A system is disclosed for measuring immunofluorescence of a thin sample layer mounted on a substrate. A sample is located in a radiation beam having a spacially-varying amplitude, for example a series of nodes and antinodes. The sample layer is small to comparison to the period over which the radiation intensity varies, but the support is large in comparison to the period. Thus, when the sample and support are moved relative to the radiation beam, the fluorescence signal from the sample layer will be significantly affected, while the signal from the support is substantially unaffected. The difference between two measurements at different relative orientations of the source and radiation beam can provide a measure of the fluorescence of the sample layer independent of any background scatter of fluorescence of the support. In a preferred embodiment, relative movement between the sample and radiation beam is provided to subject the sample to a time-varying radiation amplitude, and the fluorescence of the sample is obtained from a varying component of the detected signal.