Inadequacy of high K+/nigericin for calibrating BCECF. I. Estimating steady-state intracellular pH.
AuthorsBoyarsky G, Hanssen C, Clyne LA
JournalAm J Physiol
PubMed ID8897819
'Intracellular pH (pHi) was measured in single vascular smooth muscle (VSM) cells, cultured from rabbit abdominal aorta, using 2'',7''-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) on a microscope-based fluorescence system. Three lines of evidence are presented that using nigericin along with high external K+ to calibrate intracellular BCECF produces systematic errors in pHi. 1) The ... More
Spectral and photophysical studies of benzo[c]xanthene dyes: dual emission pH sensors.
AuthorsWhitaker JE, Haugland RP, Prendergast FG
JournalAnal Biochem
PubMed ID1862936
'A series of fluorescent, long-wavelength, benzo[c]-xanthene dyes has been characterized for pH measurement in both excitation and emission ratio applications. The two general classes of these indicators are seminaphthofluoresceins (SNAFLs) and seminaphthorhodafluors (SNARFs) which are substituted at the 10-position with oxygen or nitrogen, respectively. These probes show separate emissions from ... More
Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance.
AuthorsCarlsson K, Liljeborg A, Andersson RM, Brismar H
JournalJ Microsc
PubMed ID10947903
'We investigate the performance of confocal pH imaging when using phase fluorometry and fluorophores with pH-dependent lifetimes. In these experiments, the specimen is illuminated by a laser beam, whose intensity is sinusoidally modulated. The lifetime-dependent phase shift in the fluorescent signal is detected by a lock-in amplifier, and converted into ... More
Fluorescence lifetime-resolved pH imaging of living cells.
AuthorsLin HJ, Herman P, Lakowicz JR
JournalCytometry A
PubMed ID12655651
BACKGROUND: The regulation and maintenance of intracellular pH are critical to diverse metabolic functions of the living cells. Fluorescence time-resolved techniques and instrumentations have advanced rapidly and enabled the imaging of intracellular pH based on the fluorescence lifetimes. METHODS: The frequency-domain fluorescence lifetime imaging microscopy (FLIM) and fluorophores displaying appropriate ... More