Simultaneous dual-region two-photon imaging of biological dynamics spanning over 9 mm in vivo

Biodynamical processes, especially in system biology, that occur far apart in space may be highly correlated. To study such biodynamics, simultaneous imaging over a large span at high spatio-temporal resolutions is highly desired. For example, large-scale recording of neural network activities over various brain regions is indispensable in neuroscience. However, limited by the field-of-view (FoV) of conventional microscopes, simultaneous recording of laterally distant regions at high spatio-temporal resolutions is highly challenging. Here, we propose to extend the distance of simultaneous recording regions with a custom micro-mirror unit, taking advantage of the long working distance of the objective and spatio-temporal multiplexing. We demonstrate simultaneous dual-region two-photon imaging, spanning as large as 9 mm, which is 4 times larger than the nominal FoV of the objective. We verify the system performance in in vivo imaging of neural activities and vascular dilations, simultaneously, at two regions in mouse brains as well as in spinal cords, respectively. The adoption of our proposed scheme will promote the study of systematic biology, such as system neuroscience and system immunology.