Enhancing light contrast for brightfield microscope with easy to made filters to create darkfield, oblique, Rheinberg and polarizing light illumination

In addition to the lens, the illumination system of the microscope plays an important role for the clarity of the image viewed under a microscope.  A regular brightfield microscope relies on the light source directly under the specimen to provide illumination.  The direction of the light makes the image looks flat and lacks of contrasts. It is difficult for the eyes to see the boundaries of structures if two structures are in similar color.  The use of chemical staining is commonly used by microscopists to enhance the viewing of specimen.  Unfortunately, the chemical staining typically kills the organisms.  The main focus of this post is to demonstrate the use of filters in the optical train to enhance the contrast of the specimen without using chemical staining (i.e. not killing the organism). 

An important component of your microscope is the condenser which located between the light source and the specimen under the mechanical stage.  The Abbe condenser is mounted under the mechanical stage.  If you are lucky, you can find a small ring that can be swing out at the bottom of the condenser.  

The condenser ring can be removed and served as the housing for the filter.  The size of the filter should be larger than the inner circle of the ring and slightly smaller than the outer circle. The filter should be snuggly fitting into the house without falling through the hole.  A regular transparency film for overhead projector can be used as the base for the filter. The transparency films are can be cut easily cut with a pair of regular scissors.  If your condenser does not have a the ring, you might be able to attached the filter onto the bottom of the condenser with a adhesive tape.

 

 

In addition to the condenser, you might also need a hollow punch set (below left).  The tool is essential for punching the through the plastic sheets for making the filter. To cut the circle, put the plastic sheet on the top of a wood block, select a hollow punch with desirable side and drive the punch through the plastic sheet with a hammer.  The plastic circle will be collected in the hollow space in the puncher.  In the picture, I was punching a circle from a cutting board to form a red filter for optical staining (The black dot is the filter for making dark field filter). 

Darkfield Filter

The principle of the darkfield microscopy is to block the light coming from the center of the light source.  It only allows the light coming from the peripheral.  The effect is the increase of the contrast to the surface of the object or structure.

The darkfield filter is the easiest one to make.  Simply drop a coin to the center of the filter base on filter housing and swing back the filter housing, you are ready to view your specimen in the darkfield.  Depending on the type of condenser you have, you may have to use the coins with different size.  In my case, I have to use dime to allow the light the pass through from the sides. It's very likely that the coin is not located in the center of the filter. Use a tweezer or tooth pick to adjust the location of the coin until it is centered.  Another adjustment that you are likely to make is the height of the condenser.  Move the condenser up and down until the light is just across below the specimen.  You should be able to see a nice dark background with bright subjects. To make a permanent filter, glue the coin to the filter base.

The alternative material for making the dark field filter is a piece of black paper or black plastic sheet. Cut of a piece of black paper or plastic to the size about the coin.  I used the plastic sheets from a two pocket folder (I bought it for 59 ¢ in back to school scale. Not a bad deal!).  The material is much harder than the transparency filter.   It's a little bit too hard with regular scissors.  I used a special tool called, hollow punch. They can be purchased in a set with different sizes.

The picture on the left shows the ibuprofen crystal in bright field microscope.  The image has very little contrast between the crystal and the background.  After applying the dark field filter, the crystal stands out from the background (right).

Oblique Light Filter

The oblique light is similar to the dark field.  Rather than using the peripheral light, the oblique light technique shifts the center of the light source to the side.  By increasing the light intensity from one side but block the light from the other side.  It increases the contrast of the image.

To make the oblique light filter, punch a black circle as the dark field filter then punch another hole off the center to make a crescent shapped piece. The picture below shows the difference in the image contrast between Bright field and oblique light illumination.

Rheinberg Illumination Filter

Rheinberg illumination is a form of optical staining.  The technique was initially demonstrated by the British microscopist Julius Rheinberg to the Royal Microscopical Society over a hundred years ago.  The Rheinberg technique is similar to the darkfield illumination. In darkfield microscopy, the substage condenser is arranged so that the rays of light coming through the condenser will pass through the specimen only at very oblique angles.  The light from the condenser cannot enter the objective directly. The only light that does enter the objective is light reflected, refracted, or diffracted by the specimen when the oblique rays from the condenser "strike" the specimen. The specimen then appears bright on an otherwise black field; hence the name darkfield illumination. The stark contrast of a bright object on a black field increases the visibility of already-resolved detail. Contrasting the darkfield illumination, the Rheinberg illumination contains color circle in the middle and another color on the outer layer rather then opaque center and transparent outer layer. This will produce the results of background in the center color while the object with the outer layer color.  The outer layer of the Rheigberg illumination filter can futher divided into two, four, six or eight sections to create even more striking color staining.

The flexible cutting board looks like a perfect material for making the Rheinberg Filters. They are soft enough to be cut with the hollow punch and transparent enough to allow the light to pass through.  The cutting board set that I purchased came with four different colors - Red, Green, Yellow and Blue.  This seems to be the perfect combination for making different combination of Rheinberg filters.  Cut a circle from the flexible cutting sheet with the hollow punch set.

If you apply the filter to the illumination, you can see the background of the image is stained with the color of the filter.  The picture on the left shows the ibuprofen crystals optically stained with red filter.

The make the green-red Rheinberg filter, punch out the center of the disc with a hollow puncher with the diameter about half of the disc. Do the same thing for a different color. Now you got two sets of circles and rings in two colors - like two sets of donut and donut hole.  You may use a sculpture knife and sand paper to shape the center piece to make it fit the ring of snuggly.  Place the center piece into the ring in different color and glue it to the filter base.

On the left is the picture of Ibuprofen crystal under the regular bright field illumination.  On the right is the picture of ibuprofen crystal using a red-green Rheinberg illumination with red color in the center.

Polarized Light Illumination

Polarized light is a contrast-enhancing technique that improves the quality of the image obtained with birefringent materials, such as rocks and minerals. The polarized light microscope must equipped with a polarizer, positioned in the light path between the light source and the specimen and a analyzer placing between the specimen and objectives.  A homemade polarized light microscopy can be easily made by using polarizing films: One below the mechanical stage and the other above the specimen.

On the left is the picture of a brightfield converted polarzing light microscope with two pieces of polarized films.  On the top of the light source and the other is on the top of the specimen.  On the right is the picture of Ibuprofen crystal viewed under polarized light microscope.

The technique demonstrated in this post was based on the article by Wim van Egmond published on Micscape Magazine.