MicrobeHunter.com » Köhler

Human hair under the microscope

Oliver — Tue, 23 Feb 2010 11:00:38 +0000

Two intertwined human hair under the microscope. The original image. The field iris diaphragm is still visible at the corners.

The power of image clean-up: dust and dirt removed.

Today I’d like to show you a nice microscopic picture, which I took several years ago of two human hair. The pictures on the right show you the original, unprocessed image at the top, and a second cleaned-up image with a nice background on the bottom. The top image shows darkened corers from the field diaphragm (from Köhler illumination). I closed the diaphragm quite a bit in order to increase image contrast (I intended to crop the image anyway). Then I did some processing in PhotoShop (or was it GIMP?). I selected the hair to remove dust and dirt and placed it on a nice blue gradient background. The most difficult part of the project was the making the hair knot and finding of an appropriate focus of the relatively thick sample. I did not do any image stacking (combining several images into one to get one final image which is sharp throughout).

Köhler illumination to reduce reflections

Oliver — Sun, 21 Feb 2010 13:50:28 +0000

Field diaphragm is wide open. Reflections from the side of the tube are very strong.

Field diaphragm is half open. The reflections are less.

Field diaphragm is closed. Only the direct light is able to reach the camera.

Taking a picture of the tube with a webcam. Any camera with a small lens would also have done the job.

The Köhler (or Koehler or Kohler) field diaphragm is located above the light source. It is responsible for controlling the width of the light beam (but not its intensity). The light source of a microscope without Köhler illumination will illuminate the whole specimen, which may be the source of stray light and excessive heating of the specimen. By closing the field diaphragm, it is possible to limit the beam of light only to the part of the specimen which is actually observed.

Advantages of Köhler illumination for photography

Köhler illumination increases the contrast of a photomicrograph because it reduces stray light and glare caused by reflections inside the microscope. On the right side you can see images taken through a trinocular head with a web cam. The more that the field diaphragm is closed, the less the reflections coming from the side of the tube. The bright spot in the center is the light which comes directly (unreflected) from the light source. In order to see a picture, it would be necessary to remove the lens from the webcam and project the image directly on the sensor of the webcam. In this case, the lens was left on to be able to see the side of the tube.

For more background info on Köhler illumination, you may be interested in the following two posts:

Adjusting Koehler Illumination

Oliver — Fri, 19 Dec 2008 20:44:16 +0000

The Koehler diaphragm is centered and in focus. The adjustment is correct.

The Koehler diaphragm is centered but out of focus. Raise or lower the condenser to focus the diaphragm.

The Koehler diaphragm is off-center. Turn the centering screws on the condenser to move the aperture into the center.

Koehler illumination ensures that the specimen receives a bright uniform light. Only those areas actually seen are illuminated.

A uniform, bright light source of the correct color is very important for obtaining high quality microscopic images. One problem is that the lamp is not able to produce a uniform light, because the filament of the lamp is brighter than its surrounding. One solution is to place a frosted glass plate above the light source as a diffuser. This reduces the light intensity and changes the color of the light, however.

Koehler illumination was developed by August Köhler (1866-1948). This illumination principle greatly enhances the quality of the microscopic images (especially photographs). The illumination principle offers the following advantages:

It illuminates the specimen uniformly without the need of a diffuser.
It only illuminates the part of the specimen which is actually observed (at a higher magnifications a smaller section of the specimen). This reduces the heating of the specimen.
It reduces internal reflections. This improves the contrast in photomicrographs.

The Koehler illumination must be adjusted before observation:

Rotate a low power objective (eg. 4x or 10x) into position. This will increase the field of view.
Insert a slide with a specimen and focus it.
Adjust the field iris diaphragm (the diaphragm of the light source) in such a way that its edges become visible. The field of view is reduced this way, only a small round part of the specimen is visible.
Raise or lower the condenser (not the stage!) and bring the edges of the field iris diaphragm (not the condenser aperture diaphragm) into focus. The focus of the specimen is not changed. Now both the edge of the iris diaphragm and and the specimen should be in focus. If the height of the condenser is not properly adjusted, then dust of the lamp will come into focus and disturb the image.
There are two condenser centering screws/knobs at the side of the condenser. Turn these knobs to bring the field into the center of view.
Now you can open the field diaphragm and start regular microscopic observation.
When doing photographic work, open the field diaphragm only as far as necessary. Opening it further will increase internal light reflections and result in a lower contrast. You need to observe the edges of the field diaphragm through the camera viewfinder. It may also be necessary to refocus the specimen when looking through the camera.

Advantages of Koehler Illumination

Oliver — Thu, 18 Dec 2008 19:11:20 +0000

An opened field iris diaphragm increases the width of the light beam. This setting is used for low magnifications (large field of view)

An closed field diaphragm decreases the width of the light beam.

A closed field diaphragm.

Koehler illumination offers a range of advantages over “critical illumination”. Illumination is more uniform, specimen heating is reduced as well as light reflections for photographic work.

Some microscopes are equipped with a field diaphragm in the light source. This Koehler illumination, while not absolutely required for simple microscopic work conducted in schools, does offer several advantages:

Uniform specimen illumination: Before the advent of Koehler illumination, a diffusing glass was placed over the light bulb. This had the disadvantage of reducing the light spectrum.
Reduction in specimen heating: A heated specimen increases evaporation of the water beneath the cover slip and also reduces the dissolved oxygen, a potential problem when viewing live organisms.
Reduction of light reflections in photographic work: Excessive light is eliminated reducing reflections inside the optical system. As a consequence the contrast of the photographic image increases.

The Koehler field diaphragm is designed to restrict the light beam only on this part of the specimen which is actually observed. Especially at high magnifications only a very small part of the specimen needs to be illuminated.