Evolve 512 emccd camera

Live-cell imaging experiments were performed in an inverted microscope Nikon Eclipse Ti-E (Nikon), equipped with a Plan Apo VC 100x NA 1.40 oil and a Plan Apo VC 60x NA 1.40 oil objective (Nikon), a Yokogawa CSU-X1-A1 spinning disk confocal unit (Roper Scientific), a Photometrics Evolve 512 EMCCD camera (Roper Scientific) and an incubation chamber (Tokai Hit) mounted on a motorized XYZ stage (Applied Scientific Instrumentation) which were all controlled using MetaMorph (Molecular Devices) software. Coverslips were mounted in metal rings and imaged using an incubation chamber that maintains temperature and CO₂ optimal for the cells (37°C and 5% CO₂). Neuron live imaging was performed in full conditioned medium and fresh medium was added to COS-7 before imaging.
Time-lapse live-cell imaging of EB3-RFP was performed with a time acquisition of 1 s. NF186-RFP alone was acquired at 2 frames per second. NPY-GFP, GFP-Rab6, Rab11-GFP or Rab5-GFP, alone or in combination with NF186-RFP were acquired at 10 frames per second.
For simultaneous imaging of green and red fluorescence, we used ET-mCherry/GFP filter set (59022; Chroma) together with the DualView (DV2; Roper) equipped with the dichroic filter 565dcxr (Chroma) and HQ530/30 m emission filter (Chroma).

Chamber preparation and microscopy were performed as described (Volkov et al., 2018 (link)). In brief, both coverslips and slides were cleaned in oxygen plasma, immediately silanized, and later assembled in a chamber using double-sided tape. The chambers were first incubated with ~0.2 µM anti-DIG antibody (Roche) and passivated with 1% Pluronic F-127, followed by GMPCPP seeds (diluted 1:200 – 1:1000) and then the reaction mix. The reaction mix contained MRB80 buffer supplemented with 8 µM tubulin (4–6% labeled with HiLyte-488), 1 mM GTP, 1 mg/ml κ-casein, 0.01% methylcellulose, 4 mM DTT, 0.2 mg/ml catalase, 0.4 mg/ml glucose oxidase and 20 mM glucose; this mix was centrifuged in Beckman Airfuge for 5 min at 30 psi before adding to the chamber.
Imaging was performed at 30°C using Nikon Ti-E microscope (Nikon) with the perfect focus system (Nikon) equipped with a Plan Apo 100 × 1.45 NA TIRF oil-immersion objective (Nikon), iLas² ring TIRF module (Roper Scientific) and a Evolve 512 EMCCD camera (Roper Scientific). Images were acquired with MetaMorph 7.8 software (Molecular Devices). The final resolution was 0.16 µm/pixel. The objective was heated to 34°C by a custom-made collar coupled with a thermostat, resulting in the flow chamber being heated to 30°C. All images were analyzed using Fiji (Schindelin et al., 2012 (link)).

Live-cell imaging experiments were performed on an inverted Nikon Eclipse Ti–E confocal microscope equipped with a perfect focus system (Nikon), a CSU–X1–A1 Spinning Disc unit (Yokogawa), a Photometrics Evolve 512 EMCCD camera (Roper Scientific) and a Plan Apo VC 100× N.A.1.40 oil objective. Coverslips were mounted in a Ludin chamber (life imaging services) and maintained in culture medium at 37°C and 5% CO₂ in a stage incubator (Tokai Hit) during image acquisition.