Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.

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Double-strand breaks (DSBs) are the most lethal DNA damages induced by ionising radiation (IR) and their efficient repair is crucial to limit genomic instability. The cellular DSB response after low IR doses is of particular interest but its examination requires the analysis of high cell numbers. Here, we present an automated DSB quantification method based on the analysis of γH2AX and 53BP1 foci as markers for DSBs. We establish a combination of object properties, combined in the object evaluation parameter (OEP), which correlates with manual object classification. Strikingly, OEP histograms show a bi-modal distribution with two maxima and a minimum in between, which correlates with the manually determined transition between background signals and foci. We used algorithms to detect the minimum, thus separating foci from background signals and automatically assessing DSB levels. To demonstrate the validity of this method, we analyzed over 600.000 cells to verify results of previous studies showing that DSBs induced by low doses are less efficiently repaired compared with DSBs induced by higher doses. Thus, the automated foci counting method, called AutoFoci, provides a valuable tool for high-throughput image analysis of thousands of cells which will prove useful for many biological screening approaches.

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Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSimaTM Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at sub-cellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinityTM), release of antibodies (REAleaseTM) or their labels (REAdyeleaseTM). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 normal tissues. One potential target pair for chimeric antigen receptor (CAR) T-cell therapies identified for ovarian carcinoma is EPCAM/THY1. Using an adapter CAR T cell approach, we show selective killing of cells only in presence of both markers. MICS represents a new high content microscopy methodology to be widely used for personalized medicine.