Feasibility of dual-contrast fluorescence imaging of pathological breast tissues

The combination of intravital dye, methylene blue (MB), with molecular cancer marker, pH low insertion peptide (pHLIP) conjugated with fluorescent Alexa532 (Alexa532-pHLIP), was evaluated for enhancing contrast of pathological breast tissue ex vivo. Fresh, thick breast specimens were stained sequentially with Alexa532-pHLIP and aqueous MB and imaged using dual-channel fluorescence microscopy. MB and Alexa532-pHLIP accumulated in the nuclei and cytoplasm of cancer cells, respectively. MB also stained nuclei of normal cells. Some Alexa532-pHLIP fluorescence emission was detected from connective tissue and benign cell membranes. Overall, Alexa532-pHLIP showed high affinity to cancer, while MB highlighted tissue morphology. The results indicate that MB and Alexa532-pHLIP provide complementary information and show promise for the detection of breast cancer.