Bowburn ConsultancyEnvironment Agency
Back to 'Setting up the light microscope'

Does it matter if the light microscope is not set up correctly?

Condenser aperture

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The photographs shown below illustrate the effect on resolution of closing down the condenser aperture (via the condenser diaphragm). Everything else in the microscope was set up optimally for Köhler illumination (correct condenser height, specimen oiled to condenser and to objective, etc). The specimen was a Frustulia valve with c. 31 striae in 10 µm (0.32 µm between dark lines). The whole image can be downloaded; the original image was enhanced using the Levels and Brightness/Contrast tools in Adobe Photoshop..

Aperture wider than optimal

1. Condenser diaphragm opened wider than is traditionally regarded as appropriate for Köhler illumination, so that superfluous light is present, introducing glare and reducing contrast. However, despite the low contrast, both longitudinal (horizontal in figure) and transverse striations are clearly resolved

Part of the original image, with no enhancement. The scale bar is 10 µm.

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Aperture wider than optima, image enhancedl

1A. As 1, but with digital enhancement of contrast./p>

The image now shows good contrast, though this has also made the image noise more obvious; the noise was introduced during image acquisition.

Approximately the same degree of enhancement was applied to the following images. In each case, the Levels tool in Photoshop was used to expand the dynamic range of the image to the full range from black to white, followed by further slight enhancement of contrast..

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Aperture optimal

2. Condenser diaphragm optimal, according to the requirements for Köhler illumination.

There is no significant difference in the definition of the striae relative to 1A. This means that opening the diaphragm further than it needs to be (as in 1 and 1A) need not adversely affect the quality of the image, if digital enhancement is possible. Not opening the diaphragm enough, however, is deleterious, as shown below.

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Aperture smaller than optimal

3. Condenser diaphragm closed down a little too much. Although the diaphragm is still quite wide open, relative to where most students would place it, there is already noticeable loss of clarity in the striations.

On the other hand, the outer wall and parallel ridges around the raphe slit appear more obvious, relative to 1A.

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Aperture small

4. Condenser diaphragm closed further, down to the position where, in my experience, most students would put it. The striae have almost disappeared (and they were even less obvious in the image before enhancement). The margin and raphe ribs are still well defined.

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Aperture etremely small

5. Condenser diaphragm closed even further. Hard to believe that anyone would choose to do this, but they do. Longitudinal striae invisible; hints of transverse striation, but the stria density couldn't be measured.

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