Label-free visualization of biological structures in lung and parathyroid biopsies using higher harmonic generation microscopy

Higher harmonic generation microscopy (HHGM) is a technique to image fresh tissue without any preparation or staining. Recently, we increased the number of simultaneously detected channels from 3 to 4, to include third harmonic generation (THG), second harmonic generation (SHG) and two- and three-photon excited autofluorescence (2PEF and 3PEF) to visualize cells, collagen, elastin and other fluorescent molecules. We used a commercialized HHG-microscope to image lung and parathyroid biopsies, which both have a clinical need for fast pathological confirmation during surgery. We compared the newly added 3PEF signals with the original THG, SHG, and 2PEF signals and we found that 3PEF visualizes fat cells, fibrin fibers and cell cytoplasm, providing additional information on biological structures in tissue and better cellular contrast.