Koehler Illumination: A Brief History and a Practical Set Up in Five Easy Steps

In the 1893 paper entitled "Ein neues Beleuchtungsverfahren für mikrophotographische Zwecke" which was published in Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik (“A New System of Illumination for Photomicrographic Purposes” in the Journal of Scientific Microscopy and Microscopic Technique) he set out the main problems microscopists faced at the time. Auer gas lights and zirconia light were the main sources of illumination available in this era, but these had the tendency to produce an unwanted ‘glare’.

The Auer gas light was named after the Austrian scientist and inventor, Baron Carl Auer von Welsbach (1858-1929). He is credited for patenting the gas mantle which was used as street lighting throughout Europe. Although Auer gas lights still produced carbonic acid and a certain amount of heat, the London Journal of Gas Lighting and Sanitary Water Supply exclaimed that the Auer light was “eminently suitable” for microscopy. 

The alternative zirconia light was an intense chemical light source produced by heating the element zirconium. However, with such light sources, Koehler declared that “it is difficult or impossible to achieve uniform illuminations”. Koehler realised that for consistency in illumination, every single component of the microscope needs to be considered and correctly aligned in relation to each other. By making adjustments to the diaphragms and condensers, he overcame the main problems of overheating and light consistency (even using an opera glass in place of a convex lens!).

Even today, his consistent illumination technique is still widely used and forms the basis of many techniques including confocal, phase contrast and differential interference contrast (DIC) microscopy. Once you know how to set up a microscope for correct Koehler illumination, it should become second nature and will transform your imaging work. It is simply one of the most important principles of light microscopy.

It takes less than a minute to set up a microscope for Koehler illumination. This will ensure that the light source evenly illuminates your specimen, produces a sharper image and will increase the contrast between areas of staining or between regions of tissue in your sections. You will also be able to collect more imaging data from each of your specimens.

If you require images for use in Powerpoint presentations, poster presentations or for submitting to journals, then Koehler illumination is a must. This simple five step technique goes a long way and, with a little practice, will easily become second nature before you capture your images.

As well as the microscope objectives, there are two other components which you should be aware of (and which are adjusted) for Koehler illumination.

The ‘field diaphragm’ is usually located in the base (in an upright microscope) of the instrument as part of the microscope body (Figure 1). Some older (or simpler) microscopes may just have an ‘on/off’ switch for the light source and there may be no field diaphragm to control the intensity of light from the source. The field diaphragm simply controls the amount of light which finally reaches the slide on the stage. By controlling the amount of light coming from the light source, you are able to reduce glare from the source and this results in images with greater contrast. The field diaphragm is usually controlled by a knurled ring around the lens.

Directly under the stage in an upright microscope (or above the stage but in front of the light source/field diaphragm in an inverted microscope), is the sub-stage condenser (Figure 1). Although it has focus wheels which are similar to the fine/coarse focus of the microscope, this component is used for focusing the light from the light source/field diaphragm and not to bring your specimen into focus. 

The ‘sub-stage condenser’ collects the majority of the light from the source and focuses it as a cone of image-forming light on the slide placed on the microscope stage. When this condenser is correctly adjusted, the light which illuminates your specimen and enters the objective is fully optimised. This results in a uniform intensity and contrast in the final image. As each of the objectives of a microscope is different, it means that the sub-stage condenser will need to be adjusted when you change magnification.

Within the sub-stage condenser there are two component parts which are adjusted in setting up correct Koehler illumination. These are the centering screws and the diaphragm. As well as the field diaphragm, the sub-stage condenser also has a light controlling diaphragm which is adjusted by either a slide control or a knurled ring around the condenser similar to the field diaphragm. This diaphragm controls the angle of the light cone which is illuminating the specimen. As I alluded to in my article on Numerical Aperture (NA), the NA of the whole microscope system is not only determined by the objectives. Indeed, when the sub-stage condenser diaphragm is opened, this produces an increase in the NA of the whole instrument resulting in greater resolution, contrast and optimal illumination. The centering screws ensure that the light used to illuminate your slide is directly in the centre of the field of view, thereby offering uniform intensity across the specimen.