Seeing Natural Images through the Eye of a Fly with Remote Focusing Two-Photon Microscopy

Visual systems of many animals, including the fruit fly Drosophila, represent the surrounding space as 2D maps, formed by populations of neurons. Advanced genetic tools make the fly visual system especially well accessible. However, in typical in vivo preparations for two-photon calcium imaging, relatively few neurons can be recorded at the same time. Here, we present an extension to a conventional two-photon microscope, based on remote focusing, which enables real-time rotation of the imaging plane, and thus flexible alignment to cellular structures, without resolution or speed trade-off. We simultaneously record from over 100 neighboring cells spanning the 2D retinotopic map. We characterize its representation of moving natural images, which we find is comparable to noise predictions. Our method increases throughput 10-fold and allows us to visualize a significant fraction of the fly's visual field. Furthermore, our system can be applied in general for a more flexible investigation of neural circuits.