Köhler illumination

Diagrams of the light paths through a light microscope that has been correctly set up to achieve Köhler illumination will open in a new window. Please call this up

The Köhler system is the usual system for illuminating specimens in normal light microscopy. Getting it right is an essential prerequisite for achieving good results in terms of detail and aesthetics.

The aim is to illuminate the specimen evenly, in such a way as to create a cone of light that matches (fills) the objective lens when focused on the object. This allows the maximum detail to be resolved (see Resolution) and it can only be achieved if the condenser is at the correct position along the optical axis, the condenser diaphragm is sufficiently open, and, in the case of immersion objectives, the appropriate fluid (normally immersion oil) is present between specimen and objective (for optimum resolution, immersion fluid should often also be present between condenser and specimen).

In essence, the light microscope comprises the following optical components:

• light source
• condenser
• specimen
• objective lens
• eyepiece lens
• eye or camera

In addition, many manufacturers incorporate lenses between objective and eyepiece, which alter the magnification (but should not affect the resolution, either positively or negatively) of the final image.

In the diagrams (in the separate window), two sets of ray paths are shown. In A, the image rays are traced. These are the rays that are in focus in the specimen plane. They are gathered from across the whole of the light source; in other words each part of the specimen is illuminated by the whole of the light source, so that the illumination is even and not affected by the fact that the light source is usually discrete and small (e.g. a filament). The specimen intercepts or alters these rays according to its properties. The rays pass on through the objective which brings them to a focus (sometimes auxiliary lenses are also present), producing a greatly magnified image, at a plane below or within the eyepiece, which is where an eyepiece graticule can be placed. The eyepiece magnifies the image further onto the retina of the observer, or onto an image -recording device.

B traces the paths of rays emanating from the light source. These are brought to a focus in various places along the optical path; the critical one to note is the focus in the plane of the iris diaphragm in the condenser. This diaphragm therefore controls the diameter of the cone of light passing through the microscope and hence also critically limits the resolution of the system.

The collector lens and field diaphragm are in effect the light source, rather than the lamp itself. In Köhler illumination it is absolutely essential that the condenser is set at the height necessary to focus a sharp image of the field diaphragm onto the focused specimen. So, with the object in focus, move the condenser up or down until the field diaphragm is in focus. Then centre the field diaphragm with respect to the optical axis of the objective, using the condenser centring screws. Finally, open the field diaphragm just enough that it is just no longer visible.

The condenser diaphragm must be set to correspond to the outer limit of the cone of light created by the focused image of the field diaphragm (B). In practice, this is most easily achieved by opening the condenser diaphragm fully (the image then usually has low contrast) and then closing it until the light begins to dim (and the contrast becomes just adequate for viewing).

Most people (1) place the condenser too low, so that the field diaphragm is not in focus, and/or (2) close down the condenser diaphragm too far. In both cases, the outcome is a contrasty image with far lower resolution (less detail) than the microscope is capable of. For ×25, ×40 or ×100 objective lenses, if the microscope has been set up with the top lens of the condenser more than c. 0.5 mm from the back of a normal microscope slide, then it has probably been set up wrongly.