CRR Histology Lab 1
Introduction to Tissue Microscopy
CRR Week 1
Principal objectives for Lab 1
Lab guidelines
1. Become acquainted with your microscope, your
boxed set of study slides, and various print and internet
resources.
Scheduled lab periods are optional opportunities for
study in the presence of instructors.
2. Learn to see cells. Appreciate how all cells share
similar structures and how distinct cell types are
differentiated from one another.
3. Begin distinguishing basic patterns of tissue
organization, particularly epithelial and connective
tissues.
Please bring slides and reference materials to labs.
Handouts are intended to stimulate, not to educate.
"Check-off boxes" enable your instructor to monitor your
learning during the unit..
For additional supporting resources, see back of page.
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LIGHT MICROSCOPY
Scalp, slide 2.
Skin, slide 3.
Blood smear, slide 98.
(EASY) Find an epidermal cell. Question--What kind of
tissue is epidermis?
(EASY) Distinguish nucleus from cytoplasm for cells of
epidermis. Question--How much of the epidermis consists
of cells (nuclei and cytoplasm together)? Question--Can
you see any extracellular space in the epidermis?
(EASY) Find the dermis. Question--What kind of tissue is
dermis?
(EASY) Find a cell without a nucleus. Question--What
type of blood cell is this? Find a few hundred more cells of
the same type. Question--How much do these cells vary in
size?
(EASY) Find a cell with its nucleus pinched into several
lobes. Question--What type of blood cell is this? Find a
few dozen more cells of the same type. Question--How
much do these cells vary in size?
(EASY) In dermis, distinguish cells from extracellular
material. Question--How much of the dermis consists of
cells? How much is extracellular material?
(EASY) Find a cell with a round nucleus. Question--What
type of blood cell is this? Find a couple dozen more cells
of this type. Question--How much do these cells vary in
size?
(EASY) Find collagen. Question--Where should you look?
(Epidermis or dermis? Within cells or outside cells?)
(HARDER) Find a large cell with a deeply dented nucleus.
Question--What kind of cell is this?
(HARDER) Try to identify special structures of skin (e.g.,
hair follicles, sweat glands, blood vessels).
(EASY) Find small bits of cytoplasm, without nuclei,
much smaller than entire cells. Question--What are these?
(REPEAT) Find epithelial cells, connective tissue cells,
and collagen in cornea (slide 25), tongue (slide 29), and
trachea (slides 61, 62).
Sickle Cell, slide 52. Repeat the exercises above.
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At your discretion, you may notify an instructor
for a brief oral evaluation on this material.
Bone marrow smear, slide 99. What is the most striking
difference between this and peripheral blood smears?
At your discretion, you may notify an instructor
for a brief oral evaluation on this material.
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ELECTRON MICROGRAPHS
Use Rhodin's Atlas of Histology (from the Learning
Resources Center) or other any other collection of electron
micrographs.
(EASY) On an image of an entire cell, identify plasma
membrane, cytoplasm, nucleus, and extracellular material.
Estimate diameter of cell and nucleus.
Below are approximate sizes (diameter) for some
commonly encountered cellular structures.
(EASY) On an image of an entire cell, identify nuclear
membrane, nucleolus, euchromatin and heterochromatin.
ribosomes -- about 0.02 µm.
mitochondria -- variable,commonly about 0.5 µm.
"typical" nucleus -- variable, commonly 5-10 µm.
"typical" cell -- variable, commonly 6-20 µm.
red blood cell -- fairly uniform, 6-8 µm.
adipocyte -- variable, commonly about 50 µm.
skeletal muscle fiber -- variable, commonly 30-60 µm.
terminal arterioles -- about 30-50 µm.
(EASY to HARD) On images showing a significant
portion of one or more cells, identify mitochondria, rough
endoplasmic reticulum, ribosomes, Golgi bodies, secretory
vesicles, lysosomes, glycogen granules.
At your discretion, you may notify an instructor
for a brief oral evaluation on this material.
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Learning Resources for CRR Histology
Scheduled Activities: www.siumed.edu/~dking2/crr/CRRsched.htm (or see CRR Unit Calendar).
