Nanoscanz nz400ce nano positioning piezo z stage

Fluorescence images were acquired with an upright microscope (BX61, Olympus) equipped with a Photometrics Coolsnap HQ2 cooled CCD camera, a 10 × objective lens (0.3 NA, UPLFLN, Olympus), two optical shutters (one for fluorescence and one for white light), an excitation filter (500/20 nm) and emission filter (535/30 nm), a z-stage system (NanoScanZ NZ400CE Nano-positioning Piezo Z Stage, Prior Scientific) and a X-CITE® Exacte light source equipped with a closed feedback-loop. Based on DIC micrographs of the first worm positioned in the imaging chamber, we defined the initial focal plane which remained the same throughout the experiment as no big variations in posture were observed among serially confined worms. For image acquisition, the exposure time was tuned to 160 ms to avoid extensive animal movement and the z-resolution was set to 2.5 μm, totaling 30 slices for each z-stack. For post-experimental image analysis, out-of-focus frames were discarded. A threshold (70% of maximum intensity) was applied to in-focus z-slices to determine the number and fluorescence intensity of foci. Fluorescence was calculated as the average intensity in the inclusion region, subtracted by the average intensity of the background (a larger region of the animal with no YFP fluorescence).