Quantum counter for correcting fluorescence excitation spectra at 320- to 800-nm wavelengths

A procedure for recording corrected fluorescence excitation spectra to wavelengths as long as 800 nm is described. The procedure involves the use of a commercial spectrofluorometer, which is modified by substituting 1,1',3,3,3',3'-hexamethylindotricarbocyanine perchlorate in place of rhodamine B as the quantum counter dye. This modification is applicable to spectrofluorometers supplied by several different manufacturers and can be accomplished by a user having only modest technical skills. A study of the fluorescence excitation spectrum of bacteriochlorophyll a is presented as an illustration of the use of the procedure. The procedure will be valuable in biological and biochemical studies that involve the use of long-wavelength fluorescent probes of either natural or synthetic origin.