Genetically-encoded gas vesicles bridge mesoscopic ultrasound and cellular-resolution microscopy

Understanding complex biological systems requires imaging strategies that connect organism-level readouts with cellular resolution. Multiscale imaging approaches typically rely on combining distinct reporter systems for each modality, rather than a single genetically-encoded structure that generates multimodal contrast from the same physical source. Gas vesicles are gas-filled protein nanostructures that have been established as ultrasound contrast agents. Here we show that the same nanostructures also generate optical contrast in linear reflection microscopy and third harmonic generation microscopy. In engineered bacteria, gas vesicles produce strong third harmonic signals with cubic power dependence. In complex tissues, third harmonic signals are comparable in intensity to endogenous sources, enabling reliable detection against a physiological background. Gas vesicles remain optically detectable after phagocytosis by macrophages, enabling cellular-resolution mapping of the tissue sources underlying mesoscopic ultrasound contrast. The gas-filled architecture further confers selective sensitivity to focused multiphoton irradiation, enabling confined microscale ablation of gas vesicle positive cells and tissue regions. Reflection signals persist through aqueous tissue clearing and allow correlation of in vivo ultrasound imaging with ex vivo light-sheet microscopy of intact tumors. Together, these findings establish gas vesicles as genetically encodable reporters that connect mesoscopic ultrasound imaging with cellular-resolution microscopy and enable integrated imaging and actuation across scales.