Anoplocephala perfoliata (Tapeworm) in Equine Cecum

The longitudinal section through a tapeworm was easily seen with a quick glance at the hematoxylin and eosin slide and the unusual specimen immediately leant itself to further exploration. When viewed with darkfield, the colors that resulted added the final touch to an arresting image. This photomicrograph won 7th place in the 1998 Small World competition.

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  Anoplocephala perfoliata (Tapeworm) in Equine Cecum

  Specimen: Hematoxylin and Eosin Section
  Technique: Darkfield Illumination
  

  Visual impact is an important first element in choosing subjects for creative microscopy. Most often, the detail can only be seen through the microscope and the microscopist faces long hours of searching for interesting images. In this case, the longitudinal section through a tapeworm was easily seen with a quick glance at the hematoxylin and eosin slide and the unusual specimen immediately leant itself to further exploration. When viewed with darkfield, the colors that resulted added the final touch to an arresting image. This photomicrograph won 7th place in the 1998 Small World competition.

Anoplocephala perfoliata (Tapeworm) Segments

This view of the tapeworm provides the scientist with a better view of the rudimentary segments, but the abstract visual pattern lends itself to other levels of artistic interpretation as well.

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  Anoplocephala perfoliata (Tapeworm) Segments

  Specimen: Hematoxylin and Eosin Section
  Technique: Darkfield Illumination
  

  As with all other forms of photography, the microscopist should not be satisfied with only one view of a subject. Even though the first attempt at photographing this tapeworm was successful at low magnification, further experimenting with magnification and composition can often result in adding to the story of the subject. This view of the tapeworm provides the scientist with a better view of the rudimentary segments, but the abstract visual pattern lends itself to other levels of artistic interpretation as well.

Danio rerio (Zebrafish) Head

As with the low magnification image of a tapeworm, this cross section through the head of a zebrafish was easily seen with a cursory glance at the histology section. This photomicrograph won 20th place in the 1999 Small World competition.

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  Danio rerio (Zebrafish) Head

  Specimen: Dividing cells in culture (triple labeled)
  Technique: Confocal Microscopy
  

  This specimen was so large that the challenge here was to be able to apply darkfield techniques using the standard turret condenser with a 1X objective. Ordinarily, such a combination would not be possible, due to the extreme low magnification creating vignetting in all corners of the image. In this case, by de-focusing the condenser somewhat, the darkfield light was able to spread a little more evenly over the center area and, because the subject is basically circular, all the vignetting fell in the black area anyway.

Danio rerio (Zebrafish) Gills

Additional exploration of sections from the zebrafish turned up additional, interesting patterns at higher magnifications. As the serial sections approached the gill area, it only took changing the composition slightly to discover even more patterns.

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  Danio rerio (Zebrafish) Gills

  Specimen: Hematoxylin and Eosin Section
  Technique: Darkfield Illumination
  

  First seen at a lower magnification, this "eagle totem" interpretation was not apparent until the view was tightly cropped. With further experimenting, the gill branches on the left side of the image provided yet another visual abstract as seen in this zebrafish image.

Danio rerio (Zebrafish) Gill Branches

Enlarged further from the previous photomicrograph, this image of tangentially sectioned gill branches in the zebrafish reminds the viewer of a row of termites, or other insect, converging on an object.

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  Danio rerio (Zebrafish) Gill Branches

  Specimen: Fluorescently labeled cell culture
  Technique: Confocal Microscopy
  

  Enlarged further from the Danio rerio (Zebrafish) Gills image, this image of tangentially sectioned gill branches in the zebrafish reminds the viewer of a row of termites, or other insect converging on an object. From a scientific point of view, the color differentiation in the section caused by using the darkfield technique provides a wonderful illustration of the structure and composition of the gill branches. Either way, it adds even more to the story already begun with the zebrafish head and zebrafish gills images.

Danio rerio (Zebrafish) Gill Branches

Photomicrographer James E. Hayden, RBP has been a consistent winner of the Small World competition for many years. Featured below is a photomicrograph of a zebrafish specimen taken with darkfield illumination

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  Danio rerio (Zebrafish) Gill Branches

  Specimen: Hematoxylin and Eosin Section
  Technique: Darkfield Illumination
  

  In yet another section of the zebrafish seen in previous images at yet a higher magnification, the gill branches now remind the viewer of tree branches. The function of the structures becomes even more apparent by seeing the red and green blood as it goes in and out of the fish's breathing apparatus.

