Michael Hooker Microscopy Facility (MHMF.ORG)

Zeiss 510 Meta Laser Scanning Confocal Microscope

Setting Up the Zeiss Axiovert200 Inverted Microscope
**Requires Zeiss 510 Meta software for microscope control

    (print version)

• Set up optics on the Zeiss Inverted
  • For transmitted light (Kohler Illumination) - if transmitted illumination is not required then go to "for fluorescence only" below
    • Log on to computer and run LSM 510 Meta software (opens ocular shutter)
      • Choose scan new image
      • Expert mode
      • Push on microscope icon in order to open Microscope Control window
    • On Microscope Control window, choose "transmitted" button
    • Open field diaphragm (silver slider; manual control only)
    • Minimize condenser aperture - important!
    • Lower objective by pressing and releasing the ▼ FOCUS button on right of scope
    • Place slide on stage with cover slip down (This is an inverted microscope!)
    • Choose required objective by selecting the objective in the Microscope Control window 
      • Generally choose a lower power dry objective, locate sample and region of interest and then move to a higher power dry or immersion objective
    • Raise objective by pressing and releasing the ▲ FOCUS button on right of scope
    • Adjust incandescent lamp power if necessary using the rocker control on the front of the microscope
    • Focus onto sample using the fine focus knob
    • Close field diaphragm until see a dark ring impinging onto field of view.  If using 63x or 100x condenser may have to be moved off center to see an edge of the field diaphragm.
    • Focus the image of the ring with the condenser focus knob, and center it with the 2 knurled screws at 45 degrees.
    • 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 and for optimum image quality redo Kohler Illumination setup
       
  • For Nomarski (DIC) - if desired
    • Set up for Kohler
    • Open condenser aperture maximally
    • Swing in polariser and setup angle to zero degrees
    • Push "DIC III" button to switch in analyser and the two Wollaston pisms
    • Note for 10x objective choose "DIC II"
    • Adjust Wollaston prism shear by turning know at base of the selected objective for a uniform brick gray color

     

  • For fluorescence only
    • Lower objectives
    • Choose required objective.  (Generally choose a lower power, locate sample and region of interest and then move to a higher power)
    • Place sample on stage cover slip down - (This is an inverted microscope!)
    • From Microscope COntrol window choos FITC, Rhodamine or Dapi filter sets (should see blue light for FITC, or green light for tritc/texas red or dim dark blue for DAPI (don't look at it)
    • Focus on sample
    • Switch to higher power objective if desired
    • Push "Dark" when not viewing sample in order to avoid bleaching the sample

     

		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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