Automated long-term two-photon imaging in head-fixed walkingDrosophila

Abstract

The brain of Drosophila shows dynamics at multiple timescales, from the millisecond range of fast voltage or calcium transients to functional and structural changes occurring over multiple days. To relate such dynamics to behavior requires monitoring neural circuits across these multiple timescales in behaving animals. Here, we develop a technique for automated long-term two-photon imaging in fruit flies, during wakefulness and sleep, navigating in virtual reality over up to seven days. The method is enabled by laser surgery, a microrobotic arm for controlling forceps for dissection assistance, an automated feeding robot, as well as volumetric, simultaneous multiplane imaging. The approach is validated in the fly's head direction system. Imaging in behaving flies over multiple timescales will be useful for understanding circadian activity, learning and long-term memory, or sleep.

Axial motion estimation and correction for simultaneous multi-plane two-photon calcium imaging

“…The algorithm was further validated with simulated data with multiple one dimensional voxels, representing regions of interests (ROIs) defined on a three dimensional sample (see next section and Methods for details). For this, neural activity in a ring attractor was simulated, similar to experimentally observed data 22,26 . A random distribution of the axial position of the different ROIs was assumed, reflecting structural variability (Fig.…”

“…Therefore, we defined a template, consisting of the 10 first images acquired during the experiment, and all images, including those of the z-stacks, were aligned with respect to this template using a phase correlation algorithm (as implemented in Skimage 27 , see Methods). After alignment, the 10 z-stacks were averaged and a total of N = 32 ROIs were defined along the dendritic toroidal structure as described previously 22 (Fig. 4A, right side).…”

“…The experimental setup is shown in Fig. 1B and was previously described 22 . Simultaneous two-plane imaging was implemented with two temporally offset beams (by 6 ns) recorded independently using temporal multiplexing 17 .…”

“…Finally, motion was corrected in a naturally moving brain during in vivo calcium imaging in a fly walking in a virtual reality setup 7,22 , without fixing the fly's proboscis or removing muscles in the head. Motion estimation and correction proceeded as described in the previous section.…”

“…The developed motion correction approach allows axial motion estimation and correction at high frame rates for isolated structures in the imaging volume in vivo, such as sparse expression patterns in the fruit fly brain.Similarly, naturally occurring fiducials such as cell bodies are often not present in the imaging volume. Further, often only a single fluorescent dye, for instance a non-ratiometric calcium indicator, is used.Alternatively to recording volume images in z-stacks, simultaneous multi-plane imaging allows recording a limited number of focal planes at the same time [17][18][19][20][21][22] . Similar to imaging in a single focal plane, multi-plane recordings are still subject to axial motion artifacts.…”