Imager two microscope

Representative images were acquired with a Zeiss LSM700 confocal microscope using a Plan-Apochromat 40×/1.4 objective. Worms were immobilized using 1.5% 1-phenoxy-2-propanol (TCI America, Portland, OR) in M9 buffer and mounted on 5% agar slides. 3D reconstructions were done using Zeiss Zen software. A Zeiss Axio Imager two microscope equipped with Chroma HQ filters was used to score AMsh migration defects and take images to measure Migration Index. Experiments were conducted in triplicates consisting of at least 50 day one adult animals each.

Imaging was performed in an upright Zeiss Axio Imager two microscope with a 470 nm LED and a GYR LED (CoolLED) with a dual-band excitation filter F59-019 and dichroic F58-019 (Chroma) in the microscope turret. Emission filters ET515/30M and ET641/75 and dichroic T565lprx-UF2 were placed in the cube of a Cairn OptoSplit II attached between the microscope and an ORCA-Flash four camera (Hamamatsu). Acquisition was performed at 20 fps. Imaging during mating was performed with a 20x long working distance objective (LD Plan-NEOFLUAR numerical aperture 0.4), placing the male on an agar pad with food and 20 hermaphrodites. The ~50 mm per side agar pad was cut out from a regular, seeded NGM plate and placed on a glass slide. The hermaphrodites were placed in a ~ 100 mm² centre region. A fresh pad was used every two recordings.
Imaging in restrained animals was performed for 2.5–3 min with a 63x objective (LD C- apochromat numerical aperture 1.15). Animals were glued with Wormglu along the body to a 5% agarose pad on a glass slide and covered with M9 or 20 mM histamine (Sigma, H7125) and a coverslip.

Bright field images were acquired on a Zeiss Axio Imager two microscope. Fluorescent images were taken using Leica TCS SP8 and TCS SPE laser confocal microscopes with either 10x, 20x, 40x or 63x objectives; all fluorescent pictures are Z-stack confocal images, unless stated otherwise. Images were processed using Fiji ImageJ or Photoshop software (Adobe) and exported to Illustrator vector-based software (Adobe) for figure generation.