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 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.
Highlights
Biochemical reactions in living cells and organisms are strongly influenced by changes in environmental variables such as pH, ionic strength, temperature and pressure
The impact of temperature and pH on chemical equilibria, activation energies and reaction volumes is well-known for many enzymatic biochemical reactions, the effect of pressure as an environmental variable of biochemical equilibria, has only been elucidated in selected enzymatic or cellular systems [2,3,4]
We found that the relative fluorescence intensity of Fluo-4 changes at a rate 0" of -0.308 ±0.017% per MPa of hydrostatic pressure (Fig 6A)
Summary
Biochemical reactions in living cells and organisms are strongly influenced by changes in environmental variables such as pH, ionic strength, temperature and pressure. The impact of temperature and pH on chemical equilibria, activation energies and reaction volumes is well-known for many enzymatic biochemical reactions, the effect of pressure as an environmental variable of biochemical equilibria, has only been elucidated in selected enzymatic or cellular systems [2,3,4]. Fluorescence and Buffer Properties of Fluo-4 and Fura Red under High-Pressure
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