Stain-free histopathology by programmable supercontinuum pulses

The preparation, staining, visualization, and interpretation of histological images of tissue is well-accepted as the gold standard process for the diagnosis of disease. These methods were developed historically, and are used ubiquitously in pathology, despite being highly time and labor intensive. Here we introduce a unique optical imaging platform and methodology for label-free multimodal multiphoton microscopy that uses a novel photonic crystal fiber source to generate tailored chemical contrast based on programmable supercontinuum pulses. We demonstrate collection of optical signatures of the tumor microenvironment, including evidence of mesoscopic biological organization, tumor cell migration, and (lymph-)angiogenesis collected directly from fresh ex vivo mammary tissue. Acquisition of these optical signatures and other cellular or extracellular features, which are largely absent from histologically processed and stained tissue, combined with an adaptable platform for optical alignment-free programmable-contrast imaging, offers the potential to translate stain-free molecular histopathology into routine clinical use.