Flash4.0 v3 scmos camera

All whole-mount immunostaining images were collected with a Nikon A1R point scanning confocal with spectral detection and resonant scanner on a Nikon Ti-E inverted microscope equipped with a Plan Apo VC ×20 objective (NA 0.75). Alexa-488, Alexa-594, Alexa-647 fluorophores coupled to secondary antibodies were excited with the 488 nm, 561 nm, and 647 nm laser lines from a Spectral Applied Research LMM-5 laser merge module with solid-state lasers (selected with an AOTF) and collected with a 405/488/561/647 quad dichroic mirror (Chroma). For time-lapse experiments, images were acquired with a Yokagawa CSU-X1 spinning disk confocal on a Nikon Ti inverted microscope equipped with a Plan Apo ×20 objective (NA 0.75) and a Hamamatsu Flash4.0 V3 sCMOS camera. Samples were grown on six-well glass-bottom multiwell plates with no. 1.5 glass (Cellvis, Cat# P06-1.5H-N) and mounted in a OkoLab 37°C, 5% CO₂ cage microscope incubator warmed to 37°C. Images were collected every 15 min using an exposure time of 800 ms. At each timepoint, 30 z-series optical sections were collected with a step size of 2 µm. Multiple-stage positions were collected using a Prior Proscan II motorized stage. Z-series are displayed as maximum z-projections, and gamma, brightness, and contrast were adjusted (identically for compared image sets) using Fiji/ImageJ (Schindelin et al., 2012 (link); https://imagej.net/Fiji).

All fluorescence microscopy experiments were performed using a DMi8 inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 100×, 1.47 numerical aperture (NA) Plan-Apochromat oil immersion lens, a Flash 4.0 v3 sCMOS camera (Hamamatsu, Shizuoka, Japan), an LED3 fluorescence illumination system, 488 nm and 561 nm lasers, a W-View Gemini image splitting optical device (Hamamatsu), compatible filter sets for fluorescence and DIC imaging and the LAS X v3.7.6.25997 software (Leica). Image acquisition parameters (illumination intensity, exposure time and camera binning) were consistently applied within each trial of an experiment to allow comparison between strains.
Images were processed following acquisition by importing them into Fiji/ImageJ2 v2.9.0/1.53t. Linear adjustments were made to minimum and maximum intensity levels for each 16-bit image within an experiment prior to conversion into 8-bit format for figure preparation.