Application of two-photon flash photolysis to reveal intercellular communication and intracellular Ca2+ movements.
Two-photon excitation makes it possible to excite molecules in volumes of much less than 1 fl. In two-photon flash photolysis (TPFP) this property is used to release effector molecules from caged precursors with high three-dimensional resolution. We describe and examine the benefits of using TPFP in model solutions and in a number of cell systems to study their spatial and temporal properties. Using TPFP of caged fluorescein, we determined the free diffusion coefficient of fluorescein (D=4 x 0(-6) cm(2)/s at 20 degrees C, which is in close agreement with published values). TPFP of caged fluorescein in lens tissue in situ revealed spatial nonuniformities in intercellular fiber cell coupling by gap junctions. At the lens periphery, intercellular transport was predominantly directed along rows of cells, but was nearly isotropic further from the periphery. To test an algorithm aiming to reconstruct the Ca(2+) release flux underlying physiological Ca(2+) signals in heart muscle cells, TPFP of DM-Nitrophen was utilized to generate artificial microscopic Ca(2+) signals with known underlying Ca(2+) release flux. In an experiment with mouse oocytes, the recently developed Ca(2+) cage dimethoxynitrophenyl-ethyleneglycol-bis-(beta-aminoethylether)-N,N,N('),N(') tetraacetic acid-4 (DMNPE-4) was released in the oocyte cytosol and inside a nucleolus. Analysis of the resulting fluorescence changes suggested that the effective diffusion coefficient within the nucleolus was half of that in the cytosol. These experiments demonstrate the utility of TPFP as a novel tool for the optical study of biomedical systems.
Soeller
C
C
University of Auckland, School of Medical and Health Sciences, Department of Physiology, Private Bag 92019, Auckland, New Zealand. c.soeller@auckland.ac.nz
Jacobs
M D
MD
Jones
K T
KT
Ellis-Davies
G C R
GC
Donaldson
P J
PJ
Cannell
M B
MB
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D020013:Calcium Signaling / Q000502:physiology* / Q000528:radiation effects; D002450:Cell Communication / Q000502:physiology / Q000528:radiation effects; D002478:Cells, Cultured; D003198:Computer Simulation; D017630:Connexins / Q000378:metabolism*; D046508:Culture Techniques; D004058:Diffusion; D005240:Feasibility Studies; D019793:Fluorescein / Q000737:chemistry / Q000528:radiation effects; D017629:Gap Junctions / Q000378:metabolism / Q000528:radiation effects; D007834:Lasers; D007908:Lens, Crystalline / Q000378:metabolism* / Q000528:radiation effects; D051379:Mice; D008810:Mice, Inbred C57BL; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D008954:Models, Biological; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000528:radiation effects; D009865:Oocytes / Q000378:metabolism* / Q000528:radiation effects; D010782:Photolysis; D051381:Rats; D014018:Tissue Distribution
D017630:Connexins; D019793:Fluorescein