INTRODUCTION TO MICROSCOPY
I. MICROSCOPE FUNCTION - Magnification and resolution of images.
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PREPARATION OF TISSUES FOR ROUTINE HISTOLOGICAL EXAMINATION
STEPS
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ARTIFACTS During
processing
1. FIXATION
Formalin to terminate cell metabolism
and prevent auto digestion.
Can cause shrinkage and swelling of cells
and tissues.
2. DEHYDRATION
Tissue washed with increasing % of
alcohol until all water is removed.
Removes lipids, glycogen, and proteins.
Can cause shrinkage or swelling of cells
and tissues.
3. CLEARING
Replaces alcohol with volatile solvents
(xylene, toluene) that will mix with
paraffin.
Can cause shrinkage or swelling of cells
and tissues
3. INFILTRATION
Tissue is placed in melted paraffin until
it is infiltrated with paraffin.
Same as above
Do not memorize this table
4. EMBEDDING
5. SECTIONING
6. STAINING
Tissue is placed in a small mold and
allowed to harden into a paraffin block.
(plastic resins can also be used)
Same as above
The block is sectioned into thin slices
(3 -10 µm) with a microtome.
The slices are placed on a glass slide
and the paraffin is removed.
Folds or scratches in section
The colorless tissue is stained with dyes
to increase contrast
Over or under-staining of cells and tissues
False precipitates
Heat can result in spaces between tissues
Do not memorize this table
Most slides are stained with hematoxylin
and eosin (H&E)
7. MOUNTING
Tissue preserved with coverslip
Tissue folds and breaks
The appearance of tissues after all these processes is considerably distorted compared to the
tissue in the living state. In histology you will learn to recognize the “normal” appearance of
cells and tissues after slide preparation as well as artifacts introduced during tissue
processing. Abnormal appearing tissues may be due to artifacts caused by poor slide
preparation and not due to pathology in the living tissue.
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FROZEN SECTIONS:
Tissues can be immediately frozen, sectioned with a cryostat (cold microtome) and then
processed through the rest of the steps in minutes versus days with paraffin sections.
This method is used for surgical biopsies to quickly determine if the tissue is benign or
malignant. Frozen sections are also used to study tissue components normally removed
(lipids, glycogen) or inactivated (enzymes) in paraffin prepared sections.
TISSUE STAINING and Descriptive terms:
Most sectioned tissues are routinely stained with 2 dyes, hematoxylin and eosin (H&E).
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Important concept: The affinity of the dyes to tissue components and staining has NO relation
to the biochemistry of the components in the LIVING state. Remember typical H&E staining is
done on tissues after they have been fixed, washed in water, alcohol, xylene, put in hot
paraffin in a vacuum, cut into tiny pieces, and washed again.
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CYTOLOGY: THE NUCLEUS
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NUCLEAR APPEARANCE – The shape, size, and staining of a nucleus is a useful tool for cell
identification. It can also give us clues about the functional activity of the cell.
The proportion of heterochromatin versus euchromatin (i.e. dark versus light staining) is
a reflection of how much of the cell’s genome is actively being transcribed.
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When fully condensed during mitosis or meiosis, heterochromatin is visible as distinct
chromosomes.
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MITOSIS
Although mitosis, the process of cell division is a continuous process, it is traditionally divided
into 4 stages: Prophase, Metaphase, Anaphase, and Telophase
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In sectioned tissue, mitotic cells can be recognized by their darkly staining clumps of
chromatin and are not staged but collectively called “mitotic figures”
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KARYOTYPING –
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CELL CYCLE - divided into cell division (mitosis) and interphase (G1, S, G2)
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Is a particular cell capable of replicating or is it terminally differentiated?
If it is unable to replicate is there a more primitive or stem cell in that tissue that can
replicate and differentiate into that type of cell?
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The ability of a cell to replicate and the number and location of mitotic cells in each type of
tissue is important for pathology.
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CYTOLOGY: THE CYTOPLASM
I. CELL MEMBRANE = unit membrane (7-10nm thick)
It consists of a semi-permeable lipid bilayer with associated proteins (integral and
peripheral) and carbohydrates. This active organelle transports substances in and out of
the cell, maintains the cell’s internal composition and has receptors.
II. CYTOSKELETAL COMPONENTS
The supporting framework of the cell consists of a variety of proteins assembled into
minute rods or filaments and tubules. The cytoskeleton is involved in cell support, shape,
movement of the cell, and movement of materials within the cell.
A. Filaments - tiny intracellular rods in 3 sizes (daddy, mommy, and baby size)
1. Microfilaments (7nm in diameter)– tiny rods composed of the protein actin.
Specialized structures with large amounts of microfilaments include:
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2. Myosin filaments (12-16nm) - also called thick filaments, are best developed
in muscle where it is involved in contraction
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3. Intermediate filaments (8-12nm)- a heterogeneous class of filaments important in
cell support and shape. They are usually very stable, with low turnover. Different
proteins form these filaments in different tissues (ex. keratin in skin) They are also a
component of desmosomes (type of junction).
