Page 29 - Fundamentals of Light Microscopy and Electronic Imaging

12 FUNDAMENTALS OF LIGHT MICROSCOPY

Exercise: Calibration of Magnification

Examine a histological specimen to practice proper focusing of the condenser and
setting of the field stop and condenser diaphragms. A 1 m thick section of pan-
creas or other tissue stained with hematoxylin and eosin is ideal. A typical histo-
logical specimen is a section of a tissue or organ that has been chemically fixed,
embedded in epoxy resin or paraffin, sectioned, and stained with dyes specific for
nucleic acids, proteins, carbohydrates, and so forth. In hematoxylin and eosin
(H&E) staining, hematoxylin stains the nucleus and cell RNA a dark blue or pur-
ple color, while eosin stains proteins (and the pancreatic secretory granules) a
bright orange-pink. When the specimen is illuminated with monochromatic light,
the contrast perceived by the eye is largely due to these stains. For this reason, a
stained histological specimen is called an amplitude specimen and is suitable for
examination under the microscope using bright field optics. A suitable magnifi-
cation is 10–40 .

Equipment and Procedure
Three items are required: a focusable eyepiece, an eyepiece reticule, and a stage
micrometer (Fig. 1-6). The eyepiece reticule is a round glass disk usually contain-
ing a 10 mm scale divided into 0.1 mm (100 m) units. The reticule is mounted in
an eyepiece and is then calibrated using a precision stage micrometer to obtain a
conversion factor ( m/reticule unit), which is used to determine the magnification
obtained for each objective lens. The reason for using this calibration procedure is
that the nominal magnification of an objective lens (found engraved on the lens
barrel) is only correct to within 5%. If precision is not of great concern, an
approximate magnification can be obtained using the eyepiece reticule alone. In
this case, simply measure the number of micrometers from the eyepiece reticule
and divide by the nominal magnification of the objective. For a specimen covering
2 reticule units (200 m), for example: 200 m/10 20 m.
The full procedure, using the stage micrometer, is performed as follows:

• To mount the eyepiece reticule, unscrew the lower barrel of the focusing eye-
piece and place the reticule on the stop ring with the scale facing upward.
The stop ring marks the position of the real intermediate image plane. Make
sure the reticule size matches the internal diameter of the eyepiece and rests
on the field stop. Carefully reassemble the eyepiece and return it to the
binocular head. Next focus the reticule scale using the focus dial on the eye-
piece and then focus on a specimen with the microscope focus dial. The
images of the specimen and reticule are conjugate and should be simultane-
ously in sharp focus.
• Examine the stage micrometer slide, rotating the eyepiece so that the microm-
eter and reticule scales are lined up and partly overlapping. The stage microm-
eter consists of a 1 or 2 mm scale divided into 10 m units, giving 100
units/mm. The micrometer slide is usually marked 1/100 mm. The conversion
factor we need to determine is simply the number of m/reticule unit. This
conversion factor can be calculated more accurately by counting the number of
micrometers contained in several reticule units in the eyepiece. The procedure