ALIGNMENT OF LIGHT MICROSCOPE

Turn on transformer for tungsten light source and set at appropriate level
Set condenser setting to brightfield

Center lamp

• Place centering disk (paper, plastic, frosted glass or centering aid) over opening in stand below stage
• Center light with centering screws on lamp housing

Place specimen on stage and focus under low power objective (10X)

Adjust eyepieces

• Adjust separation of binocular tubes for eyes (interpupillary separation)
• Adjust eyepieces for eyes if necessary
  • Close one eye or cover eyepiece with card
  • Focus fine detail in specimen with fine focus
  • Focus same fine detail with other eyepiece with diopter adjustment collar

Center substage condenser

• Close field diaphragm until you can see edge of diaphragm in field of view
• Focus border by adjusting height of substage condenser
• Adjust substage condenser with centering screws until image of field diaphragm is centered
• Open field diaphragm until it is just outside field of view

Check focus of lamp filament (when aligning after bulb change)

• Open substage aperture diaphragm
• Remove eyepiece
• Loosen lamp lock screw on lamp housing
• Rotate bulb and move back and forth until illumination is most intense and even

Adjust substage aperture diaphragm

• Adjustment will vary for specimen
• Start with aperture wide open
• Close diaphragm slowly until image has best contrast
• Use neutral density filter(s) or adjust transformer rheostat to adjust brightness of illumination during viewing.

Phase Contrast Microscopy

• Adjust microscope as above
• Open substage aperture diaphragm if closed
• Replace one eyepiece with focusing telescope
• Set condenser setting to phase ring appropriate for objective
• Adjust lens of telescope until images of light and phase rings are sharp
• Adjust phase ring until image of light ring is superimposed on image of dark ring

  Adjustment mechanism differs on different microscopes. Some use screws or wheels. The Leitz microscope in my laboratory uses centering keys placed into back of phase ring holder in condenser. Keys must be removed before changing to next phase ring (next step).

• Repeat phase ring adjustment for each objective and phase ring pair
• Replace focusing telescope with eyepiece

Epifluorescence Microscopy

• Turn on fluorescence light source and allow to warm up
• Remove objective or swing alignment aid into place
• Place piece of white paper on stage
• Center fluorescent bulb with screws on light housing until light on paper is brightest and most uniform
• Insert appropriate excitation and barrier filter combination
• Place specimen on stage and focus under a low power objective
• Close fluorescent diaphragm and center using centering screws
• Open diaphragm until edge is just out of field of view