A capillary fluorescence standard for microfluorometry

Quartz glass capillaries of about 6 pm inside diameter were filled with fluorochrome solutions of accurately known concentrations and embedded as microscopical objects. The fluorescence intensity of a constant capillary volume has been found to be directly proportional to the amount of the excited fluorochrome in this volume within the fluorochrome concentration range 1 : 10(3). The capillary volume can be determined accurately by microinterferometric measurement of the inside diameter. The capillary section irradiated by the exciting light may be considered as very small in comparison with the total length of the capillary tube. During and after irradiation the excited part of fluorochrome solution is continuously renewed by convectional flow and diffusion. Therefore photodecomposition does not affect the measured fluorescence intensity, and the standard value remains unchanged even after numerous measurements. Thus, these fluorescence standards may be used to make successive calibrations of a microfluorometer and to check the constancy and reproducibility of measuring conditions. In certain circumstances it also permits measured fluorescence intensity to be interpreted in terms of the amount of fluorochrome or fluorescent substance. The results achieved with the capillary fluorescence standard are compared with those obtained by means of conventional methods. In addition, some essential optical properties of the microfluorometer equipment required for the proper use of this standard are discussed.