Ixon ultra 888 emccd camera

U2OS cells that were cultured in 35 mm glass-bottomed dishes (14 mm, No.1.5, MatTek Corporation, Ashland, MA, USA) were treated with 2.5 µM acridine orange (Thermo Fisher Scientific, #A1301) for 15 min at 37 °C to label lysosomes. The medium was aspirated and fresh growth medium was added prior to time-lapse imaging, which was performed in a heated chamber (37 °C) using a Plan-Apochromat 63×/1.4NA (Carl Zeiss) with differential interference contrast oil objective that was mounted on an inverted Zeiss Axio Observer Z1 microscope (Marianas Imaging Workstation from Intelligent Imaging and Innovations Inc. (3i), Denver, CO, USA), that was equipped with a CSU-X1 spinning-disk confocal head (Yokogawa Corporation of America, USA) and three laser lines (488 nm, 561 nm and 640 nm). The images were acquired using an iXon Ultra 888 EM-CCD camera (Andor Technology, Belfast, UK). The images were acquired every second for 180 s. The lysosomal volume and integrity as well as membrane integrity was evaluated by assessing the green and red fluorescence.

1 × 10⁷ OT-I CTLs (5–8 days after activation) were nucleofected 24 h prior to imaging with 5 μg Lifeact-mApple and probe construct plasmids using the Mouse T Cell Nucleofector Kit (Lonza). EL4-blue target cells were pulsed with 1 μM SIINFEKL for 30 min at 37°C, washed in serum-free CO₂-independent medium (with L-glutamine) and plated onto 35-mm glass-bottom culture dishes (MatTek) coated with 0.5 μg/mL murine ICAM-1/Fc. ∼2 × 10⁶ nucleofected CTLs in CO₂-independent T cell medium were added dropwise, imaging was started within 5 min. Interactions were imaged with cells in an environmental chamber maintained at 37°C (Okolabs) on an Olympus IX81 microscope (Olympus) using the Andor Revolution spinning-disc microscope with Yokogawa CSU-X1 spinning disk, iXon Ultra 888 EMCCD camera, 2x camera adaptor (Andor Technology, Oxford instruments) and Olympus Universal Plan Super Apochromat silicone immersion objective. 12-18 z stacks (0.8-μm apart) were imaged every 12 s with fluorophores excited at 405, 488, and 561 nm in each z-plane. 4D datasets were rendered and analyzed with Imaris software (Bitplane).

Embryos subjected to in situ hybridization were mounted in a 35-mm glass-bottom dish (P35G-1.5–10 C, MatTek, Ashland, MA) in 0.8% low melting point agarose and imaged using a stereo microscope (M165 FC, Leica, Wetzlar, Germany) with a Leica DFC7000 T digital camera.
Confocal microscopy was performed using either a Leica TCS SP8 laser-scanning confocal microscope system with 63x/1.40 or 100x/1.40 oil HC PL APO CS2 oil-immersion objectives and HyD detectors in resonant scanning mode, or a spinning disk confocal microscope system (Dragonfly, Andor Technology, Belfast, UK) housed within a wrap-around incubator (Okolab, Pozzuoli, Italy) with Leica 63x/1.40 or 100x/1.40 HC PL APO objectives and an iXon Ultra 888 EMCCD camera for smFISH and live cell imaging (Andor Technology). Deconvolution was performed using either the Huygens Professional (Scientific Volume Imaging b.v., Hilversum, Netherlands) (for images captured on Leica SP8) or the Fusion software (Andor Technology) (for images captured on Andor Dragonfly).

To validate the SF9-3xGFP myosin sensor as a proxy for junction tension, we performed junction recoil measurements in stage 16 to stage 20 embryos expressing the SF9-3xGFP myosin II sensor. Laser ablations were performed using a 532nm pulse laser (>60 µJ pulses at 200Hz) at 20% power, with each ablation set for 5 ms. The ablation system was connected to a 3i spinning disk microscope with a Plan-Apochromat ×63 oil objective (N.A. = 1.4) mounted on an inverted Zeiss Axio Observer Z1 microscope (Marianas Imaging Workstation [3i—Intelligent Imaging Innovations]), equipped with a CSU-X1 spinning disk confocal head (Yokogawa) and an iXon Ultra 888 EM-CCD camera (Andor Technology). Vertex displacement was manually tracked using Fiji and the recoil velocities were calculated as previously described^{18 (link)}.

Single-molecule assays were performed using an RM21 micromirror TIRF microscope (Mad City Labs) built in a similar manner to that previously described^{70 (link)} with an Apo N TIRF 60× oil-immersion TIRF objective (NA 1.49, Olympus). Janelia Fluor 532 and LD655 were excited with a 532 nm and 637 nm laser (OBIS 532 nm LS 120 mW and OBIS 637 nm LX 100 mW, Coherent), respectively at a frame rate of around 6 fps. Residual scattered light from excitation was removed with a ZET532/640m emission filter (Chroma). Emission light was split at 635 nm (T635lpxr, Chroma) and recorded as dual-view with an iXon Ultra 888 EMCCD camera (Andor). All microscope parts were controlled using Micromanager v1.4 (ref. ^{71 (link)}) and custom Beanshell scripts.

Electrophysiological data were acquired using an Open Ephys acquisition board (http://www.open-ephys.org/) connected via an SPI interface cable (Intan) to a 32-channel head stage (Intan). A 32-channel laminar probe (Neuronexus, A1x32-Poly2-5mm-50s-177) was connected to the head stage. The iridium electrode contacts on the probe covered a linear length of 790 µm and were arranged into two columns of 16 contacts spaced 50 µm apart. The data were acquired using the Open Ephys GUI software at a sampling rate of 30 kHz and referenced to a supra-dural silver wire inserted over the right occipital cortex. Imaging data were acquired using an Andor iXon Ultra 888 EMCCD camera attached to a Navitar Zoom 6000 lens system. The data were acquired using Andor Solis data acquisition software (Andor Solis 64 bit, v4.31). The field of view was centered over the craniotomy and adjusted to encompass the full diameter of the craniotomy. Images (512 × 512 pixels, ~6 µm²/pixel) were acquired continuously at an exposure length of 1 s and an electron multiplication gain of 300. The data were acquired in units corresponding to the number of electrons recorded by a given pixel. A TTL pulse synchronized the recording of the imaging and electrophysiological data.