Using the Microscope.
Oil Immersion Objectives.
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Using the Oil Immersion Objective.
The only reason to use an oil-immersion objective is to take advantage of the highest resolving power available to the microscopist. Unless this is a necessary requirement of the work in hand, stay with the lower power dry objectives -- they are much less trouble to use, and can provide magnifications up to x600 (using a x40, 0.65 NA dry objective with x15 eyepiece) at quite acceptable resolution.
Having said that, much of the work requiring the high powers and resolution of the oil immersion objective has adapted itself to the requirements and constraints of the immersion technique. Blood films, stained bacterial films etc. are all prepared on plain microscope slides as dry films, and the drop of immersion oil becomes the only optical medium between the specimen and the frontlens of the objective.
When the examination is over, the slide with its film may be discarded or stored without even bothering to remove the immersion oil, since more will be added if the slide is examined again. In histology, thin (10 µm or less) stained sections of tissue are mounted under a coverglass in canada balsam or other resin having the same optical properties as glass, so with the addition of immersion oil, the conditions for homogeneous immersion are met. Much thicker sections than this cannot be examined, as the working distance of these objectives is very small, and the front-lens mount is soon in contact with the coverglass. Sometimes, thinner than standard coverglasses are used to allow deeper penetration of focus into the specimen. With a covered balsam mount, the oil may be removed with no risk of damage to the specimen.
Oil Immersion: Routine Technique.
The most common oil-immersion objective in use in routine microscopy is the achromatic objective of magnification x100 and NA of 1.25, used in combination with a dry two-lens Abbe substage condenser having a maximum aplanatic NA of about 0.6.
It is clear that the condenser cannot fill more the half the NA of the objective, and the resulting image will be high in contrast and showing the coarsening of detail characteristic of images formed by narrow axial cones -- but acceptable, especially for good visibility of low contrast subjects.
If you require such an image of say a balsam mounted section of stained plant or animal tissue, or a stained dried blood film on a slide, follow these steps:
Oil Immersion at the Highest Resolutions.
If higher resolutions are required, the main problem is that of supplying the objective with a sufficiently wide cone of illumination to fill its aperture and obtain best performance. A dry two-lens Abbe condenser produces an aplanatic cone of no more than 0.6 NA, and this is hardly sufficient to exploit the higher NA of the OI objective. Immersing the condenser helps a little, but is a fiddly operation providing very little gain in image quality.
The most practically satisfactory method involves replacing the Abbe condenser with a dry achromatic/aplanatic condenser with an aperture of 0.95 which will provide a 3/4 illumination cone for an objective of 1.3 NA. This extracts an optically satisfactory performance from a better than average objective without the need to immerse the condenser.
If the highest resolutions are required, the objective will need to be of apochromatic correction and will have an NA of 1.4. The condenser required will be of achromatic/aplanatic correction, and with oil immersion, provide an aplanatic illumination cone of 1.4 NA. Additionally, the microscope stand employed will require substage centreing adjustments to enable accurate centration of the condenser.
These necessarily expensive optics are capable of producing the finest possible images -- but their deployment is an exercise in critical microscopy and outside the scope of this basic tutorial.