Microscope Maintenance

Image quality depends on how well a microscope is maintained. If you are maintaining a microscope or preparing for some critical imaging experiment, a clean microscope is the first consideration. Details for cleaning are presented at the Cleaning Your Microscope web page. Other adjustments may not require daily attention for routine imaging, but should be checked periodically. While the details may differ on indivdual microscopes, the general concepts are described here.

These require greater familiarity with the specific microscope and may not be appropriate for every microscope. The following sections are ordered according to the beam path, begining with the light source.

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A well aligned bulb will produce a uniformly illuminated field of view for transmitted light images. If gradient illumination or bright areas appear in your transmitted light images, check the halogen bulb alignment. When the bulb is properly aligned, its filaments should be centered in the pupil as seen from the back aperture of the objective. Many microscopes will have a diffuser glass in front of the bulb that will have to be removed before the filaments can be seen.

Method: Swing any filters out of the path and focus on an empty sample spot with 40x or less objective. Remove an ocular and and look down the beam path at the back aperture to see the bulb coil and its mirror image. Center the lamp coils in pupil image with adjusting screws. Adjust to the most complete and even illumination in the pupil, spacing lamp coils to fill eachother's gaps.

If you have replaced a bulb, you may want to do a coarse alignment before replacing the lamp assembly. Point the light port at a nearby wall to view the projection. Do not view the bulb directly to protect your eyes. Adjust both lamp coil images in the projection to be as clearly defined as possible. Turn the lamp off, replace the diffuser and replace the lamp assembly. Use the method above for fine adjustment.

Caution! These parts can become very hot. Protect your eyes from directly viewing the bulb. Follow the manufacturers warnings regarding working near high voltage.

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Mercury bulbs should be aligned to obtain the most evenly illuminated field possible. If dark areas appear in the fluorescent image or the illumination is not uniform, check the collector focus for the mercury bulb. If uniform illumination cannot be obtained, the bulb may need to be aligned.

Method: Set the adjusting aid into position and note the spacing of the two arc images. The real image is brighter than the reflected image.

• Adjust the collector focus to get the sharpest view of the real image. Center it with adjusting screws..
• Use adjusting screws to bring the reflected (dimmer) image into focus and equal size.
• Adjust the up/down to center the reflected image.
• Adjust side-by-side so that the images touch but do not superimpose.
• Adjust the collector focus to get the brightest uniform illumination.

   

Caution! Unfiltered UV light is harmful to eyes and skin. Mercury arc lamps have high internal pressure. Know the safety precautions for use and handling.

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Carl Zeiss HBO Microscope Illuminating Systems

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HBO 100 Illuminating System Download PDF
Quick Guide HBO Self-Aligning Download PDF
Zeiss Tips #6 Download PDF

DIC images are especially sensitive to dirt on the optical surfaces. In addition to cleaning objective and condenser front lenses, DIC polarizers and prisms should be inspected for dirt and cleaned if necessary. To check the DIC alignment, the microscope should be configuring for Köhler illumination, have the polarizer and analyzer inserted and objective and condenser DIC prisims in place. Remove the objective prism. Observe the field of view at the rear aperture with phase telescope. A dark well defined diagonal (45) band should transverse the field.

Replace the objective prism while continuing to observe the rear aperture via the phase telescope. A dark extinction cross should appear with arms oriented vertically and horizontally. A small amount of light may appear at the four corners. Bright spots (birefringent areas) elswhere indicate strained glass in objective or condenser. Dust and lint particles near a conjugate plane (condenser or objective) appear bright.

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The pinhole of confocal laser scanning microscopes blocks light from fluorescing areas of the sample that are out of the focus plane to give a superior 3D imaging capability. A smaller diameter eliminates more out of focus light, but reduces intensity, and must be positioned precisely in front of the detector. When properly positioned, the typical 1 Airy unit setting should allow noticeable variation from adjustments of detector gain and offset settings to optimize the image. When out of position, less focused light reaches the detector resulting in low intensity as any gain setting.

Each combination of dichroic mirrors, filters and objectives (anything in influencing the beam path) will have its own optimal position for the pinhole. Initially , these are made by at the factory, stored by the software and used accordingly.

Different dichroic mirrors, or any changes to the beam path will usually require different positioning.

When to adjust: Any time there is loss of intensity or sharpness in the image, the pinhole position should be checked. For periodic maintenance, all of the settings should be ajdusted monthly using automated adjustment software.

Where: Adjustments made for one specific configuration are done in the Pinhole and Collimator window of the imaging software using a fast scanning speed, no averaging, with the smallest diameter that produces highest contrast. Adjustments are made to X, Y, and Z coordinates to maximize image intensity. When that position is reached, it should be stored in the software and will become the setting for all users.

Automated adjustment software is available for periodic maintenance to improve all stored combinations using a 10x objective focused on a mirror slide. Depending on the number of combinations stored, this may take some time and the procedure should not be interrupted.

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Carl Zeiss LSM 510 META Manual Rel. 3.5

Carl Zeiss LSM 5 PASCAL Manual Rel. 3.5

Carl Zeiss ConfoCor 3 Manual

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