Flash 4 scmos camera

Fully polarized CFBE cells or fully differentiated pHBE cells were mechanically injured by scraping a sterile P10 pipette tip across the cell monolayer. For cell wounding PBS was added to the apical side of the filters. After wounding the apical surface was washed twice with PBS to remove cell debris. Fresh media was added to the basolateral (on both cell types) and apical side (only on CFBE cells).
Wound closure was monitored by live cell imaging (48 h, 37 °C, 5% CO₂) with an automated Leica DMI6000 widefield microscope coupled to a Hamamatsu Flash4 sCMOS camera, using a HCX 4x W 4×/0.1 objective. Images were taken every 2–3 h. Software used for acquisition was Leica’s LAS x, and image processing was performed on ImageJ FIJI^{39 (link)}. FIJI was used to segment, and measure wound area. Wound closure was then calculated as $\mathrm{Wound}\mathrm{size}\left(\%\right)=\left(A_t/A_0\right)\times 100$ , where A_t is the area for a given time point and A₀ is the initial wound area. Wound size was plotted as a function of time (h) and was used to calculate the rate (slope) of wound closure (%/h).

Following fixation in 4% PFA overnight, half of the right kidney was cryoprotected in 30% sucrose overnight, embedded in Optimal Cutting Temperature (OCT) medium and sectioned at 10 μm. For IF staining, sections were fixed with 4% PFA for 10 min, permeabilized with 0.2% Triton X-100 for 10 min and incubated in blocking solution [1% bovine serum albumin (BSA), 2% donkey serum and 0.02% sodium azide into 1× PBS] for 30 min at room temperature. Sections were incubated in primary antibody overnight at 4°C according to the manufacturer's recommendations, washed with PBS and incubated with the appropriate secondary antibodies in blocking solution for 1 h at room temperature (primary and secondary antibodies are listed in Table S2). After the addition of secondary antibodies, nuclei were stained by Hoechst (Sigma-Aldrich), and samples were mounted using IMMU-MOUNT (Thermo Fisher Scientific). All fluorescence images were captured on a Nikon Spinning-disk confocal microscope with a Yokogawa X1 disk, using a Hamamatsu flash4 sCMOS camera. Images were processed and analyzed using NIS Elements software (Nikon) version 5.0 and ImageJ.

Cells were induced to sporulate as above. Cells were incubated 2 h in SPO medium in flasks for analysis of chromosome segregation. Alternatively, for Mam1 localisation, cells were incubated for 6 h in SPO before addition of 1 μM β-estradiol and incubated for a further 15 min to release cells from prophase I arrest. Cells were immobilised on Concanavalin A-coated cover slips in ibidi 4-well or 8-well dishes, fresh sporulation media was added to the dish and imaging commenced. Imaging was performed on a Zeiss Axio Observer Z1 (Zeiss UK, Cambridge) equipped with a Hamamatsu Flash 4 sCMOS camera, Prior motorised stage and Zen 2.3 acquisition software. Images were processed in Image J and 8 Z-stacks were projected to maximum intensity. Representative movies were generated using imaris, cells were projected to 2D using max intensities over the projection line (MIP) and contrast was adjusted to highlight florescent markers.

Tissue sections on the slides were imaged using the Nikon Ti-E microscope coupled to a Yokogawa CSU-W1 spinning disk using the Hamamatsu Flash 4 sCMOS camera. The images were captured using 60× Plan Apochromat (NA 1.4) oil objective. The sections to image were manually identified using a 10× objective (NA 1.45). The images were obtained at 100% laser power for far red 633 nm, red 561 nm, green 488 nm and DAPI 405 nm lasers. For each channel the following filters were used: DAPI, ET455/50m; green, ET525/36m; red, ET605/70m; far red, ET700/75m. A Nikon Elements Job ‘Tiler’ was used to capture the images if tiles were taken, and the image was stitched later in Fiji (https://imagej.net/software/fiji) using the grid/collection stitching plugin. The order of experiments was Lambda (z-series), so each color was imaged in z before moving to the next color. For obtaining a z-stack through the tissue section, each slice imaged was 1 µm apart and a range of 20 steps was taken. Images were obtained in the order of 633 (for the 647 probe), 561 (for the 555 probe), 488 (for the atto 488 probe) and 405 (for DAPI). On a slide, the whole tissue section was imaged, and for each slide a single row was imaged all the way across for each genotype of embryo. All images were stitched using Fiji before further processing.

For immunofluorescence, images were acquired using a Zeiss Axio Observer microscope equipped with a Yokogawa CSUX1 spinning disc head and a QuantEM camera; EC plan Neofluar (40×; Numerical Aperture (N.A.) 1.3) and Plan Apochromat (63×; N.A. 1.4) objectives were used. Z stacking was performed following Nyquist criteria.
For SBB‐ and H&E‐stained sections, images were acquired using a Nikon E800 wide field upright microscope equipped with a Leica DFC450C camera; 10× (N.A. 0.3) and 20× (N.A. 0.5) objectives were used. 8–12 fields were taken per tissue.
For immuno‐FISH‐stained sections, images were acquired using a DMi8 fluorescence inverted microscope with a 100× (N.A. 1.44) objective. Nyquist criteria z stacks were captured using a Hamamatsu Flash4 sCMOS camera. A minimum of 50 nuclei were imaged per tissue. Image acquisition was performed using LASX software (Leica) and deconvolved with Huygens software (SVI).
To assess senescence marker in relation to injection sites, tiling scans were generated using the tiling function of a DMi8 equipped with a motorized stage, collecting high‐resolution images over large areas to enable identification of injection sites and analysis of close and distant bystander cells.