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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). (Color of lights….)
    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

The Microscope

Your first task will be to get your sample in focus and in the correct position.  This is done with the microscope and through the controls on the microscope itself.

  1. Apply oil to the lens if needed (60X, 100X).  (The lenses are parafocal, so you can start at lower magnifications first.)
  2. Place your slide with the coverslip down.
  3. Use the X-Y controlled to get close to position.
  4. Set up the appropriate light path (below).
  5. Use the microscope to adjust focus and position (below).

Brightfield Light Path and Controls

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

If you cannot see anything in the eyepieces, check your light path again.

Fluorescent Light Path and Controls

  1. Light path (on front panel) 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

XYZ Controls

The stage XY control is the joystick.

The Z control is the 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 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.

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: will still be red.
  7. Combined: will still be red.

The fluorescence source is not part of the interlocks.  You still need to block its path to get a good image.  Use the lever under the filter wheel to block the fluorscence source.  Set the fluorescence filter to ----.

Start New Session

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.

Overview button will do a quick scan.  Loop will do continuous quick scans.  This is good for focus and placement.

Gallery: opens a new tab with the images from this session.

Acquire will do a normal scan.  Loop will do continuous normal scans.

Stop: Stop the scan

Auto: I think that this is auto save new images to the gallery.

Finish Session:

Acquisition Mode: Can turn on Confocal and/or STED

Confocal: Excitation Power and Acquisition Speed

STED: Excitation Power, Resolution and Acquisition Speed

Z:  You can control the focus or Z position of the scan either with the focus knob on the slider to the left of the image.

Channel display:  At the top is a list of the channels.  The channel being displayed is underlined.  If you are using more than one channel, make sure the correct one is underlined.

Z-Stack:

Acquire z-slice and select z-stack: The z-slice can be useful in selecting the z-stack and in setting the laser power based on all the depths.

Lenses

Magnification Immersion Numerical Aperature Working Distance
4X Air    
10X Air    
20X Air    
40X - Not There      
60X Oil    
100X Oil    

Save Your Data

In the gallery, you can edit what data you want to save.

Select images by clicking on the check box beside them - or use the Select all button.

Selected images can be downloaded, or you can download all.

Alternately, when you click Finish Session, you will have the option to download or delete the session images.

 

Where do the images go?  

Your data ends up in two places:

  • The C drive downloads folder as a .zip file named:  yyyy-mm-dd session name.zip
  • The S drive as a folder: yyyy-mm-dd session name

Power Off

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

  1. Finish Session.
  2. Click Shut Down the system in the StedyCon software.  Wait for it to shut down.  Status will now be Standby.
  3. Close the software.
  4. 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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