Foundational Knowledge

Koehler Illumination

Principle and Alignment Procedure

11 September 2023 · 15 min read
  • Foundational Knowledge
  • Widefield Light Microscopy

Abstract

The Koehler Illumination achieves a homogeneously illuminated object plane imaged with optimal contrast and resolution. This illumination approach was first introduced in 1893 by August Koehler and manufactured by the leading microscope manufacturer Carl Zeiss Jena as a method of providing the optimum specimen illumination with optimal contrast and structural resolution.

Key Learnings:

  • The appropriate use of the adjustable aperture stop diaphragm is significant to achieve suitable resolution, optimal contrast, and depth of field.
  • Experienced microscopists can adjust the condenser aperture stop diaphragm accurately by observing the image without the need to view the diaphragm in the objective's back focal plane.
  • Koehler Illumination aligns the light source for homogenous illumination and contrast. It uses a collector lens system, field stop diaphragm function, and condenser aperture stop diaphragm.

Condenser Aperture Stop Diaphragm

Before looking in detail at how to establish Koehler Illumination, let us have a look at the Condenser Aperture Stop Diaphragm Function. The angle of the light cone produced by the condenser corresponds to the actual illumination aperture of the condenser in transmitted light. It is modulated by the diameter of the aperture stop diaphragm. A fully open condenser aperture stop diaphragm will allow for the maximum possible numerical aperture of the given condenser. The appropriate use of the adjustable aperture stop diaphragm is of significant importance to achieve suitable resolution, optimal contrast and depth of field.

Adjusting the Condenser Aperture Diaphragm for Optimum Contrast

Adjusting the Condenser Aperture Diaphragm for Optimum Contrast

Adjusting the Condenser Aperture Diaphragm for Optimum Contrast

Adjusting the Condenser Aperture Diaphragm for Optimum Contrast

When the condenser aperture diaphragm is closed too far, diffraction artifacts will cause visible fringes. Opposite, when opening the condenser aperture too wide, a significant loss of contrast is the result. The correct setting of the aperture diaphragm will vary from specimen to specimen, and the experienced microscopist will soon learn to accurately adjust the condenser aperture diaphragm by observing the image without having to view the condenser aperture diaphragm in the back focal plane of the objective.

Tutorial Guide

The tutorial initializes with a focused sample image on the left-hand side of the tutorial. Along with an animation of the condenser aperture stop diaphragm size adjacent to the specimen as it appears when viewing the objective’s back focal plane through a Bertrand lens or by removing one of the eyepieces and peering into the observation tube. In order to operate the tutorial, use the Aperture Diameter slider to adjust the aperture stop diameter, and thus modulate the contrast level of the image.

Koehler Illumination Procedure

Since then, manufacturers have outfitted the modern microscope with the necessary hardware for Koehler Illumination: A so-called collector lens system, a field stop iris diaphragm and condensers housing an aperture stop diaphragm. Usually, the collector lens system is built into the base of the microscope. The collector lens system will project an enlarged and focused image of the light source (e.g. halogen lamp filament, LED-array) into the plane of the aperture diaphragm of a properly height positioned condenser. The correct height of the condenser is achieved when the image of the field stop appears with maximum sharpness in the focused object plane. By doing so, the light source can never become focused into the specimen plane (instead the light source image is projected into the objective’s back focal plane). As a result, the focused object plane is illuminated very homogenously. The proper adjustment of the field stop diameter will reduce the amount of contrast- diminishing straylight. Closing or opening the condenser aperture diaphragm controls the angle of the light rays emerging from the condenser. By doing so, the optimum ratio between maximum resolution and strongest contrast can be individually set.

Now, this interactive tutorial explores how to establish Koehler illumination on a transmitted light microscope.

Tutorial Guide

The tutorial starts with the display of a transparent specimen in the transmitted light brightfield illumination: A thin bone marrow smear stained with Wright/ Giemsa. The workflow of this tutorial will guide you through the correct sequence of all Koehler alignment steps. The correct position of all sliders is indicated by a green color and check mark.

Align Koehler Illumination

Align Filament

  1. Use the slider to focus the specimen.
  2. By changing the condenser height, the field stop diaphragm will be imaged into the focal plane of the specimen.
  3. Center the field stop image by moving the sliders for the right and left screw.
  4. Close the field stop fully by operating the slider. Then, open the field stop diameter until its image is no longer visible in the object field.
  5. Adjust the condenser aperture iris diameter until its diameter restricts the diameter of the objective´s back focal plane by approximately 25%. By doing so an optimal compromise between maximum resolution and strongest contrast has been achieved.
  1. Click on the radio button to disengage the diffusing disk.
  2. Bring the filament into focus by adjusting the slider.
  3. Change the vertical position of the filament until the images overlap.
  4. Adjust the horizontal position of the filament until the images overlap ca. 50%.
  5. Adapt the lamp intensity for ideal brightness

The above tutorial workflow describes the Koehler Illumination alignment for transmitted brightfield illumination with higher objective magnifications (10x and higher). If the condenser contains phase ring stops, DIC- prisms, other masks, or filters, rotate the condenser turret to the free brightfield position. The best specimens to practice Koehler illumination alignment are well-stained brightfield samples, such as very thin cytological/hematological smears or tissue sections (8 micrometer or less).
Please note, that Koehler Illumination is done for the objective magnifications of 10x – 100 (150)x and must be repeated for each objective in transmitted light. For the lower objective magnifications (1x – 5x), the Koehler Illumination is first aligned for the 10x objective. Afterwards the condenser front lens is swung out (achromatic aplanatic condenser) or a low-power illumination lens is used below the condenser (abbe condenser). The sample is in focus and the condenser diaphragm is fully opened. The already centered field stop diaphragm will now act as aperture diaphragm, which is used for contrast/ resolution modulation as usually.

