D52 MOLECULAR INSIGHT INTO PULMONARY HYPERTENSION: Simultaneous, Real Time, Measurement Of 3d Camp And Ca2+ Signals Using Hyperspectral Imaging And Analysis Approaches

摘要: Methods: FRET-based cAMP probes and the RCaMP Ca2+ probe were transiently expressed in either HEK-293 cells or PMVECs. Cells were counterstained with DRAQ5 to identify nuclei. Emission scan-based hyperspectral imaging approaches were used to measure cAMP-mediated changes in FRET signals as well as Ca2+-mediated changes in RCaMP emission intensity in 3D and 4D using either a Nikon A1R confocal or custom hyperspectral imaging systems. Custom analysis scripts to monitor FRET signals in 3D (x, y, z) and 4D (x, y, z, t) were written in the matlab programming environment.Results: Hyperspectral imaging approaches allow measurement of spatial gradients in FRET efficiency (cAMP) and RCaMP emission intensity (Ca2+). In addition, we detected forskolin (50 µM), isoproterenol (1 µM), and PGE (1 µM) induced cAMP signals and thapsigargin 1 (0.1 or 1 µM) induced Ca2+ signals …