Page 28 - Fundamentals of Light Microscopy and Electronic Imaging

PRECAUTIONS FOR HANDLING OPTICAL EQUIPMENT 11

Note: Focusing Oil Immersion Objectives

The working distance—that is, the distance between the front lens element and the
surface of the coverslip—of an oil immersion lens is so small ( 60 m for some oil
immersion lenses) that the two optical surfaces nearly touch each other when the
specimen is in focus. Due to such close tolerances, it is unavoidable that the lens and
coverslip will occasionally make contact, but this is usually of little consequence.
The outermost lens elements are mounted in a spring-loaded cap, so the lens can be
compressed a bit by the specimen slide without damaging the optics. The lens sur-
face is also recessed and not coplanar with the surface of the metal lens cap, which
prevents accidental scratching and abrasion.
Begin focusing by bringing the lens in contact with the drop of oil on the coverslip.
The drop of oil expands as the lens is brought toward focus, and at contact (essen-
tially the desired focus position) the oil drop stops expanding. If overfocused, the
microscope slide is pushed up off the stage by a small amount on an inverted micro-
scope; on an upright microscope the spring-loaded element of the objective com-
presses a bit. Retract the lens to the true focal position and then examine the
specimen. In normal viewing mode it should only be necessary to change the focus
by a very small amount to find the specimen. It can help to move the specimen stage
controls with the other hand to identify the shadows or fluorescence of a conspicu-
ous object, which may serve as a guide for final focus adjustment. Notice that if
focus movements are too extreme, there is a risk that the objective (on an upright
microscope) or the condenser (on an inverted microscope) might break the micro-
scope slide, or worse, induce permanent strain in the optics. Focusing with oil
immersion optics always requires extra care and patience.
Before observing the specimen, examine the back focal plane of the objective with
an eyepiece telescope to check for lint and oil bubbles. An insufficient amount of oil
between the lens and coverslip can cause the entire back aperture to be misshapen; if
this is the case, focusing the telescope will bring the edge of the oil drop into sharp
focus. These faults should be removed or corrected, as they will significantly
degrade optical performance. Finally, when using immersion oil, never mix oils
from different companies since slight differences in refractive index will cause pro-
nounced blurring.

PRECAUTIONS FOR HANDLING OPTICAL EQUIPMENT

• Never strain, twist, or drop objectives or other optical components. Optics for
polarization microscopy are especially susceptible to failure due to mishandling.
• Never force the focus controls of the objective or condenser, and always watch lens
surfaces as they approach the specimen. This is especially important for high-
power oil immersion lenses.
• Never touch optical surfaces. In some cases, just touching an optical surface can
remove unprotected coatings and ruin filters that cost hundreds of dollars. Carefully
follow the procedures for cleaning lenses and optical devices.