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Using the Microscope. 
Basic Tutorial.  
Oil Immersion Objectives.  
Part
7 of 9

Page
1 of 1
Intro, Stands. Components Light Paths Köhler Specimen NA etc. OI Errors Settings



   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 Ám 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 achromaticAchromatic: Corrected for chromatic aberration. objective of magnification x100 and NA of 1.25, used in combination with a dry two-lens Abbe substage condenser having a maximum aplanaticAplanatic: Corrected for spherical aberration and coma. 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:

  1. Place the slide on the stage and locate the area of interest using the x40 objective in a Köhler setup.


  2. With the object of interest focussed in the centre of the field, close the lamp diaphragm to a small aperture in the centre of the field, and using the substage condenser adjustment, focus its blades sharply in the plane of the specimen. Increase the lamp intensity a little.


  3. Swing the x40 objective out of the way and place a drop of immersion oil on the coverglass or film at the spot where the subject is illuminated by the condenser.


  4. Swing the oil immersion objective into position, so that (if the objectives are parfocal) the front lens is now immersed in oil. If the objectives are not parfocal, the OI objective should be carefully racked down using the coarse adjustment until contact is made with the oil drop. In either case, whilst looking through the eyepiece, continue racking the objective down, noting the increasing brightness of the image. The coarse adjustment may be used for this operation if it is sufficiently controllable, or the fine adjustment may be used if it has sufficient travel.
    Either way, as the image approaches maximum brightness, either the specimen or the lamp field diaphragm or both will come into focus.
    If this operation is carried out in haste, it is very easy to pass rapidly through the focus before you realize it. In this case, the sound of breaking glass will alert you to what has happened.
    However, if you are able to close the lamp diaphragm to smaller than the field of the OI objective, and it has been carefully focused in the plane of the subject, the point of focus is hard to miss. Objectives with spring-loaded optics are an added insurance against disaster.


  5. Once focus has been established, open the lamp diaphragm to clear the field, and trim the substage condenser diaphragm for the best image. This will probably be with the blades fully open. That's it.
When you have finished examining the specimen, rack the objective up to break oil contact and swing it forward to enable removal of the remaining oil with a piece of soft paper tissue. A fresh piece, moistened with cigarette lighter fluid will completely remove any residual oil film. The strict rule of not using alcohol for this operation is still a good one, especially for older objectives in which the more-than-hemispherical front lens is held in place with a cement which is dissolved by alcohol.


  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.