Calorimetric, Photometric and Lifetime Determinations of Fluorescence Yields of Fluorescein Dyes*

Abstract

Abstract— The fluorescence yields and lifetimes of fluorescein and nine brominated derivatives in basic ethanol are reported. Calorimetric. photometric, and lifetime methods are used independently to measure the yields. A new and simple calorimetric method is presented for this purpose, and the accuracy of the techniques is assessed. There is good agreement between the calorimetric and photometric results. The importance of parameters such as purity, pH, and fluoresence reabsorption is illustrated. The theoretical determination of the natural radiative lifetime from the absorption spectrum is shown to have inherent ambiguities, so that only calorimetric methods provide a reliable, independent check for photometric yield measurements.

Intersystem-crossing and excited-state absorption in eosin Y solutions determined by picosecond double pulse transient absorption measurements

“…The radiative lifetimes r rad are determined by application of the Strickler-Berg formula [43,44] (for reported T RAD data of eosin Y see refs. [11,[45][46][47][48]). The fluorescence lifetime T s 1 is given by eq.…”

SspB delivery of substrates for ClpXP proteolysis probed by the design of improved degradation tags

“…Concentrations were determined by UV absorbance at 280 nM, using extinction coefficients of 19,770 M Ϫ1 ⅐cm Ϫ1 (GFP-ssrA and variants), 84,480 M Ϫ1 ⅐cm Ϫ1 (ClpX 6 ), 125,160 M Ϫ1 ⅐cm Ϫ1 (ClpP 14 ), 12,090 M Ϫ1 ⅐cm Ϫ1 (SspB and SspB-SBD), and 1,280 M Ϫ1 ⅐cm Ϫ1 (ssrA peptide). The concentration of fluorescent ssrA peptide was determined in basic ethanol (pH Ϸ10) by using an extinction coefficient at 500 nM of 92,300 M Ϫ1 ⅐cm Ϫ1 (29). Note that concentrations of SspB are reported in monomer equivalents.…”

Absolute quantum efficiencies

“…Several important factors favor calorimetric methods for the determination of absolute quantum yields: (a) The method serves as a valuable independent check of the assumptions used in photometric techniques for measuring <l>J; in particular, corrections for experimental geometry, index of refraction changes, and polarization effects are largely eliminated. (b) Using relatively crude apparatus, some workers have obtained values of Yh with a precision comparable to those obtained by other methods [29]. (c) The calorimetric method works well with samples that emit in…”

“…With the usual procedures and components, the principal disadvantages and limitations of the method have been the following: (a) The insensitivities of the calorimeters used thus far have forced workers to use highly absorbing samples; consequently, large corrections for reabsorption of the fluorescence [29,104] Recent advances in calorimetry, especially in the technology of light sources and transducers, and improvements in methods of data acquisition portend wider acceptance of the technique for obtaining quantum efficiencies. The generality, sensitivity, and ease of performance of calorimetric methods will undoubtedly improve.…”

“…An especially important fundamental problem con· cerns the limits of applicability of the Strickler·Berg equation [28] for obtaining radiative lifetimes of excited states from the inverse absorption processes [29]. Serious discrepancies exist between the measured natural radiative lives (To ), as obtained from the equation To = Tflcf>f whe~e Tf is the fluorescence decay time ~nd <P f is the fluorescence quantum yield, and those calculated by the Strickler·Berg relation.…”