The Denver Universal Microspectroradiometer (DUM)

SUMMARY This paper discusses the design and describes the construction of an accurate digital microspectrophotometer, microspectrofluorimeter and microrefractometer known as the ‘Denver Universal Microspectroradiometer’ (DUM). The instrument operates in the spectral range of between 428.3 and 1303.4 THz (700–230 nm). Microscopic objects are scanned digitally in two dimensions and absolute digital spectra of various kinds are obtained automatically. Optical path differences can be measured automatically in the same microdomains in which absorbance, fluorescence and bioluminescence are studied. Measurements on solutions in standard cuvettes, and other macroscopic objects, can be made without altering the alignment of the optical components used in microanalysis. The multi‐detector radiometric system is based on the principle of synchronous single photoelectron counting. The operation of the entire system is controlled by an integral computer which is also used for on‐line data‐processing, reduction and analysis. Automatic multiparametric and multicomponent analyses can be carried out with relative ease. The quality of the images obtained is largely diffraction‐limited, and the instrumental uncertainty of measurement is essentially that imposed by the quantum statistics of the radiation captured by the detectors. The linear dynamic range is about 10 8 . The limit of detectability of absorbance is of the order of 0.0001. The system is sensitive enough to measure paucimolecular photoemissive events, such as cellular bioluminescence, yielding about 0.1s −1 photoelectrons (equivalent photocathode current ≃3 times 10 −22 A). The absolute accuracy of the system is limited principally by uncertainties inherent in currently available methods of calibrating the spectral response of detectors.