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B. Microtubules – hollow tubes composed of the protein, tubulin.
Specialized structures with large amounts of microtubules include:
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III. MITOCHONDRIA - large motile organelles composed of 2 unit membranes, with the
inner membrane highly folded into shelves (cristae). Mitochondria are self-replicating,
having their own DNA and RNA. Mitochondria generate energy in the form of ATP
through oxidative phosphorylation. Metabolically active cells, requiring large amounts of
ATP, have large numbers of mitochondria as well as mitochondria with more inner folds
(increasing the surface area for substrates).
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IV. RIBOSOMES - small dense granules containing RNA manufactured in the nucleolus.
Ribosomes, with messenger RNA, assemble amino acids into proteins.
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*Ribosomes are BASOPHILIC in H&E. Cells that contain large numbers of ribosomes
either in the RER or free in the cytoplasm have a basophilic or intensely blue cytoplasm.
V. ENDOPLASMIC RETICULUM (ER) - an extensive system of interconnected membrane
bound cavities continuous with the nuclear membrane.
A. Rough endoplasmic reticulum (RER) – contains attached ribosomes
The RER is the site of protein synthesis for secretory, lysosomal, and integral proteins of
the cell membrane, all proteins that need to be enclosed in a membrane.
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B. Smooth endoplasmic reticulum (SER) –lacks ribosomes.
SER has diverse functions depending on the cell and location. It is abundant in cells
that synthesize large amounts of lipid and steroid hormones (ex. adrenal cortex). It
plays an important role in detoxification of alcohol, drugs, and toxins in liver cells.
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VI. GOLGI APPARATUS (BODY)
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VII. Terms associated with movements in and out of the cell:
1. EXOCYTOSIS - release of material FROM the cell usually by fusion of membrane
bound vesicles with the cell membrane.
2. ENDOCYTOSIS – entrance of material INTO the cell.
a. Pinocytosis (to drink) – ingestion of small particles and fluids
b. Phagocytosis (to eat) – ingestion of larger particles (bacteria, debris)
VIII. SECRETORY VESICLES (granules) - membrane bound “bags” containing products
destined for release from the cell by EXOCYTOSIS.
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IX. LYSOSOMES (suicide bags) – are membrane bound bags of hydrolytic enzymes. The acid
hydrolases must be segregated by a membrane or they will destroy the cell. Lysosomal
enzymes are synthesized in the RER and packaged in the Golgi apparatus forming primary
lysosomes. Cells filled with lysosomes are specialized to “destroy.”
B. Lysosomes can destroy materials INSIDE the cell. The materials can be a normal
part of the cell or it can be something brought in from outside the cell (ex. bacteria).
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X. CYTOPLASMIC INCLUSIONS – components not always present in a cell.
A. Stored foods:
1. Glycogen - a storage product of glucose, is dissolved away in standard preparations
2. Lipid – fat is stored as non-membrane bound droplets. During standard tissue
processing, the lipid is removed, leaving clear spaces in the cytoplasm.
Lipid and pigments
B. Pigments
1. Lipofuscin - yellowish-brown pigment that accumulates with age. It is composed of
residual bodies resulting from lysosomal activity.
2. Melanin - brownish-black pigment, present in membrane bound vesicles called
melanosomes.
3. Other pigments: carbon, hemosiderin, tattoo pigments, carotene
XI. CELLULAR ADAPTATIONS
Cells may alter their type or amount of organelles, vesicles, or inclusions in response to
environmental stimuli.
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REVIEW OF H&E STAINING
When examining H&E stained tissue you first scan the slide and determine how well the tissue is
stained. Generally, nuclei should be basophilic (bluish or purple) while the cytoplasm of most cells
should be lightly acidophilic (pink or orange).
Slides are commonly under-stained for hematoxylin and over-stained for eosin. In these slides,
everything appears disgustingly pink. In this case, basophilic structures will appear darker not bluer.
Once you judge how a typical cell is stained on the slide, you can then identify cells that stain differently
from the norm. Color in H&E is only one of the criteria for cell identification. The size and shape of the
cell and nucleus, contrast (dark and lightness), texture (grainy, striations) and even location are all
important identification criteria.
1. NUCLEAR STAINING: The RNA and DNA in the nucleus (chromatin and nucleolus) binds
to hematoxylin and appears basophilic. Descriptive terms used to describe the nucleus:
Dark condensed
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2. CYTOPLASMIC STAINING: The cytoplasm of most cells is slightly acidophilic.
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