A Spectrophotofluorometer for Measuring Very Weak Fluorescences From Biological Molecules

Abstract—A spectrophotometer using the photon‐counting method is described. The sample cuvette is mounted on a table which can be displaced in the X and Y directions, permitting investigation of absorption and self‐absorption processes within the cuvette. Fluorescence detection is performed at right angles to the direction of the exciting beam, the analysis grating being driven by a stepping motor. The apparent spectrum is fed into a multichannel analyser and printed. Corrections are made to the data provided by the printer. Signal‐to‐noise ratio, fluorescence spectra and fluorescence excitation spectra are discussed in more detail for the case of more concentrated solutions. This very high sensitivity device allows room‐temperature investigation of the fluorescent emission from neutral aqueous solutions of nucleic acid bases, nucleosides, nucleotides, dinucleotides etc., quantum yields for these molecules being of the order of 10‐4. A table of minimum concentration values of some commonly used fluorescent probes is given.