Mosaic digital micromirror device

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.

Cell imaging and activation intended for posterior force measurements was carried out using a Nikon Ti-E microscope, Zyla sCMOS camera (Andor, Belfast, UK), and a Plan Apo VC 60×/1.40 Oil objective (Nikon, Tokio, Japan). The microscope was equipped with an incubator that maintains the temperature at 37°C. Global cellular photoactivation was performed using a LED light source (X-Cite/XLED1, Lumen Dynamics, Canada) coupled to a Mosaic digital micromirror device (Andor). Depending on the experiment done, activation pulses were 10-20-50-100-150-200 ms long using an LED at 460 nm with a power of 256.7 μW (measured at the back focal plane of the objective). For our Apo VC 60× Nikon NA 1.4 objective, the transmission coefficient at 450 nm is 74% (data from Nikon). From this we deduce the energy sent to the cells by dividing the intensity transmitted by the objective by the surface of the digital micromirror device (DMD). The physical size of our DMD is 854 × 480 pixels for our 60× objective and this gives us an irradiance of 52.7 mW/mm². This finally gives the following energy per pulse sent to the cells of 0.527, 1.054, 2635, 5270, 7905, and 10,540 mJ/mm² for each pulse of 10, 20, 50, 100, 150, and 200 ms, respectively.