Bio 220: Microscopy

Front 1

The laboratory microscopes used in Microbiology class are ...

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compound, parfocal, binocular, mechanically staged, light microscopes.

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compound

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a compound microscope will have two magnifying lenses used at a given time for observing the image

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parfocal

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parfocal lenses or objective are capable of staying in near perfect focus when the magnification is changed. Only a mirror adjustment in the fine focus is necessary

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binocular

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two eyepieces or oculars are used for viewing

Front 5

depth of field

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refers to the distance, up or down, on either side of the final (sharpest) plane of focus that the objective can be moved where the image still remains in acceptable focus. The greatest depth of field occurs when using the low power (10x). The depth of field decreases as the magnifying lenses increases. The depth of field is at its least when the oil immersion objective (100x) is in use. The depth of field for the oil immersion objective is less than 1 micrometer. Focusing properly using oil immersion is therefore important. The chance of finding the correct plane of focus by using guesswork is slim.

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working distance

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is the space between the lowest point of the objective and the slide when in focus. Working distance is greatest when the low power objective is in use. It is appropriate to use the course adjustment under low power. Working distance decreases as magnifying lenses are increased. Do not
focus with the course adjustment when using the high dry objective (40X) or the oil immersion objective. With these objectives in place, the working distances are small and course adjustment focusing could possibly drive the objectives into the slide.

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total magnification

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the total magnification of the system is calculated by multiplying the magnification of the objective in place, times the magnification of the ocular (eyepiece). The ocular (10X) is a constant. The objectives are either at 10X if the low power objective is in place, 40X if the high dry objective is in place or 100X if the oil immersion objective is in place. As an example, if the high dry
objective is in place the total magnification is 400X.

Front 8

resolution or resolving power

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is the ability of the lenses to distinguish fine detail and structure. We can also say that resolution allows the observer to distinguish two points as two separate and distinct points. Bringing these two points closer together and still seeing these two points separately would mean that resolution has improved. Therefore resolution could be thought of as a distance between these two points, the smaller the distance the better the resolution.

Front 9

What is a numerical aperature?

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The numerical aperture refers to the cone of light that is able to enter the objective.
(the oil immersion objectives that we use have a Numerical Aperture of 1.25)

Front 10

What is the average size of a bacterial organism?

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the average bacterial organism has a diameter of 1 to 2μm, we are therefore able to visualize these bacterial cells using our light microscopes, as they can resolve down to 0.2μm

Front 11

A bacterial cell is comparable in size to a ...

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mitochondrion we see in eukaryotic cells.