Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor patient outcome often resulting from late diagnosis in advanced stages. To date methods to diagnose early-stage PDAC are limited and in vivo detection of pancreatic intraepithelial neoplasia (PanIN), a preinvasive precursor of PDAC, is impossible. Using a cathepsin-activatable near-infrared probe in combination with flexible confocal fluorescence lasermicroscopy (CFL) in a genetically defined mouse model of PDAC we were able to detect and grade murine PanIN lesions in real time in vivo. Our diagnostic approach is highly sensitive and specific and proved superior to clinically established fluorescein-enhanced imaging. Translation of this endoscopic technique into the clinic should tremendously improve detection of pancreatic neoplasia, thus reforming management of patients at risk for PDAC. is one of the deadliest human malignancies, with an extremely poor 5-y survival rate below 5% (1). Because patients generally present with symptoms relating to late stages of the disease, diagnosis of resectable PDAC is achieved in less than 15% of cases (1). However, investigators have reported that diagnosis and resection of early-stage PDAC (<2 cm in size) can result in a 4-y survival rate of up to 78% (2-5). These data suggest that early detection of PDAC can improve patient outcome. In addition, it has been shown that resection of PDAC combined with adjuvant chemotherapy can improve 5-y survival rates to 25%. Thus, increasing the chance of resection will lead to improved survival.Of the more than 33,000 new cases of PDAC diagnosed in the United States every year, ∼10% occur in families with a high prevalence of PDAC and are thus referred to as familial (6, 7). In a cohort study of twins, it was proposed that inherited factors may be responsible for up to 36% of pancreatic cancers, suggesting that the incidence of familial pancreatic cancer may be even higher than presently assumed (8). To date, several conditions associated with familial PDAC have been described and groups of individuals at low, moderate, or high risk for developing the disease have been defined (9, 10). Although clinical trials are currently testing screening protocols for the high-risk group, aimed at detecting curable precursors of PDAC such as intraductal papillary mucinous neoplasia, pancreatic intraepithelial neoplasia (PanIN), and early-stage PDAC (10-12), the results of these trials show that early lesions are often missed. Furthermore, false positive findings can lead to overtreatment of a significant fraction of the screened population (12). These findings show that better diagnostic tools for the detection of preneoplastic lesions and early-stage PDAC are urgently needed. Recent data demonstrate that preinvasive precursors progress slowly over many years to decades to invasive pancreatic cancer (13). The parental pancreatic cancer founder clone then requires more than 5 y to acquire the capacity to metastasize (13). Thus, there is a time frame of several years for the diagnosi...