Centre Optical Instrumentation Laboratory

Hg-Bulb Alignment

1. Put new bulb into bulb holder being very careful not to touch the glass part of the bulb with your fingers. Ensure you have it the right way round. 100W bulbs have a fat end which generally goes in the lower part of the holder

2. Tighten the clamp screws holding the bulb in place (use a 1pence piece to ensure they are tight.

3. Replace lamp holder in lamp housing and re-attach to microscope.

4. Switch on bulb and leave for 30mins

5. Remove an objective from the objective turret and rotate the empty space into the light path , place 1 sheet of paper onto the microscope stage ensure that it is flat as the burner image is to be projected onto it.

6. Put in a neutral density filter between the lamp and the objective

7. Select a filter (FITC or TRITC are good choices) Open fluorescence shutter

8. The image from the mercury bulb should now be projected onto the paper, ensure that a strong enough neutral density filter is in place to attenuate the light as this can lead to eye damage. It should look something like this


9. On most lamp housings there are 4 screws to adjust

Z adjustment - moves the lamp up and down
XY adjustment - moves the lamp left or right
Mirror Adjustment - Moves the mirror in/out
Mirror Focus - Focus the lamp image

Generally the XY and Z adjustment screws are all that need to be moved

10. Using the adjustment screws, adjust the image to produce whats known as the angels wings pattern on the paper (PIC) The edges should be sharp and well defined and the shape symmetrical.

11. Replace the removed objective and put a fluorescent plastic reference slide onto the stage. Focus onto a point just below the surface and collect a fluorescent image

12. The image should be uniformly illuminated. This can be checked using the line profile tool available in any image analysis software. There should be less than 10% difference between any point in the image. (Note. Not all objectives are designed to have a flat field so check what kind of objective you are using.

Flat Image with Line Profiles

Flat Image

Non Flat Image with Line Profiles

Not Flat

Flat Profile

Non Flat Profile