Canine Skin with Compound Hair Follicles

Skin sections are a favorite subject to explore because there are a lot of diverse structures that react very differently to the refractive changes of light when viewed in darkfield. This photomicrograph won 7th place in the 1991 Small World competition.

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  Canine Skin with Compound Hair Follicles

  Specimen: Hematoxylin and Eosin Section
  Technique: Darkfield Illumination
  

  Hair, being rather dense and composed of keratinized tissue, creates many visually interesting images. In this case, the cross sections through the hair follicles of the dog reveal an abstract collection of "eggs" or something similar. To the scientist, it clearly demonstrates the structure of canine hair as having compound follicles.

Decalcified Bone Section

The colors of stained sections as seen in darkfield are created by a combination of factors. Ultimately, changes in the refractive index of the various structures affect the wavelength as they pass through the specimen, creating a different color spectrum from the brightfield view. This photomicrograph was awarded an Honorable Mention in the 1997 Small World competition

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  Canine Endochondrial Bone

  Specimen: Decalcified bone section stained with Acid Fast
  Technique: Darkfield Illumination
  

  The colors of stained sections as seen in darkfield are created by a combination of factors. Ultimately, changes in the refractive index of the various structures affect the wavelength as they pass through the specimen, creating a different color spectrum from the brightfield view. Depending on the original color of the section, the composition and density of the structure in question, and the thickness of the section, the new colors that are created provide information about the content. Acid Fast stain creates a striking red and blue combination in bone structures as seen in brightfield, but not much color difference in the bone sub-structure. When viewed in darkfield, however, the calcified regions are remarkably different from the uncalcified areas. However, in this section, I must admit to mainly seeing an unfortunate reindeer with a bullseye on its side.

Mouse Tongue Papillae

This specimen of mouse tongue was essentially monochromatic when viewed in brightfield. All features have a different shade of blue and differentiation of the softer tissue and rougher areas in the papillae is not overly apparent.

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  Mouse Tongue Papillae

  Specimen: Hematoxylin stained section
  Technique: Darkfield Illumination
  

  This specimen of mouse tongue was essentially monochromatic when viewed in brightfield. Everything was a different shade of blue and differentiation of the softer tissue and rougher areas in the papillae was not overly apparent. When viewed in darkfield, the refraction of the denser keratinized areas is very different than the refraction caused by the less dense supporting structures. What started out as all blue was refracted into different wavelengths by the density of the specific structures resulting in excellent visual differentiation of the structure of the mouse tongue.

Minnow Skin with Encysted Fluke Larvae

Featured below is a photomicrograph of a minnow skin specimen taken with darkfield illumination.

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  Minnow Skin with Encysted Fluke Larvae

  Specimen: Hematoxylin and Eosin Section
  Technique: Darkfield Illumination
  

  When shown to a pathologist for an opinion, this section of fish skin reveals well-delineated cysts that, upon closer examination, contain fluke larvae (a parasite) and the diagnosis of Black Spot Disease is confirmed.

Canine Adnexal Nevus

This darkfield image shows a section through a nevus (mole) from dog skin tissue. This photomicrograph was awarded an Honorable Mention in the 1992 Small World competition.

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  Canine Adnexal Nevus

  Specimen: Hematoxylin and Eosin Section
  Technique: Darkfield Illumination
  

  When I first came upon this section through a nevus (mole) in a section of dog skin, I was immediately struck by my first interpretation of a bubble rising through the structure. When viewed in darkfield, it was even more striking. When the slides were processed, I realized that there also appeared to be a face in my image, with the nose pointed to the right, the chin at the bottom and the eye being the top right "bubble" while the middle "bubble" turned into a cheek. I showed it to someone else and asked if they saw the face in the picture and they immediately said yes - it had a pure black, round eye on the left side and it appeared to be breathing fire out of it's open mouth to the lower left. I realized that I had the microscopical equivalent of a cubist painting, reminiscent of Picasso. Art and Science never seemed closer.

