Tilt-angle stimulated Raman projection tomography.

Stimulated Raman projection tomography is a label-free volumetric chemical imaging technology allowing three-dimensional (3D) reconstruction of chemical distribution in a biological sample from the angle-dependent stimulated Raman scattering projection images. However, the projection image acquisition process requires rotating the sample contained in a capillary glass held by a complicated sample rotation stage, limiting the volumetric imaging speed, and inhibiting the study of living samples. Here, we report a tilt-angle stimulated Raman projection tomography (TSPRT) system which acquires angle-dependent projection images by utilizing tilt-angle beams to image the sample from different azimuth angles sequentially. The TSRPT system, which is free of sample rotation, enables rapid scanning of different views by a tailor-designed four-galvo-mirror scanning system. We present the design of the optical system, the theory, and calibration procedure for chemical tomographic reconstruction. 3D vibrational images of polystyrene beads and C. elegans are demonstrated in the C-H vibrational region.

Integration of sleep homeostasis and navigation in Drosophila.

During sleep, the brain undergoes dynamic and structural changes. In Drosophila, such changes have been observed in the central complex, a brain area important for sleep control and navigation. The connectivity of the central complex raises the question about how navigation, and specifically the head direction system, can operate in the face of sleep related plasticity. To address this question, we develop a model that integrates sleep homeostasis and head direction. We show that by introducing plasticity, the head direction system can function in a stable way by balancing plasticity in connected circuits that encode sleep pressure. With increasing sleep pressure, the head direction system nevertheless becomes unstable and a sleep phase with a different plasticity mechanism is introduced to reset network connectivity. The proposed integration of sleep homeostasis and head direction circuits captures features of their neural dynamics observed in flies and mice.

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

Two-photon imaging in behaving animals is typically accompanied by brain motion. For functional imaging experiments, for example with genetically encoded calcium indicators, such brain motion induces changes in fluorescence intensity. These motion-related intensity changes or motion artifacts can typically not be separated from neural activity-induced signals. While lateral motion, within the focal plane, can be corrected by computationally aligning images, axial motion, out of the focal plane, cannot easily be corrected. Here, we developed an algorithm for axial motion correction for non-ratiometric calcium indicators taking advantage of simultaneous multi-plane imaging. Using temporally multiplexed beams, recording simultaneously from at least two focal planes at different z positions, and recording a z-stack for each beam as a calibration step, the algorithm separates motion-related and neural activity-induced changes in fluorescence intensity. The algorithm is based on a maximum likelihood optimisation approach; it assumes (as a first order approximation) that no distortions of the sample occurs during axial motion and that neural activity increases uniformly along the optical axis in each region of interest. 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.

A place learning assay for tethered walking Drosophila.

BACKGROUND: Drosophila shows a range of visually guided memory and learning behaviors, including place learning. Investigating the dynamics of neural circuits underlying such behaviors requires learning assays in tethered animals, compatible with in vivo imaging experiments. NEW METHOD: Here, we introduce an assay for place learning for tethered walking flies. A cylindrical arena is rotated and translated in real time around the fly in concert with the rotational and translational walking activity measured with an air supported ball, resulting in a mechanical virtual reality (VR). RESULTS: Navigation together with heat-based operant conditioning allows flies to learn the location of a cool spot with respect to a visual landmark. Flies optimize the time and distance required to find the cool spot over a similar number of trials as observed in assays with freely moving flies. Additionally, a fraction of flies remembers the location of the cool spot also after the conditioning heat is removed. COMPARISON WITH EXISTING METHODS: Learning tasks have been implemented in tethered flying as well as walking flies. Mechanically translating and rotating an arena in concert with the fly's walking activity enables navigation in a three dimensional environment. CONCLUSION: In the developed mechanical VR flies can learn to remember the location of a cool place within an otherwise hot environment with respect to a visual landmark. Implementing place learning in a tethered walking configuration is a precondition for investigating the underlying circuit dynamics using functional imaging.

Automated long-term two-photon imaging in head-fixed walking Drosophila.

BACKGROUND: 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. NEW METHOD: Here, we develop a technique for automated long-term two-photon imaging in fruit flies, during wakefulness and extended bouts of immobility, as typically observed during 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. RESULTS: The approach is validated in the fly's head direction system and walking behavior as well a neural activity are recorded. The head direction system tracks the fly's walking direction over multiple days. COMPARISON WITH EXISTING METHODS: In comparison with previous head-fixed preparations, the time span over which tethered behavior and neural activity can be recorded at the same time is extended from hours to days. Additionally, the reproducibility and ease of dissections are improved compared with manual approaches. Different from previous laser surgery approaches, only continuous wave lasers are required. Lastly, an automated feeding system allows continuously maintaining the fly for several days in the virtual reality setup without user intervention. CONCLUSIONS: Imaging in behaving flies over multiple timescales will be useful for understanding circadian activity, learning and long-term memory, or sleep.