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5.0 TECHNICAL TIPS FOR OIL IMMERSION MICROSCOPY
The first rule in microscopy is to keep the optical elements completely free of dust, dirt, oil,
solvents, and any other contaminants. The microscope should be kept in a low vibration
smoke-free room that is clean as possible and has minimal disturbance of the circulated air.
Use a dust cover on the microscope when not in use and keep all accessories in air-tight
containers. Avoid using corrosive solvents to clean any part of the microscope, and use only
diluted soapy water to clean non-optical surfaces. Oil should be used keeping the following
techniques and precautions in mind:
•
The first step is to locate the sample with a low-power objective and position the microscope
slide so that the area of interest is squarely in the central portion of the stage opening. It is
often desirable to rotate the immersion objective (without oil) into place to ensure the correct
location of the specimen. To achieve optimum results when using the oil immersion techni-
que it is important to also apply oil to the underside of the microscope slide and to the top
lens of the substage condenser to form an oil bead between the two, as described below.
For applying oil to the front lens of the substage condenser, use the following considerations:
•
Make certain that the mechanical stage of the microscope has an opening of sufficient size
to allow scanning of the oiled specimen without spilling oil onto the bottom of the stage.
Many advanced research microscopes have a stage insert that is removable for use with oil.
Remove the specimen slide from the microscope stage before applying oil to the condenser
top lens.
•
In a manner similar to oiling an objective, place a drop of oil on the bottom of the microsco-
pe slide and apply a similar drop to the front lens of the substage condenser.
Make sure that the condenser is slightly lowered in its rack before replacing the microscope
slide on the stage. After the slide has been placed on the stage, rack the condenser back
to the proper position while carefully observing the oil droplets on the condenser lens and
microscope slide. These droplets should merge into one as the condenser is brought into
proper position.
•
Swing the immersion objective to a position in the nosepiece where it will be the next objec-
tive rotated into the optical pathway directly above the specimen.
•
Position the area to be imaged in the center of the microscope's optical path using lower
power objectives. Next, the oil immersion objective (without oil) is placed into the optical
path and the specimen area of interest is roughly brought into center of the viewfield and fo-
cused. This pre-positions all of the components of the system in preparation for the addition
of oil. Swing the immersion objective to an adjacent stop on the microscope nosepiece and
apply oil both to the objective front lens and specimen as described in the next point.
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