How does Köhler illumination work in microscopy?

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Figure 1: An image of the source includes the structure of the light emitting elements (left). Köhler illumination avoids imaging the source to the sample or sensor planes and provides uniform illumination to the sample plane (right).

A multi-element microscopy system can be aligned to provide Köhler (Koehler) illumination, in which the light collected from a light source like a lamp or light emitting diode (LED) is imaged differently than the light collected from the sample. The light source is intentionally never imaged to the sample (object) plane or to the camera sensor. The sample plane is instead uniformly illuminated, typically over the broad range of angles required for high-resolution imaging. As a result, Köhler illumination prevents superimposing an image of the lamp or LED structure onto the camera sensor.

Image of the Light Source
An image of the light source at the sample plane would not uniformly illuminate the sample (Figure 1, left, for example), since the light-emitting structures are clearly visible in images of the source. Köhler illumination instead uniformly illuminates the sample plane (Figure 1, right) by providing the light to the sample plane as bundles of parallel rays. In addition, aligning the system to provide Köhler illumination prevents the light source from being imaged at the camera sensor, which would superimpose an image of the light-emitting structures onto the image of the sample.

Illumination Light Path
The illumination path begins with the light source and passes through the sample to the camera sensor. The video animation (Video 1, below) traces rays from an extended source, like a lamp or LED, through this light path.

Each of the multiple light-emitting points on the source radiate light over a range of angles. The collector lens gathers this light and transmits a beam whose maximum diameter is limited by the field stop. The light is then incident on the field lens, which forms an image of each source point at the aperture stop. The alignment of the source image at the aperture stop is critical, since the aperture stop is positioned at the front focal plane of the condenser lens.

The image of the source at the aperture stop provides light to the condenser lens, which transmits the light as bundles of parallel rays. Each bundle of rays originates from a unique point on the source image. The angle at which a particular bundle of rays is incident on the sample plane is larger when the source point is displaced farther from the optical axis. In other words, closing the aperture stop would reduce the range of illumination angles, as well as the illumination intensity at the sample plane.

Since an image of the source is at the front focal plane of the condenser lens, only bundles of parallel rays are incident on the sample plane. No source image is formed at the sample and the illumination is uniform.

Source light transmitted through the sample plane is imaged to the objective back focal plane, which is located between the objective and tube lenses. No image of the light source is formed on the camera sensor, which preserves image quality. 

Imaging Light Path
The imaging path begins at the sample plane and ends at the camera sensor, and the video animation also traces rays through this light path. Each point on the sample is imaged to a point on the camera sensor.