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Application of two-photon flash photolysis to reveal intercellular communication and intracellular Ca2+ movements

Lookup NU author(s): Professor Keith Jones

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Abstract

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 × 0-6 cm 2/s at 20°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 Ca2+ release flux underlying physiological Ca2+ signals in heart muscle cells, TPFP of DM-Nitrophen was utilized to generate artificial microscopic Ca2+ signals with known underlying Ca2+ release flux. In an experiment with mouse oocytes, the recently developed Ca2+ cage dimethoxynitrophenyl-ethyleneglycol-bis-(β-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. © 2003 Society of Photo-Optical Instrumentation Engineers.

Publication metadata

Author(s): Soeller C, Jacobs MD, Donaldson PJ, Cannell MB, Jones KT, Ellis-Davies GCR

Publication type: Article

Publication status: Published

Journal: Journal of Biomedical Optics

Year: 2003

Volume: 8

Issue: 3

Pages: 418-427

Print publication date: 01/07/2003

ISSN (print): 1083-3668

ISSN (electronic): 1560-2281

Publisher: International Society for Optical Engineering

URL: http://dx.doi.org/10.1117/1.1582468

DOI: 10.1117/1.1582468

PubMed id: 12880347

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