P 725

Cells were imaged using a spinning-disk confocal microscope (Nikon Ti2000, Yokugawa CSU-X1) with 1.5x magnification lens and 1.2x tube lens, an EM-CCD camera (Hamamatsu), a 60x water-immersion objective (Nikon), an objective piezo stage (P-725, Physik Instrumente), and motorized x-y stage (ProScan II, Prior Scientific) controlled by μManager (Edelstein et al., 2014 (link)). A 488 nm laser and 514/30 filter were used to image mNeonGreen-Nuf2, and a 560 nm laser and 593/40 filter were used to image INCENP-mCherry. 11–15 z-slices, separated by 2 μm, were taken for each time point. Three time points, separated by a minute, were acquired before and after DMSO (for untreated data), 10 μM taxol, or 10 μM 5-ITu treatment.

Imaging experiments were carried out as previously described^{28 (link)} using a two-photon microscope with a movable stage (Thorlabs Bergamo II) and a fast piezoelectric objective scanner (Physik Instrumente P725) for volumetric imaging. We used a Chameleon Vision-S Ti-sapphire femtosecond laser tuned to 940 nm for two-photon excitation. Images were collected using a 20× 0.95-NA objective (Olympus). Emission fluorescence was filtered with a 525-nm bandpass filter (Thorlabs) and collected using a GaAsP photomultiplier tube (Hamamatsu).
For EPG neurons, the imaging region was centred on the protocerebral bridge, where EPG axons terminate. The imaging view was 256 × 128 pixels, and 8–12 slices deep in the z axis (4-6 µm per slice), resulting in a 6–9 Hz volumetric scanning rate. For ExR2 neurons, the imaging region was centred on the bulb and ellipsoid body. The imaging view was 256 × 128 pixels, and 12 slices deep in the z axis (6-8 µm per slice), resulting in a 6–7 Hz volumetric scanning rate.
Volumetric z-scanning signals from the piezoelectric objective scanner were acquired simultaneously with analog output signals from the visual panorama and/or analog outputs from FicTrac 2.1 through a NiDAQ PCI-6341 at 40 kHz. Data were acquired using ScanImage 2018 (Vidrio Technologies) with National Instruments hardware from Vidrio (NI PXIe-6341).