Measurement of very short fluorescence lifetimes by single-photon counting

Abstract

Measurement of very short fluorescence lifetimes by the single-photon technique is made possible by an improved fluorescence lifetime system. Fluorescence lifetimes of 4.94±0.07 nsec for anthracene in cyclohexane, 640±30 psec for diphenyl butadiene in cyclohexane, and 90±30 psec for erythrosin in water were determined. The use of a small wavelength shift between excitation and emission minimizes the effect of the wavelength dependence of the photomultiplier response and light pulser emission. The effects of deaeration of solutions and time averaging of the excitation profile are presented. We investigated the origin of small-amplitude early and late artifactual peaks in the light pulser and fluorescence profiles. Complications in the analysis of lifetime data introduced by intrinsic fluorescence and phosphorescence processes in commonly used absorption filters are discussed. Certain ’’blind spots’’ are found in the electronic pulse pileup rejection schemes most commonly used in photon counting.

Picosecond fluorescence kinetics and energy transfer in chloroplasts and algae

“…All experiments were performed using the dye rhodamine 6G, and laser excitation pulses were at 620 nm. The output of the photomultiplier is amplified and input to a constant fraction discriminator [27,28]. One output of the constant fraction The output of the photodiode triggers a level discriminator whose output provides a pulse to the TAC which stops the voltage ramp.…”

“…We have investigated these problems by studying the fluorescence kinetics in chloroplasts and algae with a picosecond resolution, singlephoton timing instrument [27][28][29]. ·We found three different kinetic components for open as well as for closed photosystem II reaction centers.…”

Time correlation method for measuring fluorescence decays with a cw laser

“…8 The signal from each detector is a random response, R(t), which is the convolution of the source and detector, (1) The correlation of the responses of the two detectors as a function of the difference, 6.t, between the time delays that separate them from the source, (2) is the Fourier transform ofthe power spectrum of R(t).9 A more direct and less costly approach to obtain the same information is to observe correlation between the intensity of the laser and the intensity of the fluorescence from the sample which it excites.…”

Nanosecond Fluorescence Spectroscopy

“…It was ob served that the red emission from the air gap spark discharge is broader in time and has a significantly longer tall component than the ultraviolet emission. 3 Generally, to minimize the system wavelength depend ence, which may arise from both photomultlpHer and light pulser effect, it 1s important to use the same light pulser-fllter 1n recording both the excitation and fluorescence profiles and to minimize the differ ence between the excitation and fluorescence emission wavelengths* Data Analysis, A number of theoretical approaches have been de veloped to extract the amplitude, «•(, and lifetime, Ti, values from the experimentally determined excita tion and fluorescence profiles. These approaches are discussed in more detail 1n the Numerical Analysis section of this paper.…”

“…Also, the difference between excita tion and fluorescence wavelength 1s chosen to be as small as practically possible. 3 Over the past decade most single-photon counting measuring systems have employed as a light source ei ther a relaxation-type spark gap light pulser or a synchronously pumped mode-locked dye laser. The re laxation-type light pulser systems* are generally sim pler and considerably 1 ess expensive than the modelocked laser based ones.…”