Neo scmos camera

For the images in Fig. 5e, membrane staining with E-Cadherin (BD Biosciences, # 610182) was done at 1:300 ratio in Blocking buffer for 20 h at 4 °C. DAPI staining was performed at concentration of 300 nM for 30 min at room temperature. All secondary antibodies were added at 1:300 for 1 h in room temperature. Cell nuclei were stained with 20 μg/ml DAPI (4′,6-Diamidino-2- Phenylindole, Invitrogen) in PBS for 15 min. High-throughput imaging was done with an automated spinning disk microscope from Yokogawa (CellVoyager 7000 S), with an enhanced CSU-W1 spinning disk (Microlens-enhanced dual Nipkow disk confocal scanner), a 40× (NA = 0.95) Olympus objective, and a Neo sCMOS camera (Andor, 2560 × 2160 pixels). For imaging, an intelligent imaging approach was used in the Yokogawa CV7000 (Search First module of Wako software). For each well, one field was acquired with 2× resolution in order to cover the complete well. This overview fields were then used to segment individual organoids on the fly with a custom written ImageJ macro which outputs coordinates of individual organoid positions. These coordinated were then subsequently imaged with high resolution (40×, NA = 0.95). For each site, z-planes spanning a range up to 140 μm were acquired. For the data in Fig. 5e, h and in Supplementary Fig. 9 2 μm z-steps were used.

One microliter of cells was spotted onto a 1% agarose pad with the appropriate medium, and imaged on a Nikon Eclipse Ti-E inverted fluorescence microscope with a 100X (NA 1.40) oil-immersion objective (Nikon Instruments). For microfluidic experiments, cells were loaded into B04A (CellASIC) microfluidic plates following previous protocols (Rojas et al., 2018 (link)). Phase-contrast and epifluorescence images were collected on a DU885 electron-multiplying CCD camera (Andor Technology) or a Neo sCMOS camera (Andor Technology) using μManager v. 1.4 (Edelstein et al., 2010 ). Cells were maintained at 37°C during imaging with an active-control environmental chamber (Haison Technology).

Medial and lateral ventricular walls from P10 mice were isolated in cold PBS and sectioned using a Leica VT1000S vibratome (Leica, Wetzlar, Germany). Tissue was fixed in 4% PFA in PBS O/N at 4°C, followed by blocking with 2.5% BSA, 0.2% Triton X-100 in PBS for 4 hours at RT. Primary antibodies used: N-cadherin (rabbit, 1:50; sc-7939, Santa Cruz Biotechnology, Santa Cruz, CA), γ-tubulin (mouse 1:250; ab11316, Abcam, Cambridge, MA). Samples were incubated with primary antibody for 24–48 hours at 4°C. Secondary antibody incubation was performed at 4°C for 48 hours using Alexa 488 goat anti-mouse (1:250, Thermo-Fisher, Waltham, MA) and Alexa 647 donkey anti-rabbit (1:250, Jackson ImmunoResearch, West Grove, PA). Tissues were placed on glass slides, mounted and imaged using an Andor WD Spinning Disk confocal microscope system equipped with an Andor Neo sCMOS camera (Andor, Belfast, UK).

Live imaging of cells was performed using a LCV100 (Olympus) equipped with a UAPO ×40/340 objective lens (Olympus), a LED light source, a DP30 camera (Olympus), differential interference contact (DIC) optical components, and interference filters. Live-cell imaging of MLC2-EGFP or Lifeact-EGFP was obtained using the spinning-disc laser confocal microscope IX71 (Olympus) equipped with CSU-X1 (Yokogawa), a Yokogawa YOKO R485/561 filter, a Neo sCMOS camera (ANDOR), and a UPLSAPO ×60/1.35 oil lens. The cells were set on a ChamlideTC CO₂ incubator (Live Cell Instrument) at 37 °C. Live-cell imaging of Raichu probes was performed using a spinning-disc laser confocal microscope IX81 (Olympus) equipped with CSU-W1 (Yokogawa), an ImagEM-1K camera (Hamamatsu), and a PLANAPO N ×60/1.42 oil lens. Images were processed using MetaMorph (Molecular Devices) and ImageJ (NIH).

