Michael Hooker Microscopy Facility (MHMF.ORG)

Nikon TE2000 with Perkin Elmer Yokogawa Nipkow disk confocal

Operating the System for widefield (non-confocal) microscopy

Powering up: - (if everything is off)

• If using fluorescence
  • Ensure all electronics are off
  • Turn on the XBO (Fluorescence) lamp.  IMPORTANT:  All other sensitive electronics must be turned off before igniting the XBO lamp!
  • Turn on the Nikon controller- switch lower left on microscope stand
• Turn on the power strip next to the monitor.  This will turn on components on the shelf under the table:
  • Sutter filter wheel changer (not used, but OK to be on)
  • Prior focus controller
  • Hamamatsu camera controller on lower shelf
• Ensure computer and monitor are on if imaging

Setting up for transmitted light imaging
--> If transmitted illumination is not required, go to "fluorescence light" section below

• For all transmitted light modalities, first set up Kohler Illumination
  • Set the transmitted light filters appropriately.  For most applications:
    • D is IN (left side)
    • ND is OUT (left side; push in to decrease the light intensity)
    • GIF is OUT (right side; if it is in, the light will be green)
    • NCB is IN for normal light (not critical)
  • Open field diaphragm (lift up)
  • Minimize condenser aperture (push to the left) - important!
  • Set condenser ring to A (brightfield)
  • Lower objectives (away from the stage) with coarse focus knob
  • Choose required objective.  (Generally, choose a lower power dry objective, locate sample and region of interest and then move to a higher power dry or immersion objective )
  • Set dichroic filter  using the Nikon control box
  • Slide out DIC Analyzer (note: lower (objective) Wollaston prism should be left in the slot at base of objective)
  • Ensure incandescent lamp power is on - switch on with button on left side of scope body and dial turned to sufficient power
  • Place sample on stage--cover slip down since this is an inverted microscope!
  • Focus onto sample
  • Close field iris until you see a dark ring impinging onto field of view
  • Focus the image of the ring using the condenser focus knob (above the stage)
  • Center Field iris using the 2 knurled silver "condenser centering" screws (at 45°).
  • Open field diaphragm to just fill the field of view seen through the eye pieces
  • Open condenser aperture to desired contrast. 
    Remember:  Maximum resolution is obtained with a fully open aperture, which also gives minimum depth of focus & contrast and maximum illumination intensity.
  • These steps have set up Kohler Illumination!
  • Switch to higher power objective if desired
  • For optimum image quality, re-check Kohler Illumination setup for each objective used
     
• For Nomarski (DIC) - if desired
  • Set up for Kohler as described above
  • Slide in Polarizer (push to the left; above the stage)
  • Open condenser aperture maximally (to the right)
  • Select condenser-side Wallaston prism by turning the condenser turret to ∞H
  • Push in Analyzer (below the fluorescence filtercube turret)
  • Rotate upper polarizer for optimum contrast
     
• For Phase contrast
  • Set up for Kohler as described above
  • Check that the Polarizer (above the stage) is out of the light path (to the right)
  • Open condenser aperture maximally. IMPORTANT!!!  If the condenser aperture is closed, you will get NO light!
  • Select the correct Phase Ring on the condenser turret for the objective you are using
    • 4X   - PhL    (not mounted on this microscope.  See a facility director for assistance)
    • 10X 0.30NA - Ph1
    • 20X 0.45NA ELWD corr - Ph1
    • 40X 0.60NA ELWD corr - Ph2
    • 100X 1.4NA PlanApo - Ph3* 
          *The 100X Ph3 objective must be obtained from a facility director for each use
    • All other objectives are not capable of phase contrast
  • Check that the analyzer is out of the light path
  • Check that the phase rings are aligned
    • Turn the wheel below the eyepieces from "O" (= oculars) to "B" (for Bertrand lens)
    • While looking down the eyepieces, use the little silver knob on the eyepiece wheel to focus the Bertrand lens on the phase rings.  You should see a solid dark ring and a light ring consisting of 3 segments.
      • If the light ring is completely within the dark ring
        • The phase rings are aligned. 
        • Move the eyepiece turret back to "O" and continue
      • If the light ring is NOT completely within the dark ring:
        • Find 2 red-handled Nikon screwdrivers (back of the transmitted light arm, somewhere on the table or at the other Nikon station)
        • Insert the screwdrivers into the inner screw holes of the Phase ring insert in the condenser turret.
        • Turn the screwdrivers gently until the light ring is completely within the dark ring.
        • Once the light ring is completely within the dark ring, move the eyepiece turret back to "O" and continue

