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

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

Monochromatic and short wavelength light

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Microscopes vary enormously in quality. Basically, the more you pay, the better the lenses are. Poor lenses suffer from astigmatism (small round objects appear distorted, as short lines, and change in shape as the focus is altered), spherical aberration (objects near the periphery of the field of view are blurred), or chromatic aberration (colourless objects are coloured, small round objects are surrounded by colour fringes). Good lenses are specially designed and carefully ground to minimize these problems. However, even the best planapochromatic lenses (corrected for chromatic and spherical aberration) will perform better if they are used with monochromatic light. Generally too, lenses are corrected for a particular wavelength (usually green, at c. 550 nm) and this is convenient because it is relatively easy on the eye.

However, according to theory, the shorter the wavelength, the better the resolution. For optimum resolution, therefore, we must use blue light. However, the human eye is not very happy with intense blue light, so is it worth using? Again, it depends whether that extra little bit of resolution is necessary or whether the information can be obtained some other way. The specimen shown below is the same valve of Amphipleura pellucida shown in the pictures immediately above, with 36-37 striae in 10 µm (c. 0.27 µm between dark lines). This time, however, the optics used were 'bright field' (i.e. normal transmitted light).

Note that for the blue-light image, the condenser was not oiled to the slide.

Amphipleura, photographed in blue light

Amphipleura, blue light (max transmission of the filter at 440 nm)

Condenser not oiled to slide. With a wavelength of 440 nm, the theoretical resolution, even without oiling the condenser, would be c. 0.2 µm, so it is not surprising that 0.27 µm is achieved.

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Frustulia: condenser not oiled

Amphipleura, white light: Condenser oiled to slide. The striae are visible and could be counted, but they are not well defined. Again, correct Köhler illumination, but condenser not oiled to slide.

The longitidinal and transverse striae are both resolved, but there is a noticeable loss of clarity, relative to the image produced with an oiled condenser.

Theory indicates that a resolution of c. 0.25 µm should be achievable in this case, whereas only 0.32 is needed to resolved the Frustulia striae.

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