Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and λ) Hyperspectral FRET Imaging and Analysis.

Cyclic AMP is a second messenger that is involved in a wide range of cellular and physiological activities. Several studies suggest that cAMP signals are compartmentalized, and that compartmentalization contributes to signaling specificity within the cAMP signaling pathway. The development of Fӧrster resonance energy transfer (FRET) based biosensors has furthered the ability to measure and visualize cAMP signals in cells. However, these measurements are often confined to two spatial dimensions, which may result in misinterpretation of data. To date, there have been only very limited measurements of cAMP signals in three spatial dimensions (x, y, and z), due to the technical limitations in using FRET sensors that inherently exhibit low signal to noise ratio (SNR). In addition, traditional filter-based imaging approaches are often ineffective for accurate measurement of cAMP signals in localized subcellular regions due to a range of factors, including spectral crosstalk, limited signal strength, and autofluorescence. To overcome these limitations and allow FRET-based biosensors to be used with multiple fluorophores, we have developed hyperspectral FRET imaging and analysis approaches that provide spectral specificity for calculating FRET efficiencies and the ability to spectrally separate FRET signals from confounding autofluorescence and/or signals from additional fluorescent labels. Here, we present the methodology for implementing hyperspectral FRET imaging as well as the need to construct an appropriate spectral library that is neither undersampled nor oversampled to perform spectral unmixing. While we present this methodology for measurement of three-dimensional cAMP distributions in pulmonary microvascular endothelial cells (PMVECs), this methodology could be used to study spatial distributions of cAMP in a range of cell types.