5 Dimensional Hyperspectral FRET Imaging and Analysis Approaches for Measuring cAMP Gradients

cAMP is a second messenger that regulates numerous cellular and physiological functions. A growing body of evidence suggests that cAMP signals are localized resulting in the formation of spatial cAMP gradients. Recently, Forster Resonance energy transfer (FRET) imaging has become a standard method for assessing changes in cAMP concentration. However, many FRET probes have low signal-to-noise ratios and limited dynamic range, making measurements of cAMP difficult. Our previous work suggested that hyperspectral imaging and analysis allows quantitative measurement of FRET efficiencies in 3D (x, y, and t) within single cells. However, traditional (x, y, and t) approaches of measuring FRET often lead to misinterpretation of data. Here, we present data demonstrating that hyperspectral imaging approaches can be used for 5D (x, y, z, λ and t) assessment of FRET efficiency in living cells allowing the study of spatially-localized cAMP signals. We utilized H188-2 (cAMP reporter) and AKAR4 (PKA activity reporter) FRET biosensors that were transfected into PMVECs and HEK 293 cells, respectively. Time lapse hyperspectral Z-stack images were acquired using a Nikon A1R confocal microscope. We used custom MATLAB scripts to calculate FRET efficiencies from image data. These approaches allowed the measurement of spatial cAMP gradients and PKA activity induced by agonist treatment. We found that cAMP gradients developed from the apical to the basolateral side of PMVECs when treated with isoproterenol, while gradients developed from both the basolateral and apical side when treated with PGE1. In addition, we observed an increase in localized phosphorylation upon treatment with isoproterenol and PGE1 in HEK293 cells. Thus, these studies demonstrate the feasibility for simultaneously studying cAMP gradients and PKA activity in cells.This work was supported by AHA 16PRE27130004, NIH P01HL066299, NIH S10RR027535, and the Abraham Mitchell Cancer Research Fund.