Michael Hooker Microscopy Facility (MHMF.ORG)

Nikon Microphot SA  (Upright Compound Microscope)

Location: 6129 Thurston Bowles

Notices

• Although the upright compound microscope can do fluorescence imaging, it is not as efficient as the inverted Leica and Nikon TE2000 and Nikon 80i microscopes
• DIC (Normaski) is not functioning on this scope due to a missing polarizer and objective prism.
• When imaging with the 4x or lower power objectives the wide field of view condenser needs to be used
• When capturing brightfield images with the Act-1 software sometimes flecks may be evident.  To reduce this, set camera sensitivity to normal.

Introduction

This upright microscope is well suited for transmitted light microscopy although it uses the older 160 mm tube length optics.  It has a good quality color digital video camera attached to it, the Nikon DXM 1200, which is linked directly to the Dell PC (called \\Hooke on the MHmicroscopy Windows network).  Files can be transferred to connections to other computers on the campus network or to the MHmicroscopy file server called \\Minsky, which is accessible from either Windows or Macintosh computers. 

For very low magnification pictures please use the Leica MX16FA Fluorescence Stereo Microscope/Macroscope, which has transmitted and/or overhead illumination available as well as fluorescence.

Two different programs can run the DXM 1200 camera (both programs can not be run together):

• Act-1, which is simpler to run
• C-Imaging, which is the standard acquisition and processing software on most workstations. 

Setting up the Microscope - Kohler Illumination

1. Turn on power to microscope.  Switch is on left front side of the base (1).
2. Lower stage to allow sufficient clearance to change objective
3. Choose objective
4. Place slide on stage
5. Open field diaphragm at base of microscope to maximum
6. Minimize condenser aperture
7. Set lamp power to about "9" or press green "photo" button on the front of the base
8. Move condenser up as close as possible to the slide using its focus knob
9. Set condenser ring to "0"
10. Open port to the eye pieces (use the diagrams next to the sliders for guidance)
11. Choose neutral density filter (NDF) combinations, 1/2, 1/8 or 1/32 (front 3 buttons on right base of scope) for sufficient light - typically all out for no NDF
12. Focus on sample first
13. Minimize field diaphragm
14. Focus condenser by getting the sharpest image of the field diaphragm as possible
15. Center bright spot to match center of the field of view
16. Open field diaphragm to just fill the field of view
    *When imaging with the 4x or lower power objectives, the wide field of view condenser needs to be used.  See a facility director for instruction on changing the condenser.
17. Open the condenser aperture to maximum - gives more light, less contrast, thinner depth of focus and more even illumination intensity.  This is the best setting for photography.
18. Adjust focus if necessary

Imaging

• Turn on computer if necessary
• Log on using your 1. account name, 2. password & 3. mhmicroscopy domain (NOT local computer, Hooke)
• The Nikon DXM 1200 camera can be controlled with either the Act-1 or SimplePCI software

Using the ACT-1 Software (easier to use)

• Start Act-1 software
  1. In the Photo tab choose normal sensitivity
  2. Set Mode to "Quick" or "Fine" and picture size 1280 by 1024 pixels
  3. Set format to TIFF & check Auto Save box
  4. Turn on line histogram display
  5. Choose Save tab
  6. Use Image Folder to select location where images will be saved:
     • Choose a file name Prefix
     • Choose a file name Body e.g. $## will produce a series of 2 digit numbers.  "Ex:" will show name of next image file which will be saved
     • Press "Apply"
  7. Adjust exposure
     • If the exposure is not between 1/300th ("300") and 1/12th ("12") of a second, use combinations of the 3 neutral density filters on the right of the microscope base to adjust the light intensity (do NOT adjust the brightness of the lamp--this would effect the color of your image)
  8. Choose Live tab
  9. Perform a color balance on a white rectangular area (you may need to move your sample out of the field of view for this step)
  10. Do not change lamp power after performing a white balance
  11. Adjust exposure if necessary
  12. Press the green Exposure button to take a picture
  13. White balance does not need to be reset for subsequent slides
  14. repeat steps 12 & 13 for more photographs

   

Using the SimplePCI (more complex, but on all image acquisition systems)

Acquiring Images Using SimplePCI with the the Nikon DMX1200 color camera

• Log on the computer
  • enter username (and press tab to move to-)
  • enter password (case is important)
  • choose domain MHMICROSCOPY (not local computer e.g. FRESNEL)
  • 
• Start SimplePCI
  • run SimplePCI (C-Imaging)
  • 
  •  
  • 
  • During SimplePCI startup a warning box will appear
  • 
  • Press OK or ignore messages concerning registry
  • 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 Nikon DMX1200 is chosen in top right selection box (01)
  • 
  • Number of color channels should be Three Color Image (02)
  • 
  • Ensure transmitted light is on and Photo button is pushed in at the microscope
  • Ensure the camera ports are sending light to the camera
  • 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)
  • Focus using the microscope focus knob while viewing image on monitor
  • Press Stop (05) or Abort
  • 
  • If necessary, adjust the background color using the Color Balance control in the Capture window
    • You need a blank area of your sample for this
  • Press Capture1 (06) to acquire - to ensure exposure is correct, check intensity histogram
  • 
  • The hue can be corrected by doing an autocontrast (10)
  • 
  • Uncheck locked (11)
  • 
  • Hit auto (12)
  • 
  • Image will have a better color balance.  Corrected balance will be applied to subsequently captured images until the auto contrast button (10) is clicked off
  • 

   

• Saving the image
  • To save to uncorrected image
    • Right mouse click on image (or press the floppy diskette icon, 20)
    • Choose Save Image to file ---> All components and choose file name and save as Tiff
  • To save a color corrected image
    • right mouse click on image (or press the floppy diskette icon, 20) 
    • Choose Save Image to file ---> Display Image and choose file name and save as Tiff
  •  
• Shut down SimplePCI and log off when you are finished

Lenses

• PlanApo 2x/0.1, 160/-
• Plan 4x/0.13, 160/-
• Plan 10x/0.3, Ph1 DL, 160/0.17
• Plan 20x/0.5 DIC, 160/0.17
• Plan 40x/0.7 DIC, 160/0.17
• 50x 0.85 to 0.5collar  oil 160/0.17 - remember to set collar to NA 0.85
• Plan 100x/1.25, oil DIC, 160/0.17
• Note: use only Nikon immersion oil for the 50x or 100x objectives and the condenser on this microscope

Condenser

• Dark field is available
• The condenser can optionally use Nikon immersion oil and is appropriate with the 100x lens for the ultimate in resolution
• When using the 4x or 2x objectives the condenser should be replaced with the low power condenser or removed entirely.  Failure to do this will result in an incompletely filled field of view down the scope.  Please put the condenser back for the next user.

Acquisition Software

The Nikon DXM 1200 color camera can be controlled by either Act-1 or C-Imaging SimplePCI.  Of course both programs can not be used at the same time.

• Act-1 is the native Nikon DXM 1200 camera software.  This software is quite simple to use.
• C-Imaging is the standard acquisition and image processing software on most microscope systems in the facility.  See the Leica DMIRB pages for instructions on how to perform color image acquisition.

Fluorescence Filters

• Although filters are available for DAPI, FITC & Rhodamine  please use the much more efficient inverted Leica and Nikon microscopes.

		Copyright 2001-2014  Dr. M. Chua, Program in Molecular Biology & Biotechnology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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