MEASUREMENTS OF MULTIPLE PARAMETERS IN FLUOROMETRY

FLUORESCENCE INTENSITY, LIFETIME AND ANISOTROPY

Abstract

Fluorescence measurement of intensity, anisotropy and lifetime with Hoechst 33258 (H33258) and ethidium bromide (EB) fluorochromes were applied for structural study of DNA. The fluorescence of complexes of H33258 with DNA, chromatin and nucleosome core with and without NaCl were compared. The complex with the nucleosome core showed the lowest intensity of the three complexes, but the highest anisotropy in the absence of NaCl. At the higher NaCl concentration than 1M, the complexes with the chromatin and nucleosome core showed the same fluorescence as that of DNA complex. The anisotropic fluorescence with H33258 was applied for the determination of cell number suspended in a solution. A background fluorescence due to unreacted free dye was optically removed, without removal of the dye, through the polarized component of the fluorescence. The fluorescence decay measurement was carried out to determine the molar ratio of DNA- phosphorus to dye (P/D ratio) in situ in cell nuclei stained with EB. The fluorescence lifetime of DNA-EB complex increased with the increase of the P/D ratio.