Acetic Acid as a Cell Nucleus Contrast Agent in Third-harmonic Generation Microscopy

Backward-collected higher-harmonic optical microscopy can provide histological information from unstained fresh samples, having great potential for noninvasive in vivo evaluation of microscopic structural changes that underlie disease. Second-harmonic generation (SHG) microscopy can identify distribution of extracellular matrix fibrillar components. The imaging contrast of third-harmonic generation (THG) microscopy originates from optical heterogeneities in the specimen. In order to differentiate benign from malignant disease, detailed cell nucleus morphology and its relative size compared to cytoplasm are important. Acetic acid has been utilized to increase imaging contrast in reflection confocal microscopy of epithelial tissue. In this study, we evaluated the forward and backward propagation signals in fresh lung specimens before and after application of acetic acid using higher-harmonic optical microscopy based on a Cr: forsterite femtosecond laser centered at 1230-nm. We found that acetic acid is a good contrast agent for cell nucleus in THG microscopy.