Chapter 6
Illumination
Illumination of the sample is one important controllable variable in achieving
high-quality images in microscopy. The design of a light microscope must ensure
that the light rays are organized and precisely guided through the instrument
to the sample.
Köhler Knows How to Adjust Your
Illumination
To image your samples with optimized contrast and resolution, you need to apply
Köhler illumination. This concept was introduced in 1893 by the ZEISS scientist
August Köhler. It describes a routine that brings illumination and imaging
beam paths into a defined, aligned state, providing homogeneous illumination
without stray light. You achieve this by projecting an image with a small iris diaphragm
called a field diaphragm into the plane of the sample already in focus.
When doing so, the image from the light source cannot appear in focus together
with the object plane. In Köhler illumination you homogeneously illuminate the
object field diameter which is imaged by the objective. To set up Köhler illumination,
you need to follow several alignment steps. When you change the objective
and therefore
the magnification, the alignment process must be repeated (see
»The Part of Tens«).
IN THIS CHAPTER
Light is reflected or passes the sample
What Köhler has to do with it
Comparison of different light sources
/microscopy
