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 FÓ§rster 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.

Spectral imaging of FRET-based sensors reveals sustained cAMP gradients in three spatial dimensions.

Cyclic AMP is a ubiquitous second messenger that orchestrates a variety of cellular functions over different timescales. The mechanisms underlying specificity within this signaling pathway are still not well understood. Several lines of evidence suggest the existence of spatial cAMP gradients within cells, and that compartmentalization underlies specificity within the cAMP signaling pathway. However, to date, no studies have visualized cAMP gradients in three spatial dimensions (3D: x, y, z).This is in part due to the limitations of FRET-based cAMP sensors, specifically the low signal-to-noise ratio intrinsic to all intracellular FRET probes. Here, we overcome this limitation, at least in part, by implementing spectral imaging approaches to estimate FRET efficiency when multiple fluorescent labels are used and when signals are measured from weakly expressed fluorescent proteins in the presence of background autofluorescence and stray light. Analysis of spectral image stacks in two spatial dimensions (2D) from single confocal slices indicates little or no cAMP gradients formed within pulmonary microvascular endothelial cells (PMVECs) under baseline conditions or following 10 min treatment with the adenylyl cyclase activator forskolin. However, analysis of spectral image stacks in 3D demonstrates marked cAMP gradients from the apical to basolateral face of PMVECs. Results demonstrate that spectral imaging approaches can be used to assess cAMP gradients-and in general gradients in fluorescence and FRET-within intact cells. Results also demonstrate that 2D imaging studies of localized fluorescence signals and, in particular, cAMP signals, whether using epifluorescence or confocal microscopy, may lead to erroneous conclusions about the existence and/or magnitude of gradients in either FRET or the underlying cAMP signals. Thus, with the exception of cellular structures that can be considered in one spatial dimension, such as neuronal processes, 3D measurements are required to assess mechanisms underlying compartmentalization and specificity within intracellular signaling pathways.