Larvae were anesthetized with 100 μg/mL buffered tricaine and mounted in 1% low-melting point agarose as previously described (41 (link)). Epi-fluorescence microscopy was performed using a MVX10 Olympus microscope (MVPLAPO 1X objective; XC50 camera). Confocal microscopy was performed on ZEISS LSM880 FastAiryscan, using 20X/0.8 objective, plan apochromat equipped with DIC for transmission images, resolution at 512x512 pixels. The wavelength were respectively 488nm (Argon Laser) and 561nm (DSSP Laser) for excitation. Detection was selected at 505-550nm for PMT detector and 585-620nm for GaAsP detector. The images were taken in a sequential mode by line. The 3D files generated by multi-scan acquisitions were processed by Image J. To image Ca²⁺ oscillations at the wound, we used ANDOR CSU-W1 confocal spinning disk on an inverted NIKON microscope (Ti Eclipse) with ANDOR Neo sCMOS camera (20x air/NA 0.75 objective). Image stacks for time-lapse movies were acquired at 28°C every 20 seconds (s), with z-stack of 45 μm at 3 μm intervals. To image macrophage activation in live, z-stacks of 78 μm with 3 μm intervals were acquired every 3min, in multiposition mode. The 4D files generated from time-lapse acquisitions were processed using Image J. Brightness and contrast were adjusted for maximal visibility.

2-hybrid 3-cube FRET experiments were carried out with standard protocols similarly shared by several groups^{20 (link),24 ,56 (link)}. Briefly, experiments were performed on an inverted epi-fluorescence microscope (Ti-U, Nikon), with computer-controlled filter wheels (Sutter Instrument) to coordinate with diachronic mirrors for appropriate imaging at excitation, emission, and FRET channels. The filters used in the experiments were excitation: 438/24 (FF01-438/24-25, Semrock) and 480/30 (FITC, Nikon); emission: 483/32 (FF01-483/32-25, Semrock) and 535/40 (FITC, Nikon); dichroic mirrors: 458 nm (FF458-Di02-25 × 36, Semrock) and 505 nm (FITC, Nikon). Fluorescence images were acquired by Neo sCMOS camera (Andor Technology) and analyzed with 3³-FRET algorithms coded in Matlab (Mathworks), mainly based on the following formula: $FR=1+\frac{{FR}_{\max }-1}{1+\frac{K_d}{D_{free}}}$
FR_max represents the maximum FRET ratio, and D_free denotes the equivalent free donor (CFP-tagged) concentration. K_d (effective dissociation equilibrium constant) is calculated from an iterative procedure to evaluate the binding affinity for each pair of binding partners. FRET imaging experiments were performed with HEK293 cells in Tyrode’s buffer containing 2 mM Ca²⁺.

Cells were fixed in −20°C methanol for 1-2 min. Microtubules were detected using YL1/2 rat-anti-tyrosinated tubulin (J. Kilmartin). KIF17 was detected with rabbit anti-KIF17 IgG (Sigma, K3638). ERR1 was detected with goat anti-ERR1 IgG (Santa Cruz, sc-32971). Fluorescently conjugated secondary antibodies were from Jackson ImmunoResearch. Images were acquired with a Neo sCMOS camera (6.45μm pixels, 560MHz, Andor Technology) on a Nikon TiE inverted microscope (Nikon Inc., Mellville, NY) using 40X (NA 1.0) or 60X (NA 1.4) plan apochromat oil immersion objectives. 14-16bit images were scaled linearly to highlight features of interest and converted to 8-bit copies for figure assembly. Devices were controlled by Elements software (Nikon Instruments). Line-scan analysis: The number of ERR1 and KIF17 puncta on MTs, and their colocalization shown in Figure 4A, was determined by line-scan analysis of fluorescence intensities along 10μm of individual MTs. Overlapping fluorescence peaks were scored as colocalized puncta.