Accurate whole-spectrum measurements of intracellular pH and [Na(+)].
Fluorescent measurements of intracellular H(+) and Na(+) are improved by using whole spectra of the fluorescent indicators BCECF and SBFI, respectively. The extra data in whole spectra enable both an accurate calibration and a ready detection of artifacts which are not possible to identify using a more conventional data analysis that relies upon only two wavelength "windows" in the fluorescence spectra. The whole-spectrum technique is applicable to cell suspensions in a conventional fluorimeter (as is reported here with SBFI), as well as to attached cells using a fluorimeter combined with an inverted epifluorescence microscope. The spectral method was highly reproducible in that pairs of successive pH measurements differed, on average, by only 0.01±0.02 U. Random uncertainty from sample to sample was estimated numerically from the standard deviation of measurements on ionophore-treated cells. When full-spectrum analysis was employed, this scatter showed a two-fold improvement over results obtained using the two-wavelength ratio method. Because SBFI has a relatively narrow dynamic range, whole-spectrum analysis has been applied to improve the accuracy of sodium determinations. The calibrated system measured [Na(+)]i with excellent linearity over the range 2-150 mM and with an accuracy of approximately 5 mM.
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 19107, Philadelphia, Pennsylvania.
C S
CS
Owen
M L
ML
Wahl
D B
DB
Leeper
H D
HD
Perry
S B
SB
Bobyock
M
M
Russell
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W
Woodward