Absolute Spectral Sensitivity of Phototubes and the Application to the Measurement of the Absolute Quantum Yields of Chemiluminescence and Bioluminescence*

Abstract

Methods forthe absolute measurement of thespectral sensitivity of phototubes are reviewed in detail. Several experimental arrangements are described for the independent determination of these values to insure the elimination of systematic error. On the basis of these measurements a rapid, simple technique is described for measuring the average absolute spectral sensitivity of a phototube over a defined emission spectrum, thus obviating the original tedious point-by-point methods and allowing frequent re-Standardization. The application of these standardized phototubes to the measurement of the absolute quantum yields of the chemiluminescence of luminol and the bioluminescence of luminous bacteria and the firefly is described. An absolute low-level light emission standard in the form of the luminol chemiluminescence reaction, either in aqueous or in the non-aqueous solvent dimethylsulfoxide is proposed for general use and the conditions for its use are described in detail. It should therefore be possible for any researcher to obtain an absolute calibration, in his own experimental geometry, in the blue region of the spectrum. I N T R O D U C T I O NIN THE elucidation of the molecular mechanisms of chemiluminescence and bioluminescence and for both military and commercial applications of chemiluminescent reactions, it is extremely important to know the quantum yield; that is the number of light quanta emitted per substrate molecule reacting. In general this requires the determination, aside from the chemical stoichiometry, of the emission spectrum of the light reaction, the absolute spectral photon sensitivity of the phototube used and the physical geometry of the experiment. Seliger and McElroy'l) described a measurement of the absolute spectral sensitivity of a phototube by a standard thermopile substitution technique with an overall accuracy of f 15 per cent. The phototube was then used to measure the quantum yields of the in vitro firefly bioluminescence reaction and later, the quantum yield of the luminol chemiluminescence reaction in aqueous solution.(2)

The Spectral Distribution of Firefly Light. II

“…This included 20 species, both American and J a m a i c a n , measured by photoelectric spectrometry (2,3). T h e present paper extends the d a t a to include an additional 15 American species.…”

Measurements of absolute values in biochemical fluorescence spectroscopy

“…By choosing suitable gratings and phototubes, it is sometimes possible to obtain linear response in regions of visible fluorescence as well, thus avoiding the need for point-by-point correction of spectra. The accuracy of detector calibration depends o~ such factors as the photometric accuracy of the system and the accuracy of the standard lamp calibrati:m. Lee and Seliger [17] have considered at length all the possible errors involved in absolute calibrations of detector systems. Their interest was principally the determination of chemiluminescent quantum yields, and it was necessary to know the relation between the number of photons and the detector response.…”

Thermolysis of the tricyclic 1,2-dioxetanes 3,4:3,4-dipropano-, 3,4-pentano-3,4-propano-, and 3,4-butano-3,4-propano-1,2-dioxetane

“…The four tricyclic 1,Zdioxetanes 1, 5, 6, and 7 undergo thermolysis with energies of activation 4This value is based on the luminol light standard (8). The use of the luminol standard should yield apparent excited state yields that are smaller by a factor of -2.5 (9) than those determined on the basis of the radioactive light standard (10) which is often used (le, 2a).…”