What is Kohler illumination and How it Works?

What is Kohler illumination?

Kohler illumination is a sample illumination method used for transmitted and reflected light in optical microscopy.

Kohler illumination serves to create uniform illumination of the sample and ensures that an image of the illumination source (e.g. a filament of a halogen lamp) is not visible in the resulting image.

Kohler illumination is the dominant sample illumination technique in modern scientific light microscopy. It requires additional optical elements that are more expensive and may not be present in simpler light microscopes.

This lighting method was first developed by August Kohler in 1893. Kohler illumination optimizes the optical train of a microscope to produce homogeneously bright light free from artifacts and glare.

Before Koehler illumination, critical illumination was the dominant technique for specimen illumination. The main limitation of critical illumination is that the image of the light source (typically a light bulb) falls in the same plane as the image of the sample, i.e. H. the filament of the light bulb is visible in the final image.

August Kohler, working for Carl Zeiss Corporation, introduced this method as a replacement for critical illumination techniques where the specimen was illuminated by using the converging lens to create an image of the illumination source on the specimen. The illumination was uneven and the filament of the light source bulb was placed on the sample.

While various solutions have been tried to overcome this problem, from diffusing the light to reducing the light intensity, these remedies had their own drawbacks.

Kohler’s method of properly aiming the incoming light and perfectly defocusing the image of the light source allows for the best possible imaging in an evenly diffused field of light.

Kohler illumination

Components of Kohler Illumination

Kohler illumination requires several optical components to operate:

  1. Collector lens and/or field lens
  2. Field diaphragm
  3. Condenser membrane
  4. Condenser lens

Although all major manufacturers of high-quality microscopes offer Kohler illumination, a microscope without Kohler illumination can be retrofitted provided certain criteria can be met.

A real Kohler lamp has a very large filament, but a standard lamp can be used. If your microscope does not have a field diaphragm, your instrument must allow the addition of these and the required lenses fitted over the lamp. Also, you need to be able to raise and lower the condenser.

Kohler Illumination Principle

Kohler illumination acts to generate an even illumination of the sample and ensures that an image of the illumination source (for example a halogen lamp filament) is not visible in the resulting image. Kohler illumination is the predominant technique for sample illumination in modern scientific light microscopy.

The primary limitation of critical illumination is the creation of an image of the light source in the specimen image plane.

Kohler illumination addresses this by ensuring that the image of the light source is perfectly defocused in the sample plane and its conjugate image planes. This can be seen in a ray diagram of the illumination beam path as imaging beams run parallel through the sample.

In Kohler illumination, four separate planes combine to form conjugate planes in both the illumination and imaging light paths.

The lamp filament, the aperture diaphragm, the back focal plane of the objective, and the eyepoint, which is about one centimeter above the top lens of the eyepiece, form the conjugate plane of illumination.

The conjugate planes of the imaging ray path are the field diaphragm, the specimen, the fixed diaphragm of the eyepiece, and the viewer’s retinal plane.

In Kohler illumination, the collector lens, or field diaphragm, collects light from the illumination source and focuses it onto the front focal plane of the sub-stage condenser’s aperture diaphragm, which essentially projects an image of the lamp filament onto the lens.

The condenser transmits the light to illuminate the specimen. Often the condenser needs to be adjusted to ensure that the filament image appears at the focal plane and fills the aperture.

The image of the filament must fill the aperture diaphragm and the field diaphragm, and they must share the same conjugate image planes as the specimen.

This leads to an extremely uniform illumination as the filament is completely defocused in the specimen image planes and produces a clear image of the specimen.

Closing the field diaphragm does not reduce the brightness of the image, it merely controls the width of the light beam transmitted to the condenser, confining the light to the portion of the specimen that is actually being observed.

The condenser diaphragm setting affects the angle of light transmitted to the specimen. The setting of this diaphragm and the aperture of the objective determine the actual numerical working aperture of the microscope. Resolution and contrast increase when the condenser diaphragm is opened.

How To Set Up Kohler Illumination?

Kohler Illumination is a process that provides optimal contrast and resolution by focusing and centering the light path and evenly distributing it across the field of view.

Sophisticated and well-equipped microscopes do not produce high-quality images due to improper use of the light source. The illumination of a sample should be bright, glare-free, and evenly distributed in the field of view.

In order for a microscope to be built for Koehler, it must have two adjustable irises: the aperture diaphragm on the under-stage condenser and the field iris diaphragm closer to the lamp.

The aperture iris controls the opening angle of the cone of light from the condenser, while the field iris controls the area of the circle of light that illuminates the specimen.

The substage condenser must be focusable up and down and equipped with an aperture diaphragm that can be opened and closed by a lever or knob.

The beam path must be equipped with a converging lens, a converging lens, and an openable and closable field iris diaphragm.

6 steps to set up Kohler Illumination

Focusing the condenser

1) Place a thin sample on the stage and focus it on a 4x or 10x objective.

For a suitable starting position, ensure that the condenser front lens is approximately 0.5 cm from the bottom of the coverslip.

2) While looking at your monitor, fully close the iris using the field iris control located on the front of the under-desk optic.

You will see a dark circle on the screen.

Note: If this dark circle does not fall within your field of view, you may need to use the two silver adjustment screws on the condenser arm to center your condenser.

3) Move the condenser up or down until the edge of the dark circle (the iris blades) appears sharp on the monitor. Once you have positioned the condenser correctly, switch the user interface back to position (I) to avoid accidentally repositioning the condenser.

Note: Depending on the working distance of the condenser, you may be in close proximity to the sample.

Centering the condenser

4) There are two silver adjustment screws on the condenser arm that are used to center the condenser. Thread the screws into the center of the now polygonal shape. It should now appear in the center of the field of view.

This process is facilitated in the final stage by opening the aperture almost to the edge of the monitor’s field of view.

5) After the condenser has been focused and centered in this way, the aperture can be opened so that it is just outside the field of view.

The condenser remains centered when other objectives are selected, but the field iris diaphragm must be set just outside the field of view at different magnifications.

Adjusting the aperture iris

This important step is often neglected, resulting in either suboptimal resolution and/or poor contrast.

6) Locate the aperture diaphragm control, which is often a thin silver lever protruding from the condenser. With the condenser in place, focused, and centered, the iris should be closed so that it occupies about the outer 20% of the field. This increases the contrast and makes observation easier.

Although some specimens may need a variation of 20%, be careful not to close the iris too much as the resolution will be drastically reduced.

Advantages and Disadvantages

The main advantages are high contrast and evenly distributed illumination.

In addition, less specimen heating occurs and helps prevent thermally induced changes in the specimen. Reflection and glare are eliminated by using the field diaphragm, which controls the width of the light beam.

While this lighting method works with darkfield viewing, results are poor unless the aperture is fully open. Other than that, however, this is the preferred lighting method.