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

Abstract

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.

How Flies See Motion

“…Each neuropil repeats the columnar structure of the facet eye, giving rise to a highly ordered, almost crystalline, retinotopic structure (Figure 2b). The retinotopy is evident from the anatomy (Figure 2a) as well as from recent functional experiments, in which tilting the imaging plane of a two-photon microscope provided a direct glimpse of the retinotopic activity map (Schuetzenberger & Borst 2020). Throughout the four neuropils, approximately 100 different cell types can be found within each column (Figure 2c).…”

Three-photon excited fluorescence imaging in neuroscience: From principles to applications

“…Remote focusing is an optical refocusing technique that is implemented remotely from the specimen ( Anselmi et al, 2011 ; Thériault et al, 2014 ; Cheng Z. et al, 2020 ; Jiang et al, 2020 ; Durst et al, 2021 ). As shown in Figure 4E , this module typically uses a remote objective and a movable lightweight mirror to refocus the imaging plane within the specimen ( Schuetzenberger and Borst, 2020 ). When the mirror moves perpendicularly to the light, the imaging plane will shift relative to the nominal focal plane.…”