Infiltrating Melanoma in Canine Dermis

Another dog skin image that was created with Rheinberg illumination - a technique very similar to darkfield except that, instead of blocking all transmitted light with an opaque field stop, a colored stop (in this case blue) allows some light to pass.

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  Infiltrating Melanoma in Canine Dermis

  Specimen:Hematoxylin and Eosin Section
  Technique:Blue/Yellow Rheinberg Illumination
  

  If the Canine Adnexal Nevus is a Picasso, than this image is a Van Gogh. The abstract swirl of the connective tissue, combined with the yellow and red of the melanotic cells and the deep blue background evoke comparison to "Starry Night." From a technical point of view, this image was created with Rheinberg illumination - a technique very similar to darkfield except that, instead of blocking all transmitted light with an opaque field stop, a colored stop (in this case blue) allows some light to pass. In darkfield, white light from the edges illuminates the structure, giving rise to the image. In Rheinberg, the center colored stop is surrounded with another colored filter (in this case yellow) which is the primary illumination for the structures.

Male Ostertagia ostertagi (Roundworm)

A very striking image that was created with multiple exposure darkfield illumination with the assistance of a crossed red/blue filter. When placed over the field diaphragm during darkfield, the white light that illuminates the specimen is replaced with the colored light.

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  Male Ostertagia ostertagi (Roundworm)

  Specimen:Unstained wet mount
  Technique:Multiple exposure darkfield with crossed red/blue filter
  

  If the specimen has no inherent color to start with, refraction of the white light will only provide levels of gray. Any color must be added with colored filters. In this example the filter used was a circular piece of acetate divided into four quarters. The colors of each quarter alternate blue and red. When placed over the field diaphragm during darkfield, the white light that illuminates the specimen is replaced with the colored light. Because of the arrangement of the colors, structures that are oriented in one direction refract the blue light, while structures oriented 90 degrees to that refract the red light. Where there is a chaotic orientation, the colors combine. The result can be quite striking as seen with this male roundworm. Additionally, the use of darkfield allowed me to easily use multiple exposure techniques to get both ends of the worm in one image.

Root Hairs on Cultured Arabidopsis Seedling

A main rootlet going in one direction with the root hairs projecting out about 90 degrees from the rootlet made this specimen ideal for observation in darkfield illumination with colored annular filters.

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  Specimen: Unstained wet mount
  Technique:Darkfield with crossed red/blue filter
  

  As with the image of a male roundworm and a down feather, this image was created with darkfield overlaid with a red and blue colored filter (see the male roundworm for a description of how the filter affects the image). The main rootlet going in one direction with the root hairs projecting out about 90 degrees from the rootlet made this specimen ideal for this technique.

Down Feather

Having a main feather shaft that projects in one direction with the barbules pointed outward, the image of this specimen was created with darkfield overlaid with a red and blue colored filter.

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  Down Feather

  Specimen:Unstained dry mount
  Technique:Darkfield with crossed red/blue filter
  

  As with the image of a male roundworm and a arabidopsis seedling, this image was created with darkfield overlaid with a red and blue colored filter (see the male roundworm for a description of how the filter affects the image). Having a main feather shaft that projects in one direction with the barbules pointed outward made this specimen ideal for this technique.

Porcupine Quill Tip

Keratinized structures like hair (or a porcupine quill) exhibit a property called anisotropism, in that the structure has more than one refractive index. When viewed between crossed polarized filters, these structures create a beautiful spectrum of interference colors.

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  Porcupine Quill Tip

  Specimen:Unstained dry mount
  Technique:Polarized Light
  

  Keratinized structures like hair or this porcupine quill exhibit a property called anisotropism, in that the structure has more than one refractive index. When viewed between crossed polarized filters, these structures create a beautiful spectrum of interference colors. Other biological specimens such as bone or fingernails also react this way. Many chemicals, like ethylene glycol or common table salt are also anisotropic and can be identified using polarization techniques.

Skin

Although this section of skin was unremarkable in both brightfield and darkfield, it comes alive with color under episcopic illumination with a UV fluorescence cube in place.

