Beyond fluorescence: Advanced precision imaging using genetically encoded bioharmonophores

Observing life down to a cellular resolution provides the best means for understanding the mechanisms underpinning tissue physiology, maintenance, injury response and ageing. Intravital light microscopy has revolutionised our ability to witness cellular behaviours in their native context and thus track dynamics in real time. The development of various genetically encoded fluorescent proteins has introduced powerful and versatile imaging tools. The ease of expressing fluorescently tagged proteins or obtaining fluorescently marked cell populations through straightforward genetic methods has yielded a wealth of information about the function and behaviour of many biological processes in development and disease.

Nevertheless, there remain many areas in which significant improvement is urgently needed. Biologists are keen to image more deeply into living tissues and organs, yet imaging depth is limited by both the scattering and absorption of visible light in tissue. Conventional fluorescent probes also suffer from photobleaching, which refers to the irreversible destruction of the dye when excited for extended periods or with excessive intensity. Additionally, signal saturation creates an intrinsic limitation on fluorophore brightness even under intense illumination – limiting imaging sensitivity particularly in deeper tissue where signal detection is already challenging. Furthermore, the number of cell types that can be observed simultaneously within the same tissue volume remains limited, as the broad and often overlapping signal profiles of fluorescent dyes greatly reduce specificity.

To overcome these challenges, we propose the development of transformative genetically encoded bioharmonophores as superior optical markers for precision imaging. Bioharmonophores emit a distinct signal known as second harmonic generation (SHG). This signal, which can be generated and detected using two-photon microscopy — a widely established technique in biomedical research —...