On-line study guide:
www.siumed.edu/~dking2/index.htm
../crr/index.htm
../intro/index.htm
Other internet materials:
U. of Utah, The Internet Pathology Laboratory
www-medlib.med.utah.edu/WebPath/webpath.html
U. of Iowa, Virtual Slidebox
http://www.path.uiowa.edu/virtualslidebox/
Additional resources:
http://www.siumed.edu/anatomy/histolinks.htm
Self-Assessment Questions (SAQs), at the on-line study guide, above.
SAQ slides (unlabelled "unknown" slides), available in the MRC (Medical Resource Center).
Textbooks. All students should read at least one introductory text. All of the following are roughly equivalent in
depth and coverage, appropriate for the level of understanding expected in our current year-one curriculum. Your
choice may be based on availability or on personal preference (style varies markedly among texts).
Junqueira's Basic Histology: Text & Atlas, 12th ed. (2010), Mescher.
Wheater's Functional Histology: A Text and Colour Atlas, 5th ed. (2006), Young, Lowe, Stevens & Heath (eds.).
Histology: A Text and Atlas, 5th ed. (2006), Ross & Pawlina.
Histology and Cell Biology: An Introduction to Pathology (2002), Kierszenbaum.
Human Histology, 3rd ed. (2005), Stevens & Lowe.
Elsevier’s Integrated Histology (2007), Telser et al.
Atlases. Just as with snapshots of landscapes or persons, single micrographs are seldom sufficient to catch the full
character or personality of a tissue. Since any given text typically illustrates each organ/region with only one or two
images, students are encouraged to view several sources for multiple images. Histology atlases (some examples are
listed below) are quite useful for this purpose, but personal possession of an atlas should not be necessary.
An Atlas of Histology (1976), Rhodin.
Excellent source for electron microscope images. Although out of print, multiple copies are available in the MRC.
This resource is also available online, http://projects.galter.northwestern.edu/rhodin/
Atlas of Descriptive Histology (2009), Ross, Pawlina, Barnash.
Color Atlas of Basic Histology, 3rd ed. (2003), Berman & Milikowski.
Color Atlas of Basic Histopathology (1997), Milikowski & Berman (an excellent resource for images of particular
pathologies).
References. The following are much more substantial (heavier, much more detail, ~1200 vs. ~400 pp.), better for
reference than for introductory exposure. Students should be aware that such resources exist, but personal ownership is
not necessary.
Wheater’s Basic Histopathology, 4th edition (2002), Stevens et al.
Histology for Pathologists, 3rd ed. (2007), Mills; or 2nd ed. (1998), Sternberg.
Robbins and Cotran Pathologic Basis of Disease, 8th ed. (2010), Kumar, et al.
Textbook of Histology, 12th ed. (2001), Fawcett.
KNOW YOUR MICROSCOPE
Basic terminology
Three rules for cleaning a microscope:
Light source
1. Don't! (not without proper training). It is
easy to cause permanent damage by
injudiciously cleaning a microscope.
The light source, or "illuminator", should have
adjustable brightness and may include an adjustable field
diaphragm to minimize scattered light.
Condenser (light-gathering lens)
The condenser is located beneath the stage and
includes an adjustable aperture, controlled by a diaphragm
within the condenser. Proper condenser adjustment is
essential for optimal resolution (see "Kohler illumination"
on the other side of this page).
Stage (specimen support platform)
The stage supports the microscope slide and allows
specimen movement in the x-y plane. The stage may
move up and down for specimen focus.
 Clean eyepieces and other optical surfaces only
when absolutely necessary. It is better to ignore
dirt than to cause permanent damage to the
microscope.
 Never the touch the objective lens surface, unless
you own the microscope and have been properly
trained.
2. If you see dirt when you look through the
microscope, determine by logic and
experiment where the dirt is located before
trying to eliminate it.
Specimen
 Dirt on a slide moves when you move the slide.
Note that the slide and coverslip are both parts of the
optical system. At high magnification, optimal optical
function requires that coverslip thickness be appropriate
for the objective lens.
 Dirt on the condenser goes in and out of focus
when you focus the condenser.
Objective lens (image-forming lens)
 Dirt on an objective cannot be seen while viewing
through the microscope, but manifests itself as a
decrease in image quality.
