Ld lci plan apochromat objective

Images were collected using silicone-oil immersion media with a 40x multi-immersion LD LCI Plan-Apochromat objective (Zeiss, NA = 1.2; silicone oil immersion media) on a Zeiss LSM 880 microscope. Single apical cilia were imaged at zoom 10 with 4 Airy units (146 μm pinhole), 212 x 212 frame size, 0.1 μm pixel size, 0.93 μs pixel dwell time, 16-bit bidirectional scanning, and 4 optical sections (1.8 μm) to cover the entire length of the cilium. The resulting temporal resolution was 0.25 s. The donor fluorophore, sGFP2 was excited by the 488 nm laser. Donor emission from mScarlet-I was collected with the spectral detector set to 490-550 nm. Acceptor-sensitized emission was collected with the spectral detector set to 570-650 nm.
For uncaging experiments, the following uncaging parameters were used on a 5 μm radius circle adjacent to the ciliary tip: repeat each stack x10 using the same pixel dwell time as during scanning. Two experiment blocks (400 time frames each) were used to acquire data with and without uncaging. Uncaging started at frame 20 in the first block, with no uncaging during the second block.

Hippocampal neurons expressing GRAB-HTR6-cilia and serotonergic neurons expressing hM3Dq or farnesylated SNAP-Tag:JF552 were imaged with a 40x multi-immersion LD LCI Plan-Apochromat objective (Zeiss, NA = 1.2; silicone oil was used as the immersion media) on a Zeiss LSM 880 microscope in artificial cerebral spinal fluid (NaCl 124 mM, KCl 2.5 mM, NaH₂PO₄ 1.2 mM, NaHCO₃ 24 mM, HEPES 5 mM, glucose 12.5 mM, MgSO₄ 2mM, CaCl₂ 2mM, Ting et al., 2018 (link)) at 37°C. Two-channel FAST Airyscan images were first imaged to characterize the axociliary synapses using 488 nm, 561 nm, or 594 nm excitations for GFP, JF552, and mCherry, respectively. GRAB-HTR6-cilia were then imaged at every 5 s using the 488 nm laser with z-stacks. After 1 min, 10 nM DCZ was directly added to the imaging media. A total of 60 z-stacks were acquired per cilium (3 min).