Troubleshooting Darkfield Microscopy

There are numerous common problems associated with darkfield microscopy and photomicrography or digital imaging. These range from insufficient illumination and condenser mis-alignment to using a field stop of incorrect size. Most darkfield illumination problems are associated with the substage condenser, and this should be the first suspect when things do not work properly. The following problems and solutions should be used as a guide when imaging specimens using this technique.

Problem: There is insufficient illumination to make the specimen visible, or the specimen is visible but very faint.
Solution: The lamp voltage may be set too low, or the lamp may have insufficient intensity to properly illuminate the specimen. Raise the voltage to a maximum of about 12 volts (for 12-volt, 100 watt tungsten-halogen bulbs) or replace the light source, if possible, with one of greater intensity. If this does not solve the problem, check the substage condenser to ensure that it is positioned correctly with a field stop of the appropriate size. Also, check the light path for color and/or neutral density filters, polarizers, retardation plates, or any other components that might reduce illumination intensity. If this problem occurs with high numerical aperture reflecting condensers, check to make certain that the condenser top lens is correctly oiled to the bottom of the microscope slide. If the immersion contact has been broken, re-apply the oil and rack up the condenser until the top lens is completely immersed in oil and in contact with the bottom of the slide.

Problem:
There is insufficient illumination to make the specimen visible, or the specimen is visible but very faint.

Solution: The lamp voltage may be set too low, or the lamp may have insufficient intensity to properly illuminate the specimen. Raise the voltage to a maximum of about 12 volts (for 12-volt, 100 watt tungsten-halogen bulbs) or replace the light source, if possible, with one of greater intensity. If this does not solve the problem, check the substage condenser to ensure that it is positioned correctly with a field stop of the appropriate size. Also, check the light path for color and/or neutral density filters, polarizers, retardation plates, or any other components that might reduce illumination intensity.

If this problem occurs with high numerical aperture reflecting condensers, check to make certain that the condenser top lens is correctly oiled to the bottom of the microscope slide. If the immersion contact has been broken, re-apply the oil and rack up the condenser until the top lens is completely immersed in oil and in contact with the bottom of the slide.

Problem: The viewfield has a dark spot in the center reducing illumination intensity, but objects in the periphery are well-illuminated and appear normal.
Solution: The substage condenser is either incorrectly positioned or the light stop is the wrong size for the objective. Carefully rack the condenser up and down while observing the specimen in the viewfield. Check to make certain that both the condenser aperture and field diaphragms are set to the wide open position. If this does not affect the illumination, change the field stop size making it either slightly larger or smaller and then re-check the illumination. Occasionally this problem arises when microscope slides of excessive thickness (greater than the standard one millimeter) are used. Use a micrometer or a set of dial calipers to determine the thickness of the slide and the slide/coverslip combination. If the slide is too thick, either change to a slide of the correct thickness or check the condenser to determine if it can be adjusted to compensate for slides of varying thickness.

Problem:
The viewfield has a dark spot in the center reducing illumination intensity, but objects in the periphery are well-illuminated and appear normal.

Solution: The substage condenser is either incorrectly positioned or the light stop is the wrong size for the objective. Carefully rack the condenser up and down while observing the specimen in the viewfield. Check to make certain that both the condenser aperture and field diaphragms are set to the wide open position. If this does not affect the illumination, change the field stop size making it either slightly larger or smaller and then re-check the illumination.

Occasionally this problem arises when microscope slides of excessive thickness (greater than the standard one millimeter) are used. Use a micrometer or a set of dial calipers to determine the thickness of the slide and the slide/coverslip combination. If the slide is too thick, either change to a slide of the correct thickness or check the condenser to determine if it can be adjusted to compensate for slides of varying thickness.

Problem: Lamps tend to have a very short lifetime and the bulb interior is sintered with a coating of black evaporated tungsten.
Solution: The bulbs have been used at excessive voltage for long periods, which will definitely reduce the life span. Replace the bulb and reduce lamp voltage after observation and/or photomicrography of the specimen.

Problem: The image has a brightfield periphery with or without dark regions in the center.
Solution: This problem is typical when the substage condenser is incorrectly centered or the field stop is too small. Revert to brightfield and re-establish the conditions of Köhler illumination, then repeat the darkfield procedure, making sure the condenser is centered. If this does not help, increase the size of the field stop until proper darkfield conditions are established. This problem may also arise when using an objective that has too high a numerical aperture for the darkfield condenser. If there is an internal iris diaphragm in the objective, reduce the iris opening size to determine if too much oblique illumination is entering the front lens of the objective. Also, it may help to increase the size of the field stop or change to a reflected darkfield condenser of higher numerical aperture.

