Csu x confocal scanhead

Live cell traction force measurements were performed on an inverted Nikon Ti-E microscope with a CSU-X confocal scanhead (Yokogawa), laser merge module containing 491, 561 and 642 nm laser lines (Spectral Applied Research) and an HQ2 cooled CCD camera (Roper Scientific). All hardware was controlled via Metamorph acquisition software (MDS Analytical Technologies). Traction force data was obtained at 37°C in a perfusion chamber (Warner Instruments) using a 60x 1.2 NA Plan Apo WI objective (Nikon). Cells were maintained in culture media supplemented with 10 mM HEPES and 30 μl/ml Oxyrase (Oxyrase, Inc.).

Live cell imaging was performed on a temperature-controlled inverted Nikon Ti-E microscope with a Lumen 200 Pro light source (Prior) and an HQ2 cooled CCD camera (Roper Scientific) controlled via Metamorph acquisition software (MDS Analytical Technologies). Live cell movies of transfected cells were collected on an inverted Nikon Ti-E microscope with a CSU-X confocal scanhead (Yokogawa), laser merge module containing a 491 laser line (Spectral Applied Research) and an HQ2 cooled CCD camera (Roper Scientific). Motility and protrusion data was obtained using either a 10X or 20x objective (Nikon); Immunofluorescence Images were obtained using a 40x 1.3 NA Plan Fluor objective (Nikon).

Glass coverslips were placed in a Chamlide magnetic chamber (Live Cell Instrument, Seoul, Korea) in culture media supplemented with 10 mM HEPES and 30 μl ml⁻¹ Oxyrase (Oxyrase Inc., Mansfield, OH) and maintained at 37 °C. Cells were imaged on an inverted Nikon Ti-E microscope (Nikon, Melville, NY) with a Yokogawa CSU-X confocal scanhead (Yokogawa Electric, Tokyo, Japan) and laser merge module containing 491, 561 and 642 nm laser lines (Spectral Applied Research, Ontario, Canada). Images were collected on either a CoolSNAP HQ2 CCD (Roper Scientific, Trenton, NJ) or Zyla 4.2 sCMOS Camera (Andor, Belfast, UK). Local recruitment using the optogenetic probe was performed using a 405 nm laser coupled to a Mosaic digital micromirror device (Andor). Images were collected using a 60 × 1.49 NA ApoTIRF oil immersion objective (Nikon). All hardware was controlled using the MetaMorph Automation and Image Analysis Software (Molecular Devices, Sunnyvale, CA).
Unless otherwise stated, cells were imaged in the 561 channel every 20 s for 45 min, with the first 15 min used to determine the steady state of the system, the second 15 min to perform local recruitment and the final 15 min to record any recovery. During recruitment, a local region drawn in MetaMorph was illuminated by the 405 nm laser for 960 ms at a power <1 μJ s⁻¹ immediately before the acquisition of each 561 image.