Simultaneous measurement of fluorescence and phosphorescence using synchronously gated photon counters

John C. Sutherland, George D. Cimino, Edmond J. Desmond

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

The fluorescence (prompt emission) and phosphorescence (delayed emission) from a sample can be separated and recorded simultaneously by digital processing of the photon-induced electronic pulses they generate. A single rotating sector wheel (or "chopper") periodically interrupts the beam of radiation incident upon the sample. Both prompt and delayed emissions from the sample pass through the same optical system to a photomultiplier. The electronic pulses they generate are processed by the same amplifiers and discriminator. Separation of the prompt and delayed components is a two-stage process. Pulses produced while the chopper is blocking the exciting beam are stored in one counter, the contents of which are a measure of the delayed emission. Pulses produced while the chopper is open are stored in another counter. The contents of this counter represent contributions of both prompt and delayed emissions. The contents of both counters are transferred to a computer for postexperimental deconvolution of the fluorescence and phosphorescence. Since the two components are measured simultaneously, the ratio of phosphorescence to fluorescence cannot be affected by changes in the sample as with sequential scans. The optical path, electronic amplification, and gating periods are identical so fluorescence and phosphorescence are measured with equal efficiency. This two-step method of signal processing recovers data which are lost in other arrangements.

Original languageEnglish (US)
Pages (from-to)158-165
Number of pages8
JournalAnalytical Biochemistry
Volume97
Issue number1
DOIs
StatePublished - Aug 1979
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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