dark-field, oblique and Rheinberg illumination
by Wim van Egmond, The Netherlands
Wood section in Rheinberg illumination. The vertical fibers light up blue, the horizontal fibers yellow.
|Without proper illumination it is impossible to get a good image through a microscope. With normal 'bright-field' illumination you can see very fine details but with little contrast. Many techniques have been developed to enhance the contrast of the image. These methods are often expensive. In this article I would like to give an overview of some easy to make contrast enhancement techniques.
The techniques described here all require (easy to make) filters that should be placed under the condenser of the microscope. If your microscope doesn't have such a filter holder you could try to make one. Success will depend a bit on the quality of the microscope and your patience. A condenser with a high NA will also make things easier.
The basic technique is dark-field illumination. It is relatively easy to create by adding an opaque 'patch stop' under the condenser. If the condenser has a filter holder you can put the 'patch stop' there.
does it work?: By placing the stop in the cone of light (that the condenser
directs through the subject), only peripheral light refracted from the subject
will enter the objective. This way you will see a light subject on a dark background.
|'dark-field' or 'dark-ground' illumination
can be created by adding an opaque 'patch stop' under the condenser There are
several ways to make this 'patch stop'.
You can use a piece of transparent plastic to hold the disc of black cardboard. The size of stop depends on the objective that is used. A high power lens requires a bigger patch stop because of the higher NA. The circle has to be big enough prevent the light to enter the objective. 12 mm may be a good size to try.
|If you don't have something to place the dark 'patch stop' on it's possible to make the filter from one piece of cardboard.
crescent-shaped piece of cardboard in the filter holder will produce 'oblique'
If you have a phase-contrast condenser it is easy create oblique illumination because you can shift the diaphragm sideways.
illumination is based on dark-field illumination. With coloured filters instead
of the dark 'patch stop' wonderful effects can be created. Because there is less
contrast it is easier for the eyes. Monochromatic colours will avoid chromatic
aberration. A green outer filter may help to see more details since the human
eye is especially good in seeing greens!
It is important that the central filter is much darker than the outer filter! You can use a black marker (felt pen) to make a normal filter very dark and still retain the colour.
|The use of several colours will create a distinction between different areas in the subject. You can use any colour you like. In this case I use the primary light colours. Red, green and blue. The combination of these colours with a proper subject (try mounted diatoms!) will produce all colours.
is the Rheinberg filter I would recommend! The colours will create a distinction
between directions in the structure of a subject. Plant tissues are most interesting
subjects for this type of illumination.
The top image was made with such a filter, only with different colours!
'Zoom filter' is made of rings of different colours. I made the outer ring bigger
since it depends on the aperture of the condenser if this area is shown in the
The different refractive indexes and angles in a subject will light up in a different colours. This way the colours will give information about the three dimensional aspects of the subject.
|I would suggest to experiment with combinations of these techniques. It is possible to make oblique darkfield or oblique Rheinberg!
How to make Rheinberg filters?
|Cut the filter material with a circle cutter. First the outer circle, the diameter the size of your filter holder. The inner circle may be smaller than a dark-field patch stop has to be.
|Make the inner filters dark with a black marker (felt pen). You can use 2 filters and glue them together with the blackened sides. This will make the filter darker. It is also possible to use separate dark gray (neutral density) filters to make the inner circle darker!
|Combine several filters in the required arrangement. A slight overlap of the colours will only make the filter better. It will create dark bands that may enhance the effect and prevent light gaps.
you've made your choice of colours use sticky tape to put the filter together.
Remove unwanted bits of sticky tape.
Before sticking the filters togther you can experiment with separate filters to see if the effect is o.k.
|Another interesting method I like to mention briefly is the use of Polaroid filters. It is not truly a contrast enhancing method but by placing a Polaroid filter under the condenser and above the objective it is possible to create wonderful colour effects. Crystals give very interesting results.
Click to enlarge these images for examples of the techniques
Darkfield, oblique and bright field illumination
click to see how dark field works.
All comments to the author Wim van Egmond are welcomed.
Visit Wims home page for links to his many web pages on microscopy
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