Installing and Aligning a Mercury/Xenon burner for Fluorescence Microscopy

Thanks to Emile Meylan of Serco Technical Services, Inc.

The light source for most epifluorescence microscopes is a high pressure mercury or xenon arc lamp. These lamps output a broad spectrum of light that spans from the UV through the entire visible range (Figures VI-33, VI-34). The life span of a mercury lamp is determined by the length of time it is on (shown usually as a digital meter on the lamp power source) plus the number of bulb “starts” multiplied by 4. Folklore suggests that each bulb start burns 4 h off the life of the bulb. The manufacturer’s projected life is between 200 and 300 h (Osram: HBO 100W/2 and HBO 103W/2; Ushio: USH-102D). In practice however, lamps last much longer; even up to a calculated 1000 h. As a rule replace the lamp when the meter reads 300–400 h or if the arc flickers. Overextending the use of a lamp can result in the bulb exploding or damage to the power supply. These lamps contain mercury, so dispose of them according to local environmental guidelines. The following are generic instructions for replacing and aligning arc lamps. Consult the instruction manuals for your particular microscope for details.

REPLACING THE LAMP

  1. Unplug the lamp power supply.
  2. Remove the lamp housing from the microscope and place it upside down on the bench with the collector lens facing you.
  3. Remove the collector lens or extend it to its outer position.

  4. Loosen the lamp base screw and carefully remove the base and bulb array. The bulb has a large heat sink attached to its distal end, so take care not to damage the reflector mirror within the lamp housing.
  5. If the wire connecting the distal end of the bulb is a rigid wire (e.g., Carl Zeiss, Inc.) loosen the wire’s connecting thumb screw. DO NOT apply any pressure or torque to the heat sink or bulb since the bulb will break.
  6. Loosen the pressure nut at the base of the lamp.
  7. Holding the heat sink, pull the lamp up and out.
  8. While holding the heat sink (not the lamp) use an appropriate tool and loosen the fastener holding the lamp to the heat sink, then remove the lamp.
  9. Assemble the lamp/heat sink array in the reverse order. Take care never to apply pressure or torque to the lamp or the lamp will break. When placing the new lamp back in the base be certain it seats fully into the base or alignment will be impossible.
  10. Place the base assembly into the lamp housing and tighten the fastener screw and insert the collector lens. Do not place the housing back on the microscope yet.
  11. Dispose the mercury lamp in compliance with local and Federal regulations.

ALIGNING THE LAMP

To increase the intensity of illumination, arc lamps have a mirror behind the lamp to reflect back to the specimen light that would otherwise be lost. The goal in alignment is to position the lamp and mirror to create two focused spots of light (direct and reflection) that are centered in the field of view but not overlapping.

  1. Start with the lamp off.
  2. While looking straight into the collector lens adjust the collector focus until you can see the lamp electrodes. You should also see the inverted mirror image, but it might not be in focus.
  3. Change the lamp height and lateral adjustments until the lamp electrodes (not the mirror image) are just slightly off center in the field of view. If achieving this is impossible, then disassemble the lamp housing and reinsert the lamp fully into the base.
  4. Mount the lamp housing on the microscope and turn on the lamp. The light output of an arc lamp is intensely bright. Wear good sunglasses for all following steps to protect your eyes.
  5. If the microscope has an alignment objective, mount this to the objective nosepiece. If the microscope has an integrated alignment port observe the arc here. Otherwise, remove an objective and observe the arc projected onto a piece of paper or a business card. Use an epifluorescence excitation line that is less intense (e.g., blue).
    1. Mirror Up/Down
    2. Mirror Left/Right
    3. Mirror image focus
    4. Lamp Height adjustment
    5. Lamp Left/Right
    6. Collector Focusing knob

  6. Focus the collector lens until the image of the arc (not mirror image) forms a sharp oval.
  7. Change the arc lamp adjustment screws (vertical and/or horizontal) until the image of the arc is just slightly to the left of center within the field of view.
  8. Adjust the three mirror controls until a focused, oval spot of light is approximately the same size and shape as the arc itself and positioned just to the right of center. You will have to adjust the mirror focus, then the vertical and/or horizontal positions multiple times to achieve this desired positioning.
  9. Stop off the arc light and insert an objective or swing the nosepiece into an objective position, then focus on a piece of fluorescent paper (a business card works well).
  10. Focus on the paper surface in epifluorescence mode and repeat steps 5–7 until there exists two centered ovals of light, the arc and image of the arc.
  11. Defocus the collector lens until both spots of light merge and fill the entire field of view. At this point the arc lamp is adjusted correctly.

Let the lamp "burn-in" for at least 3 h.