Fluorescence Microscopy Configuration and Photomicrography Errors

Microscope Configuration Errors

Problem No. 1:
The microscope lamp is on, but the image cannot be seen, or is darker than the expected output.
Solution: This problem could be caused if the shutter knob is not closed, or if a low transmission (very dense) neutral density filter is in use. To alleviate the error, move the shutter knob to an open aperture, and/or remove the neutral density filter from the light path. Similar problems also occur when the fluorescence cube is not correctly rotated into the light path, or if the cube in use is not suitable for the specimen (wrong excitation and/or barrier filter). If no illumination is visible after rotating the cube into the appropriate position, check the filter parameters and change to a suitable cube. An incorrect combination of the exciter and/or barrier filter could also cause dim or absent illumination in the microscope eyepieces. Check the manufacturer's data sheets on the fluorochrome to ensure the proper filters are being used. Finally, check to make sure that the substage condenser aperture iris diahragm, the field iris diaphragm, and objective iris diaphragm (if present) are opened to the proper setting. If any of these diaphragms are closed or not opened sufficiently, open them to the widest point and peer into the eyepieces to see if secondary fluorescence is being emitted by the specimen. Also check the microscope alignment to ensure it is properly adjusted for Köhler illumination.
Problem No. 2:
The specimen is emitting secondary fluorescence, but details are unclear and blurred and/or the image is lacking in sufficient contrast.
Solution: The most obvious cause of this problem would be dirty objectives or filters. Carefully clean the objective front lens and filters with soft cotton or lens tissue. Apply sparing amounts of lens cleaning solvent if persistent dust, dirt, or debris remains on the glass surface(s). If this does not cure the problem, check the exciter filter and barrier filter combination to ensure it is adequate for the fluorochrome. Poor contrast can also occur when using an objective with an iris diaphragm that is opened too far or when the aperture diaphragm in the vertical illuminator is not closed sufficiently to generate suitable contrast. Carefully check the aperture and field diaphragm and adjust if necessary. To do this, open the aperture iris diaphragm completely and close the field iris diaphragm opening until the leaves are just outside the photo frame in the eyepiece reticle. Then slowly close the aperture diaphragm while examining the specimen for contrast. Finally, make certain the fluorescence filter cube is suitable for the desired wavelength ranges.
Problem No. 3:
Parts of the image are obscured or unevenly illuminated.
Solution: Check the objectives to make sure that they are inserted into the light path correctly. Do this by rotating the revolving nosepiece until it "clicks" into place on the détente. This action should lock the objective into place. Also check to make sure that the cube is properly oriented in the light path, and that the excitation and barrier filters are suitable for the chromophore. Often, problems such as this can occur due to insufficient opening and/or alignment of the field or aperture diaphragm. Open and close these diaphragms while viewing the specimen to see if they are causing the problem. Other sources could be an extended shutter slider, an off-center mercury burner lamp, or inadequate focus adjustment. Most or all of the latter problems will be cured if the microscope is properly configured for Köhler illumination.
Problem No. 4: An image is present in the eyepieces, but it has excessive glare.
Solution: In this case, it's most likely that either the exciter filter or barrier filter has not been inserted or have been installed improperly. Consult the fluorochrome data information sheet and insert a filter cube containing the proper filters.
Problem No. 5:
The power switch indicator does not light up and the mercury burner is not lit.
Solution: Check the power cord to make sure it is connected properly. If so, check all toggle and rocker switches on the microscope. If the burner still does not light, chances are it is burned out. Install a new burner.
Problem No. 6:
The power switch indicator lights, but the mercury burner seems inoperative.
Solution: First, make sure that the mercury burner connectors have been installed correctly. Also, make certain that the lamp housing interlock is operating. Do this by tightening the bulb socket locking screw securely. If the problem persists, it could be due to a faulty auto-ignition switch. Try turning off the power supply unit and then turning it back on and continue attempts to fire the burner. Repeat as necessary, or call a qualified service technician.
Problem No. 7:
The mercury burner flickers, or is dark.
Solution: This occurs when the mercury burner is warming up to operating temperature. Wait for 10 minutes after turning on the burner for the arc lamp to stabilize. If the problem is not corrected after 20 minutes or so, the bulb life may have expired. Replace the mercury burner if the life meter reads over 200 hours.

