Imaging experiments were carried out as previously described28 (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).
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).
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