Problem:
The image has a brightfield periphery with or without dark regions in the center.

Solution: This problem is typical when the substage condenser is incorrectly centered or the field stop is too small. Revert to brightfield and re-establish the conditions of Köhler illumination, then repeat the darkfield procedure, making sure the condenser is centered. If this does not help, increase the size of the field stop until proper darkfield conditions are established.

This problem may also arise when using an objective that has too high a numerical aperture for the darkfield condenser. If there is an internal iris diaphragm in the objective, reduce the iris opening size to determine if too much oblique illumination is entering the front lens of the objective. Also, it may help to increase the size of the field stop or change to a reflected darkfield condenser of higher numerical aperture.

Problem: The periphery of the viewfield is bright on one side only.
Solution: Check the nosepiece to ensure the objective is correctly positioned in the optical axis of the microscope. There is a detent stop that should produce a positive "click" when the objective is properly positioned. The problem might also occur if the field stop is not correctly centered. Remove the eyepiece and place a phase telescope in the eye tube (or use a Bertrand lens), then use the centering screws to properly align the field stop.

Problem:
The periphery of the viewfield is bright on one side only.

Solution: Check the nosepiece to ensure the objective is correctly positioned in the optical axis of the microscope. There is a detent stop that should produce a positive "click" when the objective is properly positioned. The problem might also occur if the field stop is not correctly centered. Remove the eyepiece and place a phase telescope in the eye tube (or use a Bertrand lens), then use the centering screws to properly align the field stop.

Problem: Images of the specimen are not clear and lacking in sufficient contrast and detail.
Solution: The specimen might not be suitable for darkfield microscopy. Many stained specimens do not scatter enough light to be clearly imaged under darkfield conditions. Specimen thickness may also be a problem, because very thin specimens are often not imaged well with the oblique light rays emitted from darkfield condensers. Change to brightfield, phase contrast, DIC, or Hoffman Modulation Contrast to determine if this improves specimen contrast.

Problem:
Images of the specimen are not clear and lacking in sufficient contrast and detail.

Solution: The specimen might not be suitable for darkfield microscopy. Many stained specimens do not scatter enough light to be clearly imaged under darkfield conditions. Specimen thickness may also be a problem, because very thin specimens are often not imaged well with the oblique light rays emitted from darkfield condensers. Change to brightfield, phase contrast, DIC, or Hoffman Modulation Contrast to determine if this improves specimen contrast.

Problem: Bright areas that are out of focus obstruct viewing and/or photomicrography of darkfield specimens.
Solution: This is probably due to dust, hair, fibers, and/or dirt contamination of an optical surface somewhere above the condenser. Thoroughly clean the specimen slide with optical-grade tissue or cotton. Occasionally, this problem arises due to contamination on the objective front lens. Carefully loosen the objective from its seat in the nosepiece and slowly turn while observing the specimen through the eyepieces. If the obstruction rotates along with the objective, then it is probably due to dust on the front lens. Remove the objective and moisten the front lens by gently exhaling on it, then clean with lens tissue or a cotton swab wrapped on a long wooden rod. Replace the objective and check to make certain the obstruction has been completely removed.

Problem:
Bright areas that are out of focus obstruct viewing and/or photomicrography of darkfield specimens.

Solution: This is probably due to dust, hair, fibers, and/or dirt contamination of an optical surface somewhere above the condenser. Thoroughly clean the specimen slide with optical-grade tissue or cotton. Occasionally, this problem arises due to contamination on the objective front lens. Carefully loosen the objective from its seat in the nosepiece and slowly turn while observing the specimen through the eyepieces. If the obstruction rotates along with the objective, then it is probably due to dust on the front lens. Remove the objective and moisten the front lens by gently exhaling on it, then clean with lens tissue or a cotton swab wrapped on a long wooden rod. Replace the objective and check to make certain the obstruction has been completely removed.

