Page 74 - Fundamentals of Light Microscopy and Electronic Imaging

MICROSCOPE SLIDES AND COVERSLIPS 57

magnified but blurry. For most applications, 10 eyepieces work well. When higher
magnifications are required for a specific objective, a magnifying booster lens (in Zeiss
microscopes, an Optovar lens magnification system) can be rotated into the optical path.
Alternatively, a different set of higher-magnification eyepieces can be employed. If the
eyepiece is examined when the microscope is focused and the lamp turned on, a bright
disk can be seen floating in space a few millimeters outside the eyepiece. The disk is
called the exit pupil or Ramsden disk and represents the aperture at the back focal plane
of the objective lens. When viewing a focused specimen, the exit pupil of the eyepiece
will be found to be coincident with the entrance pupil of the eye, an adjustment that
occurs automatically as we focus the specimen.
Eyepiece specifications are engraved on the lens barrel to indicate their magnifying
power and field of view. An eyepiece engraved 10 /20 indicates a 10 magnification
and 20 mm diameter field of view. The field of view marking also provides a quick ref-
erence for determining the diameter of the field in the specimen plane as seen with a
given objective. For example, when combined with a 100 objective lens, this eyepiece
would give 20 mm/100, or 200 m for the diameter of the visible object field. Other spe-
cial design features of the ocular are designated by letter codes, the most common of
which indicate high eyepoint (distance between ocular surface and Ramsden disk) for
glasses wearers, additional corrections for color and flatness of field, and wide field or
wide angle of view. Eyepieces also come with focusable and nonfocusable eye lenses.
At least one focusable eyepiece should be included on the microscope to allow parfocal
adjustment of the optics so that the same focal plane examined by the eye will be in
focus on a camera mounted on the microscope. Oculars are based around a general
design containing two achromatic doublet lenses (the field and eye lenses) and a field
stop, a raised ridge or flange along the inside wall of the ocular that marks the site of the
intermediate image plane. In oculars of so-called Huygenian design, the field stop and
image plane are located between the field and eyepiece lenses; in Ramsden designs, the
focal plane and field stop are located in front of the field lens below the eyepiece. To use
an eyepiece reticule, the eyepiece is unscrewed and the reticule is placed in the image
plane and rests on the flange comprising the field stop.

MICROSCOPE SLIDES AND COVERSLIPS

Many objectives are designed to be used with standard (1.1 mm thick) glass slides and
coverslips of a certain thickness, usually 0.17 mm, which corresponds to thickness
grade 1.5. Other coverslip thicknesses induce spherical aberration and give poorer per-
formance, especially when used with high, dry lenses above 40 . For lenses with an
NA 0.4, coverslip thickness is not particularly important. Remember the thickness of
your slides and coverslips counts! Refer to the following chart when ordering cover-
slips:

Grade Number Thickness (mm)
0 0.083–0.13
1 0.13–0.16
1.5 0.16–0.19 (standard)
2 0.19–0.25