Photomicrography Errors

Problem No. 8:
Secondary fluorescence emitted by the specimen is weak, producing insufficient brightness to view or photograph the specimen.
Solution: This is one of the most common problems in fluorescence microscopy and has a number of causes. Check previous storage conditions for the specimen to determine if it should be replaced with a newer candidate. If photobleaching is suspected, move to a new observation area of the specimen to see if secondary fluorescence is recovered. In cases where bleaching is confirmed, use transmitted light darkfield, phase contrast, or differential interference contrast for area selection on the specimen prior to illumination with the mercury burner for observation and photomicrography. When photobleaching does not appear to be the culprit, check the objectives to ensure the numerical aperture is adequate for imaging the specimen with fluorescence illumination. Excessive magnification of the eyepieces (and photo eyepiece) will diminish specimen brightness, so check the magnification factor on these lens assemblies. Photo eyepiece magnification should not exceed 3.3x, and oculars should not have a magnification factor above 10x. When using transmitted light, check the substage condenser aperture diaphragm to ensure it is opened sufficiently to illuminate the specimen. Always use the highest possible objective numerical aperture at the lowest suitable magnification. If possible, use an immersion darkfield condenser and apply non-fluorescing immersion oil between the front lens of the condenser and the underside of the microscope slide. Remember to also place oil between the objective front lens and the microscope coverslip. Occasionally, room lights will be too bright to visualize a weakly fluorescent specimen. The simple cure is to darken the room and never use the microscope in front of an open (to daylight) window.
Problem No. 9:
Secondary fluorescence emitted by the specimen is visible but photomicrographs have very poor contrast.
Solution: Poor contrast errors in fluorescence photomicrography have a number of origins. One of the most common is non-specific staining of the specimen by fluorescent chromophores. Review sample preparation conditions to determine if the specimen has been sufficiently cleaned (washed) to remove excess dye prior to photomicrography. Also examine all other phases of the preparation procedure to ascertain whether any adjustments need to be made. If specimen preparation seems adequate, check the excitation filter bandwidth to determine if it is too broad, a common cause of contrast errors. A fluorescence cube with a narrow bandwidth should be substituted to see if contrast problems improve. When using oil immersion objectives (and/or a transmitted light darkfield condenser), check the quality of the immersion oil to ensure it does not autofluoresce. This can be done by oiling and examining a blank microscope slide with no specimen in place.
Problem No. 10:
Secondary fluorescence seems to be diminished by autofluorescence somewhere in the specimen or microscope optical train.
Solution: Autofluorescence can be a problem when imaging relatively thick tissue specimens derived from both plants and animals. It will also arise from non-specific staining by fluorescent chromophores, and will only be aggravated by inherent specimen autofluorescence. Carefully clean the specimen to remove unwanted dye and check the specificity of the stain through a control. Also check the filter block to determine if the excitation and barrier filters match the absorption and emission characteristics of the chromophore. If not, consult the literature to determine the correct filters and replace the unsatisfactory filter block with one that will do the job. Examine the immersion oil to determine if it is the source of autofluorescence, and review the manufacturer's literature on optical components in the microscope (objective, photo eyepiece, beamsplitter, condenser, etc.) to ensure they are intended for fluorescence microscopy.
Problem No. 11:
When examining specimens with double fluorochromes, green and red (for example) image details can be seen simultaneously.
Solution: This error is most likely due to use of a filter block that is unsuitable for selective observation of individual chromophore secondary fluorescence. For instance, to selectively observe FITC secondary fluorescence in a doubly stained specimen also using TRITC, an Olympus WIBBA filter cube can be used to examine only the green fluorescence emitted by FITC, while a WIG filter cube can be used to exclusively visualize red fluorescence from the TRITC chromophore. Pay careful attention to filter cube and chromophore fluorescence specifications when designing experiments with doubly and triply stained specimens. Often, with judicious choice of filter cubes, the dyes can be observed and photographed individually, in pairs, or collectively.
Problem No. 12:
Specimens are displaying a dramatic drop in fluorescence intensity during observation and photomicrography.
Solution: Photobleaching is the likely cause of this problem. Many chromophores are rapidly bleached and undergo a reduction in secondary fluorescence intensity by the energy-rich excitation radiation passing through the filter cubes and onto the specimen. When examining specimens with transmitted light or a combination of transmitted and reflected light fluorescence, use darkfield, DIC, or phase contrast in conjunction with a tungsten-halogen light source for area selection. Antifade reagents, if available, can also be used to diminish the rate of photobleaching and allow extended observation and photomicrography times. Often, neutral density filters can be utilized to reduce light intensity during observation of specimens and image composition for photomicrography. When a suitable viewfield is found, remove the neutral density filter and switch to full intensity illumination for photomicrography using film or digital imaging techniques.
Problem No. 13:
Secondary fluorescence is visible, but the background is very light and/or has a reddish cast.
Solution: Light background intensity can be caused by several factors, including incorrect use of excitation and barrier filters. The suitability of filters for specific fluorochromes should be checked and the specimen monitored for possible overstaining, which will also lead to lowered contrast with lightened backgrounds. Check to see if a red suppression filter has been inserted into the optical pathway, and install the appropriate filter if none is present. Red suppression filters block far-red emission light that is detected and recorded by film.
Problem No. 14:
Specimens appear sharp and crisp in the viewfield with sufficient fluorescence intensity, but there is uneven illumination from one side of the field to the other.
Solution: There is a good possibility that the mercury vapor lamp is not centered correctly. Use a specially designed centering screen that fits into a threaded objective mount on the nosepiece to facilitate the centering of the image of the lamp arc to the back aperture of the objective. Also check to ensure the lamp is not flickering with uneven intensity and has not exceeded its lifetime. If any problems with the lamp are detected, install a new lamp and re-center it according to the tenets of Köhler illumination. In cases where the microscope is being used with a darkfield condenser and transmitted light excitation, check the centration of the condenser and make certain it is aligned with the center of the optical path. Air bubbles in immersion oil or poor contact between the objective front lens and a pool of oil can also cause uneven illumination. Carefully examine the oil drop contact between the coverslip and objective front lens when conducting high magnification fluorescence experiments.
Problem No. 15:
The specimen image suddenly becomes unsharp or falls out of focus during observation.
Solution: This problem is often caused by a "floating cover glass", which occurs when immersion objectives are being used to image a specimen and the cover glass is inadvertently pulled up by the objective during focusing. The solution is to remount the cover glass on the microscope slide with mounting medium (watch for autofluorescence), nail polish or a commercial cover glass cement.
Problem No. 16:
Extremely fine specimen details and contours appear unsharp when using certain fluorochromes and specimens such as quinacrine mustard on human chromosomes.
Solution: Lens flare is probably the culprit in this case. If possible, switch to objectives with an internal iris diaphragm, and reduce the objective numerical aperture by closing the diaphragm until the flare has disappeared.

Contributing Authors

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.

Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.