High Performance Phase Fluorometer Constructed from Commercial Subunits

Abstract

Using new commercial equipment including a Pockels-effect light modulating device, a phase fluorometer with the following characteristics has been constructed: high intensity light with a high degree of modulation (typically m=43% at 1 kV modulating voltage and 462 mμ); pure sine modulation of variable frequency (14.0110, 21.0170, 27.0040 Mc); narrow wavelength regions for excitation and emission; very small contamination of detected light with exciting light (<1%); spectral range of the modulated light from 200 to 1000 mμ. The phase difference between fluorescent light and scattered light is measured with a high precision wide band phasemeter employing fixed and variable coaxial delay lines. The output of the phase detector is recorded and precise phasenull found graphically. Maximum time resolution is 0.02 nsec. The average over-all precision in general use is Δτ/τ=2% in the range from 2 to 63 nsec; a wider range can be measured with slightly reduced precision. Systematic errors caused particularly by electron transit time in the photomultipliers depending on voltage, exposed area of photocathode, wavelength, and photocurrent are investigated for two photomultipliers (EMI 9558B and RCA 6217), and procedures to control these errors are described. Measurements of fluorescence liftetimes of five organic compounds at very low concentrations show a lack of dependence of the results on type of photomultiplier tube, photocurrent, multiplier voltage, type of scatterer, modulating frequency, and emission wavelength. This suggests that the accuracy of the measurements is very nearly equal to precision and is limited by the experimental error in sample preparation rather than the accuracy of the measurements themselves. Some lifetime results reported are 10−6 M fluorescein in 0.01 M KOH, τ=(3.83±0.01) nsec; 10−5 M 9,10-diphenylanthracene in 95% ethanol, τ=(7.91±0.02) nsec; acridone in water (0.01×saturated solubility), τ=(15.54±0.05) nsec; 1.3×10−6 M safranin T in pyridine containing 5% water, τ=(2.29±0.01) nsec; 3×10−7 M chlorophyll-a in 95% ethanol, τ=(5.55±0.03) nsec. Some effects of oxygen quenching and concentration are also reported. The instrument allows analysis of complex decay kinetics by varying the excitation modulation frequency and selecting narrow wavelength intervals in the emission. A wide range of excitation and emission intensities can be employed for studies of intensity dependence of liftetimes as is necessary in studies of natural photosynthesis.