Lwd 16x 0.8na water immersion objective

For live confocal imaging between 4 and 11 dpf larvae were anesthetized in 0.016% tricaine and embedded in 2% low-melting-point agarose. Imaging was performed on a Nikon C2 confocal microscope equipped with solid state lasers for excitation of EGFP (488 nm) and mCherry/TagRFP (555 nm). Whole-brain imaging of larvae was performed using a Nikon LWD 16x 0.8NA water immersion objective using 1-1.5 µm z-steps. Larvae with single labeled neurons were imaged using a Nikon 60x 1.0NA water immersion objective and 0.375-0.5 µm zsteps. For timelapse recordings, laser power was lowered to <1% and z-stacks with 0.6-1µm zsteps were acquired every 10 min for 4 hrs, yielding 25 image volumes. For PSD95-EGFP/DsRed imaging, z-stacks were acquired every 20 min for 4 hrs to prevent photodamage due to the increased number of scans per z-stack due to two-channel acquisition. Additionally, prior to each timelapse acquisition, laser power and detector gain for both channels were adjusted to yield images in which spines with bright PSD95-EGFP puncta had PSD95-EGFP/DsRed intensity ratios close to 1.0. This circumvented the need to normalize ratio values when combining data from multiple neurons (Figure 4F-G).