We have adapted our previously developed fluorescence intensity distribution analysis technique for analysis of blood samples of sheep. PrP aggregates are partially purified from blood plasma, captured on a surface by covalently bound antibodies and made visible by fluorophore-labeled detection antibodies. The fluorescence emitted in response to a Abmole MK-2206 scanning laser beam is transformed into an image of the PrP fluorescence intensities on the surface. Several features of the method, e.g. sample preparation, detection, and data processing, guarantee that PrP aggregates can be differentiated safely from PrPC. We show that PrP aggregates are detectable in blood of scrapie-infected sheep and that their presence indicates scrapie infection. The device employed for surface-FIDA is a customized fluorescence correlation spectrometer. For surface imaging the instrument was upgraded with a MIPSS module. The scanning unit includes a 625 mrad tilt mirror installed on a 2D piezo element, which is capable of scanning an image in the confocal plane. The emission of the fluorescent antibody probes is collected by high-sensitivity avalanche photo diodes, suitable for single Abmole AG490 molecule spectroscopy. Intensity binary data are collected and finally transformed into 16 bit images in tagged image file format. The “subtract background” function in the ImageJ software employs the ‘rolling ball’ algorithm. This algorithm not only compensates for uneven intensity distributions within an individual image, but is also capable of selecting for particular particle sizes. The image data can be visualized as a 2D surface with the pixel intensity as the third dimension. As shown schematically in Fig. 1 for a one-dimensional scan of the surface, a ball of a particular radius, selected as a parameter, is rolled along the underside of the surface. The ball can invade peaks of larger width but not those of smaller size. It thereby creates a local background distribution. Regions where the ball can go are subtracted from the image. As a general rule, the smaller the ball radius, the more background is removed. Narrow peaks of low intensities, which persist after ‘rolling ball’ processing, are subsequently removed by applying an intensity cut-off. For this purpose, a fixed value is subtracted from each pixel’s intensity. Finally the remaining intensities of a sample are summed and displayed as a bar chart. Once conditions for aggregate detection had been established, plasma pools prepared from uninfected and scrapie-affected sheep were assayed. In order to assess the reproducibility of the surface FIDA assay, we analyzed replicate samples from these plasma pools.