title: Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa.
abstract: The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ 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 Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (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 molecules [Formula: see text] 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 Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence.
authors.affiliation: Institute of Medical Biotechnology, Friedrich-Alexander-University Erlangen-Nuernberg, 91052 Erlangen, Bavaria, Germany.|Erlangen Graduate School in Advanced Optical Technologies (SAOT), Friedrich-Alexander-University Erlangen-Nuernberg, 91052 Erlangen, Bavaria, Germany.
authors.forename: D
authors.identifier: http://orcid.org/0000-0001-7351-6428
authors.initials: D
authors.lastname: Schneidereit
authors.affiliation: School of Physics and Astronomy, University of Edinburgh, Edinburgh EH9 3FD, Scotland, United Kingdom.
authors.forename: H
authors.initials: H
authors.lastname: Vass
authors.affiliation: Institute of Medical Biotechnology, Friedrich-Alexander-University Erlangen-Nuernberg, 91052 Erlangen, Bavaria, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Reischl
authors.affiliation: School of Physics and Astronomy, University of Edinburgh, Edinburgh EH9 3FD, Scotland, United Kingdom.
authors.forename: R J
authors.initials: RJ
authors.lastname: Allen
authors.affiliation: Institute of Medical Biotechnology, Friedrich-Alexander-University Erlangen-Nuernberg, 91052 Erlangen, Bavaria, Germany.|Erlangen Graduate School in Advanced Optical Technologies (SAOT), Friedrich-Alexander-University Erlangen-Nuernberg, 91052 Erlangen, Bavaria, Germany.
authors.forename: O
authors.initials: O
authors.lastname: Friedrich
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry*; D001572:Benzofurans / Q000737:chemistry; D002021:Buffers; D002118:Calcium / Q000032:analysis* / Q000737:chemistry; D004533:Egtazic Acid / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006874:Hydrostatic Pressure; D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D009994:Osmolar Concentration; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014966:Xanthenes / Q000737:chemistry*
chemical_list: D000814:Aniline Compounds; D001572:Benzofurans; D002021:Buffers; C409648:Fluo 4; D005456:Fluorescent Dyes; D007093:Imidazoles; D014966:Xanthenes; C082877:fura red; D004533:Egtazic Acid; D002118:Calcium