The Determination of the Fluorescence Lifetimes of Dissolved Substances by a Phase Shift Method

Abstract

The lifetimes of fluorescent substances are determined by exciting the fluorescence with light modulated at a high frequency and measuring the difference in phase between the exciting light and the resulting fluorescence. The exciting light is modulated by an ultrasonic standing wave in a liquid which causes the latter to act as an intermittent diffraction grating. Photomultiplier tubes are used to pick up the exciting and fluorescent light. Tuned circuits are used to isolate a single harmonic of the signal from the phototub~s and the phase shift determined by comparison with a calibrated phase shifting circuit. The phase angle q,isrelated to the lifetime bytanq,=wT. In whichw is 2,.. times the frequency of modulation and T is the lifetime of the excited state. The data show that lifetimes can be determined with a precision of 1 to 2X 10-10 second.The lifetimes of acridone in neutral solution (saturated to 10 percent saturated) and quinine sulfate (4X 10-6 to 4X 10-o M) in 0.01 M nitric acid are independent of the concentration. The values found are acridone 1.59±0.01XlO-s second, and quinine sulfate 2.28XO.02XlO-s second. The lifetime of fluorescein in 0.OO5M potassium hydroxide increases with concentration over the range 10-6 to 5X lO-4M. This fact can be explained by assuming that some of the fluorescent light is absorbed and re-emitted. The lifetime of fluorescein extrapolated to zero concentration is 4.5±0.lX10-9 second.

Phase shift cavity ring down absorption spectroscopy

“…It was already shown in 1933 [ 15] that if this intensity modulated light beam were to excite an atomie Al ready in the mid-fifties lifetimes of ftuorescing substances in the ns range were measured via this phase shift metbod with 1% or better precision [ 16,17]. Nowadays the phase shift metbod is still in use to distinguish species on the basis of their lifetime in twodimensional ftuorescence imaging [ 18].…”

A calibration equation for oxygen optodes based on physical properties of the sensing foil

“…Lifetime of the excited fluorophores in the foil relative to the excitation light is a much better measurement of oxygen quenching than intensity. Lifetime is measured, indirectly, by the phase delay between the excitation and emission (Bailey and Rollefson 1953). An additional red LED is included as a non-excitation reference as a means to compensate for potential drift in the electronics of the transmitter and receiver circuit.…”

Recent Developments in Frequency-Domain Fluorometry

“…This is somewhat surprising in that phase-modulation fluorometers were constructed prior to the development of time-domain instruments [10][11][12][13][14].…”