Open AccessCalcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa
Author(s) -
Dominik Schneidereit,
H. Vass,
Barbara Reischl,
Rosalind J. Allen,
Oliver Friedrich
Publication year - 2016
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0164509
Subject(s) - fluorescence , calcium , hydrostatic pressure , fura 2 , chemistry , ionic strength , analytical chemistry (journal) , biophysics , chromatography , aqueous solution , biochemistry , optics , organic chemistry , physics , cytosol , biology , thermodynamics , enzyme
The fluorescent Ca 2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca 2+ fluctuations in vitro as well as in situ . The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca 2+ -buffered solutions at different fixed concentrations of free Ca 2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye moleculesΔ d v ¯is determined to -17.8 ml mol -1 for Fluo-4 and -21.3 ml mol -1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca 2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence.