ZOOL 409, Week 1
Objective 1:
Lab Notebook.
Objective 2:
Meet your microscope
Drawing is one of the most powerful study-aids for
Basic elements (italics indicates user-adjustable
learning visual information, such as the microscopic
appearance of tissues.
features)
Each lab period should receive a notebook entry for each
slide that you observe, including:
 Date, time, slide number and slide label for each slide
that is observed.
 A list of expected observations. From your prior study
(text, atlas, lecture), what are you looking for?
 Sketches of your actual observations.
As an aid to sketching, use the the circular field of
view in your microscope as a frame. Imagine a circle
drawn halfway between the center of this field and the
edge. Then imagine the circle as a clock-face, with
four quarters and each quarter divided into three parts.
Your sketches might usefully include:
 A map (overview) of the specimen, as a guide for
locating (and relocating) specific features.
o Anything visible by naked eye should be included in
this map.
 Examples of features which you can identify, with
appropriate labels.
o Honestly, from your own observation, what features
do you actually see? (Real specimens do not always
display all typical features.)
o Which of those can you confidently identify?
 Examples of features which you cannot identify
(labelled as "unidentified").
 A list of features that you expected to find (e.g., based on
reference materials) but could not see (and/or could not
reliably identify).
If you have a question about some feature that you
have found, the best way to ask is by way of a sketch
that shows its location in the field of view and its
relationship to other features. Thus, "What is this, that
I have drawn here?" works much better than, "What's
the red thing we see in the microscope?" Your
instructor may refuse to address your question until
you have made such a sketch.
A well-kept notebook, with recognizable and accurately
labelled drawings, may be submitted for grade
consideration.
 Illuminator (light source)
The illuminator may include adjustable field
diaphragm.
 Condenser (light gathering lens)
Proper condenser adjustment is important for optimal
performance.
The condenser includes an adjustable condenser
diaphragm.
The condenser may include focus adjustment.
 Stage (platform between condenser and objective)
The stage supports the specimen (microscope slide).
The stage allows specimen to move in x-y plane.
The stage may move up and down for specimen focus.
 Specimen
Note that the specimen mount (slide and coverslip) is
part of the optical system.
At high magnification, optimal optical adjustment
requires that coverslip thickness be appropriate for the
objective lens.
 Objective lens (principal image-forming lens)
Functionally, the objective lens is the most critical
(and most expensive) element of the microscope.
Objectives are interchangeable (usually via a
swivelling "nosepiece") to change magnification.
Objectives may move up and down for specimen
focus.
 Eyepiece (enlarging lens, paired in binocular m'scopes)
If paired, interpupillary distance is adjustable; at least
one eyepiece should be independently focussable.
For more information on optimal adjustment, see
http://www.siumed.edu/~dking2/intro/kohler.htm.
Care of microscope (always follow these rules!)
 Three rules for carrying a microscope:
1. Your full attention is on the job, just as if you were
carrying a baby.
2. Use both hands.
3. Always carry the microscope upright (otherwise the
eyepieces can fall out).
 Three rules for cleaning a microscope:
1. Don't! It is too easy to cause damage
 Never the touch the objective lens surface, unless
you have bought and paid for the microscope .
 Clean eyepieces and other optical surfaces only
when absolutely necessary. It is better to ignore
dirt than to cause permanent damage to the
microscope.
2. Locate the dirt; use logic and experiment to determine
where in the optical path the dirt is located.
 Dirt on a slide moves when you move the slide.
 Dirt on the condenser will go in and out of focus
when you move the condenser up or down.
 Dirt on an eyepiece will move when you rotate the
eyepiece.
 Dirt on an objective cannot be seen while viewing
through the microscope, but manifests itself as a
decrease in image quality.
3. If dirt cannot be ignored, use the least-intrusive
procedure to make it go away.
 Some dirt can blown away without touching the
surface. Use this procedure freely, but blow gently
and be careful not to spit.
 Dirt on slides may be wiped off at will, using the
same care you would use for eyeglasses.
 Dirt on an eyepiece is often accentuated by having
the condenser aperture too small; such dirt may
become inconspicuous when the diaphragm is
opened to the correct setting.
 Dirt on the condenser may become inconspicuous
if you lower the condenser (i.e., move the dirt out
of focus).
 If dirt simply must be removed -o Begin by simply blowing dust away.
o Use clean lens paper and moisture from breath.
o Wipe gently in one direction. Do not use a
circular motion; do not scrub.
o Do not try to clean an objective lens; ask for
help.
Magnification
o For the microscopes used in this course:
Object.
lens
Field of
view
(diameter)
Eyepiece
pointer
(diameter)
Ocular
micrometer
(numbered units)
4x
10 x
40 x
4.5 mm
1.8 mm
450 µm
50 µm
20 µm
5 µm
260 µm
100 µm
25 µm
o You may check these values with the calibration slide
available from your instructor.
o Use these values to estimate the size of the objects
provided.
 Even with an uncalibrated microscope, sizes of tissue
features can be estimated by reference to familiar objects
of consistent size, such as red blood cells. (Mammalian
RBCs are approx. 6-8 µm in diameter.)
Resolution and numerical aperture
 "Resolution" refers to the "resolving power" of the
microscope, i.e., the 'scope's ability to "resolve", or
distinguish as separate, two nearby objects. Resolution
is at least as important as magnification. An increase in
magnification that is not accompanied by an increase in
resolution is termed empty magnification, with the
implication that nothing is really gained (i.e., you cannot
see any additional detail by an empty increase in
magnification.
 Magnification is determined in principle by multiplying
the power of the Objective Lens by the power of the
Eyepiece.
 Resolution is determined by optical quality and
fundamentally limited by the wavelength of light and by
the numerical aperture of the objective/condenser
combination.
 Magnification is determined in practice by calibration
with a specimen of known size.
For more information on microscopes and microscopy,
consult the following websites.
o For research purposes, calibration is done with a
reference standard, either a "calibration slide" (e.g.., a
glass slide engraved with a very finely-calibrated
ruler) or with standardized objects (e.g.,
microspheres).
www.micrographia.com/tutoria/micbasic/micbpt01/micb0100.htm
o Some microscopes include an "eyepiece micrometer",
essentially a small, transparent ruler mounted within
the eyepiece that is used to measure specimens. Such
an eyepiece micrometer must initially be calibrated
(at each magnification) using a specimen of known
size (see preceding item).
Objective 3:
o Without an eyepiece micrometer, sizes can still be
estimated in relation to the field of view or to the
thickness of an eyepiece pointer (if there is one).
Each of these reference standards must be initially
calibrated at each magnification.
On each of the following slides, find some cells. Estimate
the size (diameter) for the whole cell and for its nucleus.
www.microscopy.fsu.edu/primer/anatomy/anatomy.html
___________________________________
Meet some cells
Recognizing that all animal tissues (plants, too) are
composed of cells and cell products was a major
accomplishment in the history of scientific observation.
Slide number:
18, 35, 55, 91
11 July 2011