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Basics

Normal operation of the StedyCon microscope

These documents are to reinforce, not replace, hands on training on the microscope.  All users MUST recieve training from Cell Imaging Staff prior to use of the instrument.

Stop: This is a first draft in progress. The information is not/may not be accurate.

Power On

  1. Laser Box:  
    1. The laser box is normally in standby (There are two lights on: Power and Standby/Ready and two lights off: Emission and Scanning).
    2. Turn on the Laser Key.  Vertical is Standby, Horizontal is on.  The lights do not change.  

    3. Is there a power switch or is that just the power strip?

  2. There are 4 boxes under the table: 
    1. Piezoconcept: Turn on switch on front left.  A green LED on the front will turn on.
    2. Tango Desktop: Turn on switch in back top left.  A green LED on the front will turn on.
    3. Nikon Brightfield Lamp Power Supply: Turn on switch on front right.  A green LED on the front will turn on.
    4. Fluorescent Lamp Power Supply:
      1. Turn on the power switch, lower left.  The green Power LED will turn on.
      2. Press and release the Ignition button above the power switch.  The Orange Lamp Ready LED will turn on.
  3. Microscope:  Turn on the microscope with the switch on the lower right side near the back.  The microscope will make noise and the front display should be on.
  4. Computer and software: 
    1. If the computer is off, press the button on the front of the box.
    2. If the monitor is off, there is a power button on right bottom.
    3. Double click the StedyCon icon on the left side of the screen.  This should bring up the Welcome Screen

 Status and Interlocks

Click on the Status Info button and a Status window will pop up in a new tab.

Double click on the Interlock line in the STEDYCON State section.  This will pop up a new tab with the Interlocks status.

There are 7 interlocks.  The first 5 should be Green before starting a scan.

  1. Key: Is the key on the laser box on?
  2. Head:
  3. Interlock 1:
  4. TL arm & Condenser:  There are two interlocks:
    1. TL arm: is the transmitted light arm (tower) forward?
    2. Condenser: is the dial set so that the path
  5. Beam path: On the front of the microscope, switch the path to L100
  6. Software:
  7. Combined: 

To set up the 

 

Click on Startup.  The start process will take a few minutes.  The laser box Standby/Ready light will turn green.  Eventually, the status will be Ready for Imaging.

Wide field

To locate and focus on your sample, you will need to image in wide field (either bright field or epifluorescence).  This is all done with the controls on the microscope.  This will set off the interlocks on the StedyCon software, so you will have to restore light paths back to StedyCon mode before you start imaging.

Light sources:

Bright Field:

To use Brightfield:

  1. Light path, set to Eye
  2. Condenser dial, set to not block the upper beam.
  3. EPI Shutter: set to PSF Out --- (or you will get a combination of Bright and Fluorescent)  Control is on the right side panel.
  4. Light control is on the left side panel on the microscope.  On/Off is for the brightfield light.  The knob is for intensity.

Epifluorescence:

  1. Light path, set to Eye
  2. Condenser dial set to block the upper beam, or turn the brightfield off on the left panel.
  3. Lever on the right side of the filter turret must be back.  This is a beam block.
  4. Just to the left of the lamp, there are a number of filters.  These do get moved to the wrong spots.  Left to right, they are:
    1. Iris: Should be pulled out.
    2. A.S. Two knobs
    3. ND4: Neutral Density Filter: should be pulled out for maximum intensity
    4. ND8: Neutral Density Filter: should be pulled out for maximum intensity
    5. Filter: Should be pushed in.  If out, it will block the path.  Can be used as a beam block.
  5. EPI Shutter: set to one of the epifluorescent channels: 
    1. DAPI
    2. FITC
    3. TxRed
    4. D-F-T

Light Path:

Focus Knob:

Top towards you is up.

Acquisition

On the welcome screen, press Start Up.  The process will take a few minutes.  Wait for the status to change from Starting Up... to Ready for Imaging.

Click on Start New Session.  This will bring up the main window and the Define New Session dialog.

  • Session Name:
  • Sample Name:
  • Objective Magnification:
  • Objective NA:
  • Choose Your Dyes:
    • Dye Preset: pick from drop down
    • Display Name
    • Colormap
    • Colormap in Overlay
  • Load Settings: pull up settings from ?
  • Let's Go

You can get back to this with the Session Settings button top left.

Power Off

The power off procedure is basically the reverse of the power on.

  1. Shut Down the system in the StedyCon software.  Wait for it to shut down.
  2. Close the software.
  3. Reverse order on the switches.

 

  1. Turn off the laser key.
  2. Normally, we do not turn off the laser box.
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