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  Skin

  Specimen: Hematoxylin and Eosin Section
  Technique: Ultraviolet induced Fluorescence
  

  This section of skin was unremarkable in both brightfield and darkfield. However, the pattern in the image was intriguing and I happened to try looking at it under episcopic illumination with a UV fluorescence cube in place. The green "fire" and blue "bubbles" finally added the color I was looking for. The color comes from the autofluorescence of the specimen and the dyes it is stained with. The lesson in this image is not to give up on a promising pattern - you never know what technique may surprise you with a great result.

Dematiaceous Mold on Cultured Root Tip

Featured below is a photomicrograph of a cultured root tip specimen taken with modified brightfield illumination. In terms of this image, "digital manipulation" means holding a finger between the field diaphragm and the condenser at just the right point to provide a shadowing effect to the specimen.

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  Dematiaceous Mold on Cultured Root Tip

  Specimen:Unstained wet mount
  Technique:Modified Brightfield
  

  In this age of computers, "digital microscopy" generally means imaging with a computer interface. In terms of this image, "digital manipulation" means holding a finger between the field diaphragm and the condenser at just the right point to provide a shadowing effect to the specimen. With a turret condenser, this can also be done by de-centering and offsetting the aperture diaphragm. The color in this image comes from a colored filter also placed in the light path.

Seminiferous Tubules in Transgenic Rat

Because of the relative thickness exhibited by this translucent specimen, light from a darkfield technique gives the best illumination to show the seminiferous tubules. This photomicrograph won 15th place in the 1996 Small World competition.

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  Seminiferous Tubules in Transgenic Rat

  Specimen: X-gal stained wet mount
  Technique: Modified Darkfield
  

  The shadowing effect described in the cultured root tip specimen with brightfield, can also be applied to darkfield. Because of this relatively thick, translucent specimen, the light from a darkfield technique gives the best illumination to show these seminiferous tubules. However, the lighting is rather flat and doesn't show the roundness of the tubules without the added shadowing caused by offsetting the darkfield stop to produce a more directional, oblique illumination.

Mouse Seminiferous Tubule at the Rete Testis

Darkfield illumination combined with a little shadowing makes this specimen of seminiferous tubule entering the rete testis appear similar to a tornado.

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  Mouse Seminiferous Tubule at the Rete Testis

  Specimen: X-gal stained wet mount
  Technique: Modified Darkfield
  

  As with the rat tubules, darkfield illumination combined with a little shadowing makes this seminiferous tubule entering the rete testis appear similar to a tornado. If that interpretation doesn't interest you, the value of seeing the location of the blue-stained cells in the different parts of the structure was of primary importance in this image.

Mouse Testis

In this example of "bare bulb illumination" a microscope bulb from an older model was clamped to a ring stand next to the microscope and the point light provided by the bulb gave excellent shadowing to the raised mounds of tubules.

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  Mouse Testis

  Specimen: X-gal stained wet mount
  Technique: External Bare Bulb Illumination
  

  Occasionally, it is important to obtain interesting images of subjects that are very difficult to make interesting. This mouse testis provided just such a problem. By using the low magnification objectives, the surface looked interesting, but the transillumination from the microscope in brightfield or darkfield created only silhouettes of the tubules. To provide light to the surface, a microscope bulb from an old model was clamped to a ring stand next to the microscope and the point light provided by the bulb gave excellent shadowing to the raised mounds of tubules.

Arabidopsis Seedling Pushing Through Soil

The seedling, growing in agar, was taken out and laid down on a microscope slide with the wet agar still providing moisture through the root, then photographed with a form of modified Rheinberg illumination.

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  Arabidopsis Seedling Pushing Through Soil

  Specimen:Unstained seedling and soil
  Technique:Blue/White Rheinberg Illumination plus External Bare Bulb
  

  The object of this image was to re-create what it would look like to see this small arabidopsis seedling forcing its way up through the soil. The experiment this was a part of caused the curl in the plant. The seedling, growing in agar, was taken out and laid down on a microscope slide with the wet agar still providing moisture through the root. Small bits of soil were pushed into place with the tip of a paintbrush and Rheinberg illumination was used to create the bit of blue in the background, while still transilluminating the bottom of the seedling with white light from the edges. An external microscope bulb (see the mouse testis photomicrograph) provided top light and shadowing as if from the sun coming from above.