Problem: The background is evenly illuminated but is not completely dark or appears gray in color.
Solution: This problem usually occurs when the opaque field stop is too small for the objective. Remove the eyepiece and view the back focal plane of the objective using either a phase telescope or a Bertrand lens without a specimen in the light path. The entire back focal plane should be dark without any rim of bright light appearing around the periphery. If an arc or circle of light is observed in the back focal plane of the objective, change to a slightly larger field stop. Also, reducing the size of the field diaphragm will aid in suppressing glare and producing a jet-black background (be careful not to close this iris aperture diaphragm too much).

Problem:
The background is evenly illuminated but is not completely dark or appears gray in color.

Solution: This problem usually occurs when the opaque field stop is too small for the objective. Remove the eyepiece and view the back focal plane of the objective using either a phase telescope or a Bertrand lens without a specimen in the light path. The entire back focal plane should be dark without any rim of bright light appearing around the periphery. If an arc or circle of light is observed in the back focal plane of the objective, change to a slightly larger field stop. Also, reducing the size of the field diaphragm will aid in suppressing glare and producing a jet-black background (be careful not to close this iris aperture diaphragm too much).

Problem: The image of the lamp condenser partially fills the viewfield, obscuring details of the specimen.
Solution: The slide may be too thin or the condenser may be adjusted too high. Measure the thickness of the microscope slide and check to make certain the oil contact is not broken. If difficulty is encountered keeping the bottom of the slide oiled, try using a thicker slide.

Problem: Colors appear in the background, or the background is unevenly lit with a gray cast.
Solution: This problem often occurs with whole mounts that are very thick or when too many light-scattering contaminating artifacts are captured in the mounting medium. Close down the field diaphragm and rotate the specimen slide to locate the best orientation, then determine whether the problem has been corrected. The specimen may have to be re-mounted in a thinner mount or the mounting medium may need to be filtered. Carefully clean the entire microscope slide and coverslip with a soft lens tissue or cotton swab.

Problem:
Colors appear in the background, or the background is unevenly lit with a gray cast.

Solution: This problem often occurs with whole mounts that are very thick or when too many light-scattering contaminating artifacts are captured in the mounting medium. Close down the field diaphragm and rotate the specimen slide to locate the best orientation, then determine whether the problem has been corrected. The specimen may have to be re-mounted in a thinner mount or the mounting medium may need to be filtered. Carefully clean the entire microscope slide and coverslip with a soft lens tissue or cotton swab.

Problem: When viewing aquatic organisms in a aqueous mount, specimens drift constantly in a single direction, making observation and photomicrography difficult.
Solution: Convection currents are being created due to slow evaporation at the edges of the coverslip. Place a bead of petroleum jelly or Cytoseal mounting medium around the periphery of the coverslip to seal it firmly onto the microscope slide. Commercial products are available that can be added to the water to slow the movement of these minute organisms, making them easier to photograph.

Problem: When viewing the specimen through a 10x objective, there is a oblong spot of light in the center of the viewfield.
Solution: The lamp condenser may be incorrectly focused or the diffusion filter may be missing, leading to an image of the lamp filament appearing in the viewfield. Re-focus the condenser and make certain there is a diffusion filter in the light path.

Problem: The viewfield has bright spots and there are problems with achieving sharp focus of the specimen.
Solution: Small air bubbles may be trapped in the oil between the top of the condenser and the bottom of the microscope slide. Remove the eyepiece (or insert a Bertrand lens) to observe the back focal plane of the objective, where the bubbles will be visible. To remedy, break the oil contact and remove any residual bubbles with a cotton swab. Clean excess oil from the condenser front lens and the microscope slide, then apply a clean drop of oil to the lens. If the problem persists, thoroughly clean all optical surfaces before re-applying the oil.

Problem:
The viewfield has bright spots and there are problems with achieving sharp focus of the specimen.

Solution: Small air bubbles may be trapped in the oil between the top of the condenser and the bottom of the microscope slide. Remove the eyepiece (or insert a Bertrand lens) to observe the back focal plane of the objective, where the bubbles will be visible. To remedy, break the oil contact and remove any residual bubbles with a cotton swab. Clean excess oil from the condenser front lens and the microscope slide, then apply a clean drop of oil to the lens. If the problem persists, thoroughly clean all optical surfaces before re-applying the oil.

Problem: At high magnifications, colloidal particles display incomplete Newton rings and appear to be unevenly illuminated.
Solution: The condenser is probably off-center. Change to a lower power objective and re-center the condenser. Carefully check to determine whether the higher power objective has a different center, and if so, center the objective to the microscope optical axis.

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.

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