 nm lasers and emission was collected from 424 nm -724 nm at 10 nm intervals. Tissues including lungs, heart, mesentery, kidney, expressed the H187 FRET sensor. In conclusion, transgenic rats provide a platform to visualize cAMP signals in-vitro and in-situ.