A Label-Free Ultraviolet Photoacoustic Microscopy Enables Nuclear Imaging and Toxicity Assessment in an Intact Brain...

Fetal alcohol spectrum disorders (FASDs) are caused by prenatal exposure to ethanol (EtOH), leading to developmental brain abnormalities. Cortical organoids derived from human-induced pluripotent stem cells provide physiologically relevant models to study such neurotoxic effects. However, accurate imaging of nuclear morphology, a key indicator of cytotoxicity and developmental impairment, remains difficult. Traditional fluorescence-based techniques rely on staining and sectioning, limiting throughput and potentially altering native structures. Herein, we present an ultraviolet photoacoustic microscopy system that targets endogenous nucleic acids at 266 nm excitation, resulting in a lateral resolution of 278 nm, which is sufficient to resolve individual nuclei in situ. The system integrates precise z-axis scanning for focal-plane alignment, enabling high-resolution depth-resolved imaging of subvolumes within 3D intact live organoids without physical sectioning. Using EtOH-treated cortical organoids as a model of FASD-associated neurotoxicity, we observed significant reductions in nuclear area, diameter, and circularity by 46.1%, 20.8%, and 6.0%, respectively, indicating structural damage consistent with apoptosis and impaired neurodevelopment. This method is the first to demonstrate label-free nuclear imaging in intact live brain organoids, providing a robust and preparation-free platform for probing disease-relevant phenotypes, accelerating drug screening, and enabling early toxicity assessments in neurodevelopmental disorder models.