How to Make Rheinberg Illumination Filters for Colorful Contrast

What Rheinberg illumination is

Rheinberg illumination is a colored version of darkfield. In ordinary darkfield, an opaque disc blocks the center of the condenser’s light and the background turns black. In Rheinberg illumination, that opaque disc is replaced with a transparent colored disc, and the outer ring of light passes through a second color. The result is a specimen in one color sitting on a background of another, sometimes called “optical staining.”

It was described by the British microscopist Julius Rheinberg in the 1890s, and it remains popular with amateurs because the filters are cheap and easy to make. A filter costs little more than a few colored gel offcuts and a piece of clear plastic.

In short, to make a Rheinberg filter you:

1. Measure the filter holder under your condenser.
2. Work out how large the central disc needs to be for your objective.
3. Cut a colored ring (annulus) and a colored central disc from lighting gel.
4. Mount them on a clear base and drop the filter into the holder.

The rest of this article covers each step in detail.

How it works and why the center size matters

The filter sits in or near the condenser’s front focal plane, which is where the filter holder below an Abbe condenser usually is. Light passing through different parts of the filter reaches the specimen at different angles:

• Central disc → background color. Light through the center reaches the specimen at shallow angles and goes straight into the objective. Where there’s no specimen, this is all you see, so the central color becomes the background.
• Outer ring → specimen color. Light through the annulus arrives at steep angles and, like darkfield light, misses the objective. Only when an edge or particle scatters some of it does it enter the objective, so specimen detail glows in the ring color.

For this to work, the central disc must be at least large enough to cover the cone of light the objective can accept (set by its numerical aperture). If the central disc is too small, some ring-colored light enters the objective directly, and the background becomes a muddy mix of both colors. If it’s far too large, very little ring light reaches the specimen at usable angles and the specimen turns dim.

Because each objective accepts a different cone of light, a filter that works well with a 10x objective usually won’t be right for a 4x or a 40x. Most people make a small set, one per objective. Rheinberg works most easily at low and medium magnifications, for the same reasons as patch-stop darkfield.

What you need

• Colored lighting gels. Theatrical and photographic lighting filters, such as those sold by Lee and Rosco, are the usual choice. Their sample swatch books are inexpensive and contain dozens of colors in small pieces, which is plenty for microscope filters. Colored cellophane and craft acetate can work too, though their color is often less even.
• A clear base. Thin clear acetate, a transparency sheet, clean clear plastic packaging or an old clear glass filter that fits your holder.
• Cutting tools. Sharp scissors or a craft knife, a compass or circle cutter, and a set of hole punches in different sizes. Leather punches and craft punches are handy for clean central discs.
• A ruler or calipers to measure the filter holder and iris.
• Adhesive. A tiny amount of clear glue, glue dots or thin clear tape applied only at the outer rim, outside the light path.
• A fine marker to mark centers and label filters.

An inkjet printer and transparency film can also be used to print filters. That makes it easy to try many designs, but printed colors are usually paler and less clean than gels.

Step 1: Measure your filter holder

Swing out the filter holder below the condenser and measure its inside diameter. If your microscope came with a blue or frosted filter, measure that instead, since your Rheinberg filter will replace it. Filter holder sizes vary between makes and models, so measure rather than guessing.

Cut your clear base to that diameter. A disc that fits snugly helps keep the central spot centered.

Some microscopes have no filter holder. In that case, see the alternatives in our guide to making a darkfield patch stop, which apply equally to Rheinberg filters.

Step 2: Find the right central disc size

You can find the size by trial and error, but it’s faster to measure. This uses the same approach as sizing a darkfield stop:

1. Put a slide on the stage and focus with the objective you want to use, for example 10x. If your microscope allows it, set up Köhler illumination first.
2. Remove one eyepiece and look down the tube from a short distance. You’ll see the bright circle of the objective’s back aperture.
3. Slowly close the condenser’s aperture diaphragm until you see its edge just start to move inward across that bright circle. Open it again until the edge just disappears.
4. At that setting, the iris opening is roughly the size of the cone the objective accepts. Measure the iris diameter as best you can with a ruler or calipers from below.

Your central disc should be the same as or slightly larger than that iris opening. Because measuring an iris is imprecise, cut several central discs in small steps around your measurement, then test each one. Label each filter with the objective it suits.

Note that the iris must be fully open when you actually use the Rheinberg filter. You only close it here to measure.

