An extension module for a two-photon microscope enables flexible in vivo imaging and all-optical physiology.

Two-photon imaging is essential for revealing the structure, function, and interaction of various cell types. However, conventional two-photon microscopes have limitations in sample accessibility and optogenetic manipulation during imaging. Here, we present Ex2p/Ex2pO, an open-source extension module that adds an objective lens rotation axis and one-photon optical stimulation synchronized with the non-imaging intervals of resonant scanning to a two-photon microscope. The Ex2p/Ex2pO can be seamlessly integrated into existing microscopes by simply replacing the standard objective lens assembly without any modifications. It enables imaging of curved structures while maintaining the subject's natural posture and simultaneous imaging and one-photon stimulation with low crosstalk between stimulation and fluorescent light. We demonstrate one-photon stimulation during two-photon imaging of mice behaving in a virtual reality environment and pluripotent stem cell-derived cortical organoids. Thus, Ex2p/Ex2pO expands the versatility to investigate neural circuit motifs in behaving animals and organoids, enabling all-optical physiology in existing two-photon imaging systems.