Functionally, the objective lens is the most critical (and
most expensive) element of the microscope. Objectives
may move up and down for specimen focus. Never touch
the optical surface of the objective lens.
Eyepiece (enlarging lens)
If eyepieces are paired, then interpupillary distance is
adjustable. At least one eyepiece should be independently
focussable. Eyepieces are removeable; when held
upsidedown, an eyepiece can serve as a hand lens.
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Three rules for carrying a microscope:
1. Always carry the microscope upright
(otherwise the eyepieces can fall out).
2. Use both hands.
3. Give the job your full attention, just as if you
were carrying a baby.
 Dirt on an eyepiece will move when you rotate the
eyepiece.
3. If dirt is objectionable, use the leastintrusive 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 much less conspicuous when the
condenser diaphragm is set correctly (see "Kohler
illumination" on the other side of this page).
 Dirt on the condenser may become inconspicuous
if you lower the condenser (i.e., move the dirt out
of focus).
 If dirt must be removed manually, use clean lens
paper and moisture from breath. Wipe gently in
one direction. Do not scrub with a circular
motion. Do not try to clean an objective lens.
Kohler Illumination
Magnification
The following instructions apply to most research
microscopes, including the multi-viewer microscopes in
Lindegren Room 206. Condenser adjustment may be
simplified on student microscopes.
 Magnification is determined in principle by multiplying
the power of the Objective Lens by the power of the
Eyepiece.
Initial set up: Turn on the lamp, place a slide on the
 Magnification is determined in practice by calibration
with a specimen of known size.
stage, focus on the specimen, adjust interocular distance to
match that of your own eyes.
For research purposes, calibration is done with a
reference standard, such as a "calibration slide",
engraved with a very finely-calibrated ruler.
1. Focus the condenser.
Close the field diaphragm located on the base of the
microscope. This will reduce the illuminated field of
view, as seen through the eyepieces, to a small circle of
light. Then adjust the substage condenser up or down
until the edge of this circle appears sharp.
2. Center the condenser.
Open the field diaphragm until the illuminated
circle almost fills the viewing area. Then adjust the
screws at either side of the condenser until this circle is
precisely centered in your field of view. Now open the
field diaphragm completely to restore illumination over
the entire field of view.
3. Adjust the condenser diaphragm.
Remove one eyepiece and look into the open tube.
You should see an illuminated field of view whose size
is controlled by the diaphragm within the condenser.
Adjust the condenser diaphragm until its edge just
barely encroaches on the illuminated field of view.
Then replace the eyepiece.
This adjustment should be repeated whenever the
objective lens is changed (i.e., whenever magnification
is changed).
4. Adjust illuminator brightness.
Use the voltage regulator on the base of the microscope
to adjust image brightness to suit your eyes.
5. Repeat steps 3 and 4 whenever you change
objectives.
An "eyepiece micrometer" (a small, transparent ruler
mounted within the eyepiece) can be used to measure
specimens, after calibration with a specimen of known
size (see preceding item).
 In routine practice, magnification is estimated with
reference to familiar objects of known size, such as red
blood cells (the diameter of human RBCs is
approximately 6-8µm).
CALIBRATE YOUR OWN MICROSCOPE.
 A good calibration standard can be made by using a
size-reducing photocopier to make a miniature copy of a
millimeter ruler. Measure the actual size of this
miniature ruler, so you know the length between marks.
 Put your calibration standard on a microscope slide and
view it through the microscope. Measure edge-to-edge
across the field of view. Repeat for each magnification
(i.e., each different objective lens). Or, measure once
with lowest power and use arithmetic to compute the
change when magnification is increased. (For example,
if magnification is increased from 4x to 40x, which is a
factor of 10, the real distance measured across the field
of view will decrease by a factor of 10.)
 If your microscope has an eyepiece pointer, you can
also measure the thickness of the pointer and use that
number as a standard for estimating the size of objects
much smaller than the field of view.
Congratulations. Your microscope should now
be adjusted for proper Kohler illumination.
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Additional details may be found at http://www.siumed.edu/~dking2/intro/mscope.htm
For yet more information . . .
http://www.micrographia.com/tutoria/micbasic/micbpt01/micb0100.htm
http://www.microscopy.fsu.edu/primer/anatomy/anatomy.html