Sectioning-free virtual H&E histology with fiber-based two-photon microscopy

The routine pathology workflow relies on cutting tissue into single-cell layer thick slices using paraffin or frozen sectioning. We propose a fast method to obtain sections of equivalent quality optically using the strong sectioning capabilities of two-photon microscopy (TPM). Hematoxylin and eosin (HE) equivalent staining of the tissue is achieved using acridine orange and sulforhodamine 101. We improved our previously presented pulsed fiber laser to deliver adjustable pulse durations of ~30ps at repetition rates of up to 16MHz and kW peak power. We can now image up to one square centimeter of tissue with sub-micrometer resolution within 15 minutes.