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Start-up procedures#

Start-up#

  1. Turn on surge protector (switch on the right)
  2. Turn on laser box "F XL" by rotating key clockwise
  3. Turn on microscope controller (white Nikon box to right of laser box)
  4. Turn on CSU-X1 unit (connected to the left port of microscope) by rotating key clockwise

  5. Uncover and turn on scope body by pushing a switch on the right side of the scope at the back. (1)

    1. Sometimes the scope doesn't turn on properly. You'll know this is the case if it starts beeping and/or the joystick pad doesn't light up. Don't worry--just shut off the scope body (wait for the beeps to finish), and try starting again.

  6. Sit at computer and start NIS-Elements by double-clicking on the NIS-Elements AR icon.

  7. Select your account from the drop-down menu (default: no password).
  8. Choose Front Fusion for single-camera imaging or Dual Fusion for dual-camera imaging.
  9. Check the drawer to see the TwinCam DM's status. Ensure it's out for single-camera imaging and in for dual-camera imaging.

Single cam mode

"Front Fusion" / single camera mode highlighted

Video showing start-up steps 1-5

Live-cell checklist#

Allow CO2 flow and start Tokai Hit unit#

  • Open the Apollo valve for CO2 by rotating counter-clockwise 90 degrees until the yellow handle is parallel to the tubing.
Photo of CO2 line

CO2-tank

  • Turn on the Tokai Hit incubator control box by pushing the orange button
  • Check the airflow indicator and ensure the ball is at the 150 ML/MIN line

Objective Heater (only for immersion objectives!)#

Bioptechs Objective Heater

Bioptechs Objective Heater with labels
  • Remove the stage adapter for easier access.
  • Turn on the Bioptechs Controller by pushing the Power On/Off Button on the bottom right.
  • Take the Bioptechs Objective Heater out of its protective case.
  • Loosen the heater band by turning counter-clockwise with either a 5/64 or 2mm Allen driver
  • Give more slack to the monofilament tail by pushing on the button and feeding the monofilament loop through
  • Carefully move the objective heater towards the objectives by going underneath the motorized stage.
  • A blue LED will indicate that the heating band is not heating. Secure the objective heater by the heating band with slow clockwise turns (using the 5/64 or 2mm Allen driver) of the screw labelled "Heater Band Adjustment. Do not overtighten--as soon as the blue LED shuts off, stop turning.
  • For securing the monofilament loop, press the button until it depresses and then gently pull on the monofilament loop.
  • Adjust the correction collar.
  • Add a drop of Nikon Type F immersion oil to the objective.

Set-up Tokai Hit stage-top incubator#

  • Ensure the Tokai Hit stage adapter is installed (do not overtighten the screws!)
  • Put the Tokai Hit stage top incubator onto the stage while lifting the cables/tubing with your right hand to prevent undesirable tension on the cables/tubing.
  • Add 20mL of distilled/DI water to the reservoir
  • Inspect the sample adapter for oil (and clean if needed) before inserting into the Tokai Hit stage top incubator.
  • Insert your sample (or a clean dish/slide), and attach the Tokai Hit dish/slide lid. Make sure the magnetic lid is flat or your sample will be tilted.
  • If using an immersion objective, raise the objectives so that the oil and the bottom of the dish make contact.
  • Put the stage top incubator lid on.
  • Turn off lights, and close the door to the microscope room.

Important

  • Wait >60 minutes for temperatures to stabilize before imaging.
  • If using dual camera acquisition, please see Dual Camera Alignment for next steps.