Conjugate Planes in Microscopy – Achieving Homogeneous Illumination

Explore the essential role of conjugate planes in microscopy through our interactive tutorial. Understanding the beam path and its conjugate planes is key to mastering Koehler Illumination and achieving optimal image quality. The conjugate planes also help identify contamination in the beam path.

Key Learnings:

• The correct alignment of the Koehler Illumination establishes two independent sets of optical planes, the so-called conjugate planes.
• Conjugate planes are critical for homogeneous, straylight-free illumination.
• Optical imperfections often relate to the illuminating beam path, developed during prolonged use .

Illuminating beam path:

• Light source
• Condenser front focal plane (aperture diaphragm)
• Objective’s back focal plane
• Eyepiece exit pupil (observer’s eye position)

Conjugate planes are critical for homogeneous, straylight-free illumination, with optimal contrast-to-resolution-ratio in the microscope.

Using the sliders in the animation below, the conjugate planes within the image forming beam path can be brought simultaneously into focus using the sliders.
Correspondingly, the conjugate planes in the illuminating beam path are visible together.

The tutorial initializes with a specimen appearing out of focus in the eyepiece view with the microscope intentionally set out of alignment.
To operate the tutorial, move the sliders to an ideal position:

1. Focus the specimen. 
   
2. Adjust the opening of the field stop diaphragm.
3. Modify the condenser height.
4. Focus the eyepiece reticle.

In transmitted brightfield microscopy the light source and condenser aperture stop are imaged together. Field stop and specimen are imaged together in the in the intermediate image plane which is located within the eyepiece’s field diaphragm.

Contaminations in the microscope’s optical path can significantly affect image quality. The concept of conjugate planes helps identify and locate such issues efficiently.

If artifacts appear in focus together with the specimen, they are likely located on or near one of the following surfaces:

• Outer lens surface of the light exit opening
• Front lens of the condenser
• Specimen itself, including the cover glass
• Front lens surface of the objective (most critical!)
• Outer surface of the eyepiece field lens
• Camera sensor protection glass

These components belong to the image-forming set of conjugate planes. Dirt or damage here will be visible in the same focal plane as the sample.

Over time, imperfections may develop in the illuminating beam path due to sub-optimal conditions. These include:

• Delamination of optical cement
• Fungal contamination in objectives, condenser optics, or binocular tube prisms

Such issues are linked to the illuminating set of conjugate planes and can be diagnosed using:

• A Bertrand lens system
• An auxiliary microscope
• Observation of the objective’s back focal plane or condenser lens system

To pinpoint the origin of dirt or optical artifacts:

1. Rotate the suspected component (e.g. objective lens, camera adapter, camera housing).
2. Move the component (e.g. adjust condenser height, reposition specimen).
3. Observe whether the contamination moves with the component.

If it does, the contamination is located on that part.
Exception: If the camera housing rotates independently of the adapter, dirt on the sensor protection glass will remain stationary.