Coolsnap hq2 ccd

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.

Chlorella cells were infected with PBCV-1 at MOI of 1 for 3h and fixed with 4% paraformaldehyde (EMS) for 15 min at RT. Cells were washed in PBS and transferred to Poly-Lysine coated glass microwell dishes (Mat-Tek corp.). Cells were then blocked with 4% BSA-PBS solution for 30 min at RT and exposed to anti-capsid antibody diluted in 4% BSA-PBS for 1h at RT. Following washes in PBS, a secondary antibody, goat anti-mouse IgG conjugated to Alexa488 (Life Technologies) diluted in 4% BSA-PBS solution was added for 30–45 min. Cells were then counterstained with 500 nM SYTOX Orange (Life Technologies) in DDW for 15 min. Fluorescence images were visualized and photographed using a Deltavision system (Applied Precision) equipped with X100 UPlanSApo NA 1.40 objective. Fluorochromes were excited at 490 nm for Alexa488, 555 nm for SYTOX Orange and 640nm for chlorophyll (auto-fluorescence). Images were acquired with a photometrics coolSNAP HQ2 CCD (Roper Scientific) and de-convoluted with SoftWorx package using high noise filtering and 10 iterations. Image analysis and processing were conducted with Image J and Adobe Photoshop CS4-extended softwares.