Confidence in fluorescence lifetime determinations: a ratio correction for the photomultiplier time response with wavelength

Abstract

Can. J. Chem. 54,3246 (1976).The photomultipliers used in single photon counting fluorescence lifetime systems have a time response function which varies with wavelength. This variation results in a reduced confidence in the kinetic parameters obtained after deconvolution of the lamp intensity decay profile measured a t the excitation wavelength from the sample emission decay profile measured a t the emission wavelength. Moreover in the case of multi-component decays, inaccurate kinetic parameters are calculated because of this PMT variation. Using weighted residual plots, we demonstrate how one may make a satisfactory subjective appraisal of the adequacy of the deconvolution and the physical model of the decay processes. We have developed a ratio correction technique which accounts for the PMT time response variation with wavelength. This technique requires no a priori assun~ption of the nature of the PMT effect. Only when the data are corrected using the ratio correction technique can sufficient confidence in the accuracy of the singlet excited state parameters and the validity of the physical model be obtained. Chem. 54, 3246 (1976).Les photomultiplicateurs utilisis dans les systkmes de comptage des photons uniques pour determiner le temps de vie de fluorescence ont une fonction de temps de rCponse qui varie avec la longueur d'onde. Cette variation conduit B une confiance rCduite dans Ies paramktres cinCtiques obtenus aprks dCconvolution du profil de decroissance de I'intensitC de la lampe mesure a ia longueur d'onde d'excitation, B partir du profil de decroissance de I'Cmission de 1'Cchantillon mesurC B la longueur d'onde d'dmission. De plus dans le cas de dCcroissance B plusieurs composants, les parametres cinitiques qui ne sont pas justes sont calculCs 2 cause de cette variation PMT. Faisant appel B des courbes rCsiduelles pondCrCes, on dCmontre comment on peut faire une Cvaluation subjective satisfaisante de l3adCquation de la dCconvolution et du modkle physique des processus de dCcroissance. On a developpk une technique de correction par des rapports qui tient compte de la variation du temps de rCponse PMT avec la longueur d'onde. Cette technique ne requiert aucune hypothese a priori sur la nature de l'effet PMT. Ce n'est que lorsque les donnCes sont corrigCes en faisant appel B la technique de correction par des rapports que l'on peut obtenir une confiance suffisante dans I'exactitude des paramktres d'Ctats excitCs singulets et dans de la validit6 du modele physique.[Traduit par le journal]The increase in souhistication of the ~h o t o -multi-com~onent fluorescence decavs are likelv physical interpretation of excited state kinetics to be observed, demand a high degree of con-(1-4) and in the utilization of fluorescence life-fidence in the experimental determination of the times to study biological systems (5-lo), where form of the fluorescence decay function.In ~u l s e fluorimeters the fluorescence decay of INRCC 115449.

Excited state acid–base equilibrium of tyrosine

“…In aqueous tyrosine solutions of p H 13 Cornog and Adams (1 5) have observed weak fluorescence, quantum yield 0.006 $-0.001 (adjusted t o Chen's (1 1) value for tyrosine), which can be attributed to the tyrosinate species (12). In water -dimethylsulphoxide-ethylene glycol solvent mixture the quantum yield for tyrosinate is given as 0.03 compared with 0.23 for tyrosine (10).…”

“…Tyrosine fluorescence quantuni yields were calculated from fluorescence intensity measurements taken at a fixed wavelength by assuming Chen's (1 1) value of 0.14 for the quantum yield of tyrosine in water at neutral pH. Tyrosinate fluorescence quantum yields were calculated in a similar manner relative to the value of Cornog and Adanis for tyrosinate (12) revised to 0.006 to be relative to Chen's value for tyrosine. Where necessary intensity measurements were first corrected for background fluorescence measured from blank solutions identical to the sample solutions in every respect save the presence of tyrosine.…”

Photothermal Lensing Signal Enhancement by the Transient Absorption of Photoexcited States in Liquid Solutions

“…It is apparent that by changing the timing delay, we can optimize the large gain, although we did not do so in the present experiment. The proportionality constant (χ) was calculated with reference values having lifetimes of 1.3 ns for POPOP in ethanol 19 and 5.0 ns for anthracene in ethanol 19,20 to be 3.6 × 10 -2 and 1.4 × 10 -1 (M cm mJ -1 ), respectively. These values are used in a later discussion.…”

Multiplexed single-photon counting. I. A time-correlated fluorescence lifetime camera

“…The earliest approach to overcoming this restriction attempted to reduce flashlamp drift, a common source of error in the reconvolution process. [23][24][25] The recording of the instrumental profile and decay was alternated at short intervals by mechanically rotating the scatter medium and the sample into the optical path, and collecting each in a different MCA memory segment. A recent refinement to this approach left the sample position fixed and switched the light path between scatter medium and sample using prisms.…”