Step 3: Cut and assemble the filter

There are two common ways to build the filter.

Method A: ring with a punched hole (recommended)

1. Cut a disc of your ring color to the same diameter as the clear base.
2. Mark its exact center and punch a hole matching your chosen central disc size.
3. Punch a disc of your central color with the same punch.
4. Lay the ring on the clear base, then drop the central disc into the hole so the two colors meet edge to edge.
5. Secure the ring at its outer rim with a small dab of glue or a sliver of clear tape. Fix the central disc with the smallest possible amount of clear adhesive, or sandwich the whole filter between two clear discs so nothing needs to be glued in the light path.

Method B: central disc laid on top

Cut a full disc of the ring color and simply stick a central disc of the second color on top. It’s quicker, but the center now has light passing through both gels. The two colors combine and usually give a darker, different color than either gel alone. Some people like the effect, but it’s harder to predict, so start with Method A if you want a specific background color.

Tips for clean filters

• Handle gels by the edges. Fingerprints, scratches and creases scatter light and show up as uneven patches.
• Keep edges clean and round. Ragged edges on the central disc can give a fringe of mixed color around the background.
• Keep the gel flat. Wrinkled gel produces streaks.
• Write the colors and objective on the rim, outside the light path.

Choosing colors

Color choice is mostly a matter of taste, but a few guidelines help:

• Use clearly different colors. The greater the difference between center and ring, the stronger the contrast. Blue or green backgrounds with red, orange or yellow specimens are classic combinations.
• The central color controls background brightness. Deep, saturated central colors give a dark background that makes the specimen stand out, much like darkfield. Pale central colors give a bright background and lower contrast.
• The ring color controls specimen brightness. Pale, bright ring colors such as yellow let more light through and make specimens brighter. Very dense ring colors can make the specimen too dim to see well.
• Your lamp matters. Halogen light is warm, and white LEDs vary in their color. The same gel can look slightly different on two microscopes, so test colors on your own instrument.

A good starter set is three filters: dark blue center with yellow ring, dark green center with red ring, and red center with blue or green ring.

Multi-color and segmented designs

Once a two-color filter works, you can try more elaborate designs:

• Segmented ring. Divide the annulus into segments and color opposite segments the same. For example, make the top and bottom quarters one color and the left and right quarters another. Edges running in one direction pick up one color, and edges running the other way pick up the other. This works well on diatoms and other specimens with regular structure.
• Three colors. A segmented ring in two colors plus a third central color gives three-color images.
• Colored darkfield. Keep an opaque black central disc but make the ring colored. The background stays black and the specimen takes on the ring color.

Segmented filters are fiddly to assemble. Cut segments slightly oversize and trim them together on the clear base, or print a template to cut along.

Using the filters on the microscope

1. Open the aperture diaphragm fully. A partly closed iris cuts off the ring light, and you’ll see only the central color.
2. Insert the filter in the holder below the condenser, colored side up or down, as long as it lies flat.
3. Raise the condenser close to its top position, then adjust its height slightly for the most even background and the best specimen color. Small changes in height can shift the color balance noticeably.
4. Center the filter. If the background shows a crescent of the ring color on one side, the central disc is off-center. Nudge the filter or, if your condenser can be centered, adjust the centering screws.
5. Turn up the lamp. Rheinberg filters absorb a good deal of light, especially dense colors, so you’ll usually need more brightness than in brightfield.
6. Use the matching filter for each objective when you change magnification.

If you have a halogen lamp that runs hot, avoid leaving gels resting directly against the lamp housing. In the usual filter holder position below the condenser, they’re normally well away from the bulb.

Troubleshooting

At higher magnifications, the condenser may simply not be able to supply light at steep enough angles for the ring to work. This is the same limitation that affects simple darkfield setups, and it’s normal.

Best specimens for Rheinberg

Rheinberg suits the same kinds of subjects as darkfield: thin, transparent and with plenty of edges.

• Diatoms and radiolarians, especially with segmented filters
• Pond life, such as rotifers, small crustaceans and filamentous algae
• Hairs, fibers and feathers (see what everyday things look like under a microscope)
• Insect parts, such as wings, legs and scales
• Crystals, including dried salt or sugar solutions
• Thin plant sections and pollen

Rheinberg illumination is mainly an aesthetic technique. It makes edges and outlines stand out in color, but it doesn’t add information that darkfield or other contrast methods can’t provide. For photography and for showing specimens to others, though, few techniques give so much impact for so little cost.