Binding of the chimeric, humanized anti-CD20 mAb Rituximab (RTX) to B lymphocytes activates complement and promotes covalent deposition of C3 fragments (C3b/iC3b) on cells. Previous fluorescence microscopy studies, based on examination of B cell lines and of blood samples from RTX-treated CLL patients, suggest that C3b/iC3b is closely associated with cell-bound RTX. We examined Raji cells opsonized with serum and RTX with the ImageStream imaging flow cytometer. Cells were stained with fluorescently-labeled RTX and mAbs specific for C3b/iC3b fragments or for human IgG, and then imaged using the ImageStream cytometer and analyzed with an algorithm (Similarity Bright Detail Score, SBDS) which tests for co-localization of fluorescent probes. SBDS, calculated on 10,000 cells, verified that the majority of deposited C3b/iC3b is co-localized with bound RTX. In contrast, when cells were first opsonized in serum alone, washed and then reacted with RTX, SBDS confirmed that RTX and C3b/iC3b are poorly co-localized, thus demonstrating that cell-bound RTX directs deposition of C3b. In addition, a sulfhydryl-specific probe, maleimide conjugated to AF488, exhibited substantial co-localization with an anti-C3b/iC3b mAb on Raji cells opsonized with RTX and serum, thus validating maleimide labeling as an alternative for detecting cell-bound C3b/iC3b. The digital imaging method described should have wide applicability for quantitative analysis of co-localization.