Thursday, 17 July 2014

Basics of Light Microscopy II: Koehler Illumination

A high quality illumination is regarded as a key issue for a perfect information transfer from specimen to target (human eye/camera chip). The illumination angle, definable by the condenser diaphragm, directly affects the resolution power of the microscope system. The final illumination setup should create a homogenous image background with a high dosage of sample details “on top”.

Unfortunately the human eye is an incorrigible liar. It compensates illumination defects and pretends depth of focus. On the contrary, the microscope camera is brutally honest to a suboptimal setup and reveals any deficit in illumination. No coincidence that August Karl Johann Valentin Koehler (1866-1948) developed an optimized microscope illumination while working on photomicrography problems. 

Following A. Koehler, a perfect microscope illumination has to fulfil the following requirements:

  • The illumination aperture (=angle) should be adaptable to the NA (=opening angle) of the objective in use.
  • In order to reduce stray light, the illuminated object area should be definable.
  • Illumination aperture and illuminated area should be adjustable independently.
  • Illumination for each image point has to be identical.

Field and aperture diaphragm are the important variables of the microscope illumination and enable the user to follow Koehler’s requirements.

In Motic’s BA310 Elite, the necessary “hardware” can be found here:

How to do a proper Koehler setup? The first 4 steps have to be taken by using the field diaphragm:

The final adjustment has to be done with the aperture diaphragm:

Especially unstained specimen (native smears, water samples) require a stronger closure of the aperture diaphragm to achieve contrast, while stained histological sections are less demanding. The following chart may help to understand the consequences of the aperture diaphragm setup:

Using the aperture diaphragm will balance the image parameters (contrast, resolution, depth of field, brightness), always depending on the sample characteristics. Please do not use the condenser diaphragm to reduce the image brightness (do you place a brick on the fuel pedal of your car, while regulating the speed with the brake pedal?). The light setting in most cases is too high to observe delicate structures, be careful not to outshine them.

Take the best possible profit from the microscope hardware in front of you! Follow August Koehler, and you will install best preconditions for your maximal understanding of the sample.

If you wish, you can take a look to our video tutorial about how to adjust the Koehler illumination:

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