       

Setting up for fluorescence light imaging

• Lower objectives (only if you have not already focused on the sample using transmitted light)
• Ensure analyzer slider on right side of scope below filtercube turret
• Choose required objective.  (Generally choose a lower power, locate sample and region of interest and then move to a higher power)
• Ensure incandescent lamp is off at the switch on the left side of the scope OR the transmitted shutter is closed
• Place sample on stage, cover slip down (This is an inverted microscope!)
• Choose dichroic filter set with Nikon controller epi-filter:  closed, UV, B (fitc), Y (texas red), R ( far red))
• Select light path (see illustration on Nikon controller-- light path)
• Focus on sample
• Switch to higher power objective if desired

For imaging:

• The camera must be mounted on the right side of the microscope.  If it is not, see a facility director to have it switched.
• Set the microscope light path to the right camera (see Nikon controller-- light path)
• Set the eyepiece turret (just below the eyepieces) to the "C" position

Acquiring Images Using SimplePCI with widefield microscopy on the Spinning-Disc Confocal

• Log on to the computer
  • enter username (and press tab to move to-)
  • enter password (case is important)
  • choose domain MHMICROSCOPY (not local computer e.g. Nipkow)
    
     
• Start SimplePCI
  • run SimplePCI (C-Imaging)
    
    
    
     
  • During SimplePCI startup, 2 identical warning boxes will appear
    
  • Press OK or ignore messages concerning registry (report any other error messages to facility management and in the log book)
     
  • Main SimplePCI window will appear
    
     
• Start Capture mode in SimplePCI
  • Click on camera icon
    
     
  • Two additional windows will open
    • The  capture box window (for camera control)
      
       
    • The Image Display window
      
       
• Ensure desired camera is chosen in top right selection box (01)
  • Hamamatsu 1394 ORCA-ER
    
• Choose number of color channels (02)
   
  • 
     
• Select proper Filter Setting for each color channel using the drop down menus
  • For single channel imaging, choose Default Idle
  • For imaging multiple channels with widefield, use the manXXXX setting(s) (for manual changing)
                   
             
     
• Press Focus button, which starts rapid acquisition of an image to the display window (03)
           
   
• Increase or decrease exposure time in order to get an image (03)
  • for fluorescence it may be desirable to increase camera gain to maximum (04) and decrease exposure time in order to make focusing easier.  Note that image may be noisy.  Black level should not be changed.
     
• Focus while viewing image on monitor
   
• For multiple wavelengths, click on the "Red", "Green" or "Blue" tabs to change which channel is displayed in the Focus window
  •         [Insert Screen Image with tabs highlighted here]
     
• If desired, reduce camera gain (04) and increase exposure time (03) in order to reduce noise
   
• Press Stop (05) or Abort
           
   
• Press Capture 1 (06) to acquire a single image - to ensure exposure is correct, check intensity histogram on right side of image window
  •         [ISample image of fluorescent sample missing]
     
• Save the image
  • Right click on the image
  • Select "Save Image to File"
    • For Monochrome (single channel) images
      • ... "Original Image" -- saves the image as monochrome
      • ... "Display Image" -- saves the image as index color
        • only available if you have changed the color display in the image window
    • For Multi-channel images
      • ... "All Components" -- saves all components to a single image, each channel is the color in which it was acquired
      • ... "Red/Green/Blue Component" -- saves a monochrome image of the color channel selected
      • ... "Display Image" -- saves a 24bit color image of what is displayed in the Image window

• Shut down SimplePCI and log off when you are finished

Power down procedure:

1. Exit the SimplePCI software
2. Turn off the switch on the power strip next to the monitor.  This turns off the:
   • Sutter filter wheel changer
   • Prior focus controller
   • Hamamatsu camera controller
3. Turn off the microscope incandescent light (left side, front button)
4. Turn off the XBO (Fluorescence) lamp (always last!!)

		Copyright 2001-2015  Dr. M. Chua, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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