A method for measuring the concentration of a free ion in a cell with the use of a fluorescence - probes dye and apparatus for carrying out the method

摘要

Intracellular ion concn. is measured using a fluorescent dye probe (I) which is introduced into the cell and free ion concn. measured from the intensity of fluorescence excited. The process comprises: (1) measuring intensity of fluorescence excited, at 3 different wavelengths, in a set of solns. contg. (I) and free ion at various concns.; (2) measurements, as in (1), for a set of solns. contg. (I) and an interfering biosubstance (II), other than the free ion, which binds with (II) and ion causing a change in fluorescent intensity; (3) measurement a set of solns. of (II), (I) and free ion; (4) establishing initial values of the 3 types of equilibrium constants (reactions of (I) with free ion, (II) and intracellular complexes) and of the 12 types of fluorescent coefficients (for calculating fluorescent intensities) used to provide relations between (I), free ion, (II) and complexes; estimating error value, between estimated and measured fluorescence intensities and iterative asymptotic correction the modified simplex method until the error values are within the permitted range; and (5) measuring intensity of fluorescence excited in cells at the same 3 wavelengths and solving the simultaneous equations for fluorescence intensity (by inserting the measured values) and the equilibrium reaction equations to give a value for the free ion concn. USE/ADVANTAGE - Esp. used to measure distribution and variation in Ca or Mg ion concn. within cells. Compared with conventional 2-wavelength methods, the workload involved is reduced. It can account for errors caused by interaction of (I) with components other than the analyte.