Open AccessThree-dimensional measurement of cAMP gradients using hyperspectral confocal microscopy
Author(s) -
Thomas C. Rich,
Naga S. Annamdevula,
Andrea L. Britain,
Samuel Mayes,
Peter F. Favreau,
Silas J. Leavesley
Publication year - 2016
Publication title -
proceedings of spie, the international society for optical engineering/proceedings of spie
Language(s) - English
Resource type - Conference proceedings
SCImago Journal Rank - 0.192
H-Index - 176
eISSN - 1996-756X
pISSN - 0277-786X
DOI - 10.1117/12.2213273
Subject(s) - confocal microscopy , compartmentalization (fire protection) , confocal , hyperspectral imaging , microscopy , biophysics , microbiology and biotechnology , fluorescence microscope , microscope , chemistry , materials science , fluorescence , biology , optics , biochemistry , physics , computer science , artificial intelligence , enzyme
Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions over a wide range of timescales. Several lines of evidence have suggested that the distribution of cAMP within cells is not uniform, and that cAMP compartmentalization is largely responsible for signaling specificity within the cAMP signaling pathway. However, to date, no studies have experimentally measured three dimensional (3D) cAMP distributions within cells. Here we use both 2D and 3D hyperspectral microscopy to visualize cAMP gradients in endothelial cells from the pulmonary microvasculature (PMVECs). cAMP levels were measured using a FRET-based cAMP sensor comprised of a cAMP binding domain from EPAC sandwiched between FRET donors and acceptors - Turquoise and Venus fluorescent proteins. Data were acquired using either a Nikon A1R spectral confocal microscope or custom spectral microscopy system. Analysis of hyperspectral image stacks from a single confocal slice or from summed images of all slices (2D analysis) indicated little or no cAMP gradients were formed within PMVECs under basal conditions or following agonist treatment. However, analysis of hyperspectral image stacks from 3D cellular geometries (z stacks) demonstrate marked cAMP gradients from the apical to basolateral membrane of PMVECs. These results strongly suggest that 2D imaging studies of cAMP compartmentalization - whether epifluorescence or confocal microscopy - may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D studies are required to assess mechanisms of signaling specificity.