Introduction

This activity introduces your students to 2 lighting techniques that will increase their understanding of the microscope and help them discover the hidden beauty of the microscopic world. Using these 2 techniques, dark field and Rheinberg illumination, your students explore and highlight the beautifully intricate features of diatomaceous earth. This activity is designed for high school students and meets the following national content standards for grades 9–12: use of technology to improve investigations, the cell, and understanding science and technology.

Materials

• Black construction paper
• Colored permanent or transparency markers
• Compound microscope with a substage condenser
• Coverslips
• Diatomaceous earth
• Glue or a glue stick
• Hole punch
• Microscope slides
• Transparency film

Background

Most of your students have probably used bright field illumination during their introduction to the microscope. However, bright field illumination, which uses direct white light from the  condenser of the microscope to produce a dark image against a white field (background), can obscure the details of some specimens, especially those that are translucent or nearly translucent. To get a better idea of how this works, try looking for dust motes suspended in the air in a well-lit room.

Now recall how easily they are seen within an oblique shaft of light passing through a dark room such as that cast by a projector. This is because in a dark room you only see the light reflected or refracted by the dust motes, making them shine against the dark background. Dark field illumination works on the same principle. Direct light from the condenser of the microscope is blocked, and only the oblique light reflected or refracted by the specimen is seen. This results in a bright image against a black or dark field, a tremendous advantage when viewing translucent or nearly translucent specimens.

To create dark field illumination, an opaque circle called a patchstop is placed in the  condenser of the microscope. The patchstop prevents direct light from reaching the objective lens of the microscope. The only light that does reach the lens is reflected or refracted by the specimen. For greater detail about this technique and some amazing example images, visit Florida State University's Microscopy Primer Web site.

Rheinberg illumination can be considered a specialized type of dark field illumination. The difference between the 2 techniques is that in Rheinberg illumination the patchstop consists of 2 concentric, differently colored circles. The resulting image, in the color of the oblique light passing through the patchstop's outer circle, is set against a field the color of the light passing through the patchstop's inner circle. This produces an image in the color of the oblique light passing through the patchstop's outer circle against a field the color of the light passing through the patchstop's inner circle.

Rheinberg illumination can give dazzling color to the specimen and field and open lots of possibilities for experimentation. For example, a black patchstop with a colored outer circle can be used for a combination of dark field and Rheinberg illumination. Or, try making the patchstop's outer circle out of segments of different colors for fascinating effects. For more detail on this technique and some example images, visit Florida State University's Microscopy Primer Web site.

Preparation

1. Check your microscope to make sure it has a condenser with a diaphragm and filter holder. The filter holder is an empty ring that swings out from under the diaphragm. If your microscope does not have a filter holder, you can still do the activity but you will have to contend with the inconvenience of taping the patchstops to the underside of the condenser.
2. You need to remove the eyepiece (or one of the eyepieces if there are 2) from the microscope to measure and position the patchstops you make. Check to make sure that the eyepiece in your microscope can be easily removed. If it does not slide out easily it may be held in place by a small screw.

Procedure

How to make a patchstop for dark field illumination

1. Place your microscope in an appropriate work area.
2. Select the low-power objective.
3. Remove the eyepiece and place it somewhere clean and safe.
4. Open the diaphragm as far as possible while looking through the opening left by the missing eyepiece. Now slowly close the diaphragm until it just impinges on the visible circle of light.
5. Lean over or squat down so that you can see the diaphragm from below. The diameter of its opening is slightly smaller than the patchstop you will make.
6. Use the hole punch to make several circles of black construction paper. Compare one to the size of the diaphragm's opening. If the circle is more than 10% larger than the opening, trim it so that it is about 10% larger than the opening. If the circle is smaller than the opening, cut out a larger circle.
7. Cut out a square piece of transparency film measuring 5 cm on each side.
8. Glue the circle of construction paper about 2 cm from one corner of the piece of transparency film.
9. Label the film with the magnification of the objective. The patchstop is ready for use.
10. Select the medium-power objective and repeat steps 4–9 to create a patchstop for it.
11. Select the high-power objective and repeat steps 4–9 to create a patchstop for it only if it is not an oil-immersion type.
12. Replace the microscope's eyepiece.

How to view a slide using dark field illumination

1. Make a wet mount slide of diatomaceous earth and view it using the low-power objective.
2. Select the patchstop for the low-power objective. Slide it between the filter holder and the rest of the condenser, starting at the corner with the black circle. Note: If your microscope does not have a filter holder, hold the patchstop beneath the condenser.
3. Remove the eyepiece and set it somewhere clean and safe.
4. Open the diaphragm completely and look through the opening left by the missing eyepiece. Adjust the patchstop until the light is completely blocked. Note: If your microscope does not have a condenser, you must secure the patchstop in place with tape once you have it properly adjusted.
5. Replace the eyepiece.
6. View the slide and compare the image to the original.
7. Switch to the medium-power objective. How does it affect the image?
8. Repeat steps 1–6 with the same slide but using the medium and high-power objectives and patchstops. Can you explain what you have observed?

How to make a patchstop for Rheinberg illumination

1. Cut out a square piece of transparency film measuring 5 cm on each side.
2. Make a blue patchstop by tracing one of the black patchstops onto the new piece of transparency film using a blue marker. Make the blue circle as dark as possible.
3. Use a red marker to add a red ring around the blue circle, being sure that the colors touch.
4. Select the proper objective for the patchstop you have just made. For example, if you used the low-power black patchstop as your model, select the low-power objective. Note: As in dark field illumination, you need to make a unique patchstop for each objective you intend to use.
5. Remove the eyepiece and position the blue patchstop so that only the blue is visible when you look through the opening left by the missing eyepiece.
6. Replace the eyepiece and view the specimen.
7. Experiment with different color combinations. For a more complex effect, try using segments of different colors in the patchstop's outer ring.

Internet resources

There is plenty of information on the Internet about the microscope, diatoms, and other related topics. Here are a few recommended sites.

• Bowling Green State University's Center for Algal Microscopy and Image Digitization has a diverse collection of light and scanning electron micrographs of diatoms.
• Great Lakes Diatoms catalogs diatoms from the Great Lakes and was supported by an NSF PEET grant.
• Indiana University's Diatom Home Page has an extensive list of relevant links.
• Micscape is a free monthly online microscopy magazine dedicated to "exploring the miniature world."
• The Diatom Image Archive provides images of numerous diatom genera and includes examples of natural variations.
• "Those Who Live in Glass Houses" is an article that provides an overview of diatom biology.

Carolina microscope

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Living cultures

• Cyclotella. Small marine centric diatom.
• Diatom Mixture. Several genera.
• Marine Diatom Mixture. Special mixture of Cyclotella and Thalassiosira shipped in a 2-oz bottle.
• Navicula. Pinnate diatom.

Prepared slides

• Freshwater and Marine Diatoms, w.m.
• Diatoms Self-Study Unit, w.m.
• Diatomaceous Earth, w.m. Entire frustules or portions of frustules of fossil diatoms.
• Marine Diatoms, w.m. Various forms, protoplasts stained.
• Mixed Diatoms, w.m. Various freshwater forms, protoplasts stained.