The aperture-defined microvolume (ADM) method: automated measurements of enzyme activity using an inverted fluorescence microscope.

The aperture-defined microvolume (ADM) method is based on the relatively constant absorbance or fluorescence of a microvolume of homogeneously coloured material, which is defined by the numerical aperture of the objective. This paper describes the principle of the method and discusses the equipment needed. The main applications reported so far for the measurement of enzyme activity are reviewed. Among these are the quantification of ELISA and DASS tests used in immunology, kinetic studies of enzymes in solution using fluorogenic substrates, and the measurement of enzyme activity in single cells or cell fractions that have been isolated by flow sorting. Typical characteristics of automated ADM measurements include a coefficient of variation of less than 3%, a lower detection limit of a few nanogrammes of fluorescing dye (e.g. 4-methylumbelliferone) and a linear relationship between fluorescence yield and fluorophore concentration over a range of 0.01 to 2.5 nmol. The scanning of Terasaki-type trays and 96-well microtitration plates can be completely automated and requires approximately one minute.