Foundational Knowledge

Why Conjugate Planes are Important for Homogeneous Illumination

8 May 2024 · 8 min read
  • Foundational Knowledge
  • Widefield Light Microscopy

Abstract

Dive into our interactive tutorial to explore the essential role of conjugate planes in microscopy. The knowledge of the conjugate planes in the microscope beam path plays an important role in understanding Koehler Illumination and image formation in the microscope. The conjugate planes are also of great practical importance when it comes to detecting any dirt in the beam path.

Key Learnings:

  • The correct alignment of the Koehler Illumination establishes two independent sets of optical planes that are called the “conjugate planes”.
  • Conjugate planes are critical for homogeneous, straylight-free illumination.
  • Optical imperfections may develop during prolonged use and are linked to the conjugate planes of the illuminating beam path.
Homogeneous Illumination

Why Conjugate Planes are Important for Homogeneous Illumination

The correct alignment of the Koehler Illumination establishes two independent sets of optical planes that are called the “conjugate planes”.
One set of conjugate planes within the image forming beam path consists of

  • the field stop diaphragm,
  • the specimen plane,
  • the intermediate image plane (where the eyepiece reticule is positioned),
  • and the retina of the human eye or the sensor plane of the camera.

The other set of conjugate planes within the illuminating beam path consists of

  • the light source,
  • the front focal plane of the condenser (position of the condenser aperture diaphragm),
  • the objective’s back focal plane, 
  • and the eye point of the eyepiece, which is located approximately 1/2 inch (1 cm) above the top eye-lens of the eyepiece,
    at the point where the observer places the front of the eye during the observation. It is also called the “exit pupil".

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.

Tutorial Guide

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.

Troubleshooting Microscope Contaminations with Conjugate Planes

The image forming beam path set of conjugate planes is often useful in troubleshooting a microscope for contaminations visible together with the sample. If these artifacts are in focus with the specimen plane, they must be located on or near a surface that is part of the image forming set of conjugate planes. Examples are the outer lens surface of the light exit opening, the surface of the condenser front lens, the specimen (including the cover glass surface), the front lens surface of the objective (most important!), the outer surface of the eyepiece field lens, and dust on the camera sensor protection glass.

Rare imperfections in some of the optical elements may develop during prolonged use under sub-optimal conditions (e.g. delamination issues of the optical cement or fungus contamination mainly in objectives, condenser optics, and binocular tube head prisms). They are linked to the conjugate planes of the illuminating beam path. Usually, they can be detected by removing one eyepiece, using an auxiliary microscope or Bertrand lens system observing the condenser lens system or the objective’s back focal plane.

To locate any contamination, the suspected component is either carefully rotated in its mount (e.g. objective lens in its nosepiece thread, camera adapter, camera housing) or moved (e.g. condenser height, condenser front lens, specimen). If the dirt follows the movement of the suspect optical component, it is located. One exception: If the camera hosing is rotated separately from the camera adapter, the dirt on the camera sensor protection glass will not rotate. 


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