McClean: Nikon TI Basic Use

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This is a work in progress! Please comment and fix things as needed!

  1. Sign the microscope log book. This is mandatory.
  2. Turning the Microscope ON (1 hour before experiment!!!)
    • The microscope components must be turned on in a particular order to ensure that power surges caused by the lamp do not affect other equipment and that the controller on the microscope body recognizes other components of the scope. The components are labeled with the appropriate number. ALWAYS give the microscope 1 hour to warm up before your experiment and NEVER turn the microscope on/off in less than 30 minutes. If the next user is starting <2 hour after you, please leave the microscope on. Turn the microscope on in the following order:
    1. Remove dust cover
    2. Power supply A (#1)
    3. Power supply B (#2)
    4. Intensilight (#3)
    5. Clara Camera (#4)
    6. Nikon Eclipse Ti (#5)
    7. Lambda Shutter A (#6)
    8. Lambda Shutter B (#7)
    9. Turn on the computer (it should normally be left on) and open the NIS Elements software (short-cut on desktop)
  3. Measuring lamp intensity
    • The lamp intensity must be measured EVERY time you do an experiment if you care about comparing results from one day to another. Lamp intensity should be monitored during the warm-up hour to ensure it reaches a steady state before you start your experiment.
    1. Hook the ThorLabs USB cable to the microscope computer.
    2. Assembling the detector:
      1. Hit “escape” to bring the nosepiece (holds the objectives) all the way down to its home position
      2. Remove the stage adaptor to allow easy access to the nosepiece.
      3. Rotate the objective turret (carefully) to expose an empty port (covered with a black plastic cap). Remove the black cap.
      4. Screw the detector head and adaptor lens into the empty objective position.
      5. Adjust the optical diaphragm so that it is set appropriately given the objective that you are going to use. For the 100x objective, set it to 7mm (this corresponds to the back aperature of the objective). (Note: If you are trying to identify the lamp intensity for a particular objective, this diaphragm should be the same size as the back aperature of the objective. See Grunwald, et al 2008 Nature Protocols. If you just want to maintain lamp intensity between experiments then the diaphragm needs to be consistent between measurements—completely open is the easiest to maintain consistently).
      6. Attach the sensor to the power meter
      7. With everything closed (shutters, etc) re-zero the powermeter. Go to Meas Config and adjust the beam diameter (to 7mm) and then click zero-adjust to adjust the zero-setting of the power meter.
      8. Start a log. Allow it to run until the reading reaches steady state to get a zero-reading and an idea of the fluctuations due only to temperature changes. Then zero-adjust the meter again.
      9. Open the fluorescence shutter. Put in the ND filters that you will be using the experiment.
  4. Calibrating scope performance with fluorescence beads:
    1. Use Invitrogen FocalCheck test slide #1
    2. More to come.....
  5. Performing an ND experiment
  6. Performing Photobleaching Controls
  7. Storing Files and Updating the Microscopy Database
    1. Remind here that backup files need to be stored on McCleanLabShare under original microscopy (with a README) and that the times that files were created needs to be written down
  8. Complete your entry in the logbook. Note down any problems (software crashes, lamp surges, hardware failures) or unique occurrences (removed objective, cleaned objective, changed lamp, etc).
  9. Turn everything off in reverse order (#7->#1)