Steps in Establishing Koehler Illumination

Koehler Illumination Alignment Steps in Transmitted Light

  • Focus the sample

    Step 1

  • Close the field stop diaphragm fully

    Step 2

  • Change condenser hght

    Step 3

  • Align the field stop diaphragm

    Step 4

  • Setting of the condenser aperture diameter

    Step 5

  • Focus the sample

    1. Focus sample

    Switch on the light source. Use a transmitted light sample for brightfield (e.g. cytological smear, thin stained tissue section). Without a sample, Koehler Illumination cannot be aligned. To practice Koehler Illumination start with an objective magnification of 10x or 20x. This allows You to see the field stop diaphragm image with best clarity. Start with fully open condenser aperture diaphragm.

    Focus the sample. Leave this focal position of the sample unchanged during all other following steps!

  • Close the field stop diaphragm fully

    2. Close the field stop diaphragm fully

    If the image is totally dark, open the field stop diaphragm slightly and center it with the condenser centering screws. This is rarely necessary, especially if the condenser was removed from the microscope.

  • Change the condenser height

    3. By changing the condenser height, the image of the field stop diaphragm is focused into the already focused sample plane.

    Please note, that in some microscopes, the upper condenser height position can be limited mechanically. Make sure that the condenser can move high enough to produce a sharp field stop diaphragm within the sample plane. Take care not to move the condenser to high. Otherwise, the slide will be catapulted out of the specimen holder, which can easily damage the objective front lens.

  • Align the field stop diaphragm

    4. If the focused field stop diaphragm is off- center, it can be carefully aligned with the condenser centering screws.

    As a result, its image will be concentric with the field of view.  
    In some routine microscopes tools may be needed (e.g. SW 1.5 hex keys).
    Open the centered field stop diaphragm, until its image has just disappeared. Please do not fully open the field stop diaphragm, as otherwise straylight will be produced. This will reduce the image contrast.
    By looking at the sample, close the condenser aperture diaphragm until You have reached the best compromise between maximum resolution (condenser aperture fully open) and strongest contrast (condenser aperture diaphragm fully closed).

  • Correct setting of the condenser aperture diameter

    5. Correct setting of the condenser aperture diameter can be found by two methods

    Most commonly, one eyepiece is removed (Figure 5) giving full view of the objectives back focal plane (seen as a bright circle). When closing the condenser aperture diaphragm, its sharp image will appear in the objectives back focal plane. As a rule-of-thumb, for visual microscopy, the condenser aperture stop is closed by 20% - 25% of the back focal plane diameter (75% - 80% of the back focal plane diameter is still open). For digital imaging, the condenser aperture stop is opened more to achieve a better resolution.

    The second method is to close the condenser aperture diaphragm to the position, where the visual image brightness will drop significantly and suddenly. Both methods will give similar results.

Lamp Filament

The pre-condition for a homogeneous Koehler Illumination is the alignment of the light source (e.g. halogen lamp filament images) and the appropriate use of diffusing disks (if available). Otherwise the image background will not have the optimal illumination homogeneity.
Please be aware that many modern light sources can no longer be aligned by the microscopist (factory pre- aligned light sources and fixed diffusing disks in the illuminating beam path).
In this tutorial, the correct alignment of a halogen lamp in transmitted light brightfield is explained. Typically, this procedure is required after changing the halogen lamp bulb.
  

Preparation for Lamp Filament Alignment

First, set your microscope for transmitted light brightfield and align Koehler Illumination. Use the brightfield position of the condenser modulator disk (“H” or “BF”), preferably work with a non- Ph objective. The image of the light source can best be seen employing a 40x dry objective. Removing one eyepiece, allows to inspect the full light source image in the objective´s back focal plane. Make yourself familiar with the mechanical alignment options of your lamp.
Switch on the light source. Set the lamp intensity to a convenient brightness level. Remove all diffusing disks from the beam path. Focus an empty area of a slide. Remove one eyepiece. With a 40x objective magnification the image of the light source can be seen in the objectives back focal plane if Koehler Illumination was carried out beforehand. A halogen lamp house usually has a concave mirror to produce a second image of the light source for better light efficiency. Both halogen lamp filament images are mechanically adjustable in x/y (position) and z (focus).

Alignment steps of the lamp filament

1. Focus

Focus the lamp filament images.

2. Vertical Adjustment

Align both images vertically, so that they will overlap by approximately 50%. This is a compromize that works for all objective magnifications.

3. Horizontal Adjustment

By changing the horizontal filament position, make sure that the overlapping images will display the minimum of gaps in between the filament structures.

4. Diffusing Disk

A diffusing disk will further increase the homogeneity of the illumination. It is employed after the lamp filament is properly adjusted.

5. Lamp Intensity

The light intensity of a halogen lamp influences the colour temperature of the lamp spectrum (warm to neutral). If a colour temperature similar to daylight is needed, the lamp voltage is usually set resulting in a reproducable color temperature (e.g. 3200 K). Matching conversion blue filters will shift the spectrum to daylight color temperature of approximately 5500 K. Additional neutral density filters will adapt the light intensity again to optimum values for visual microscopy as well as digital imaging. LED light sources usually come with their own dedicated color balancing brown filters.

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