CHAPTER 1 (Book) Flashcards
Histology and its Methods of Study
the study of the tissues of the body and
how these tissues are arranged to constitute organs.
Histology
Tissues have two interacting components:
cells and extracellular matrix (ECM)
supports the cells and contains the fluid transporting nutrients to the cells, and carrying away their wastes and secretory products
ECM
The most common procedure used in histologic research is the
preparation of tissue slices or “sections”
To preserve tissue structure and prevent degradation by enzymes released from the cells or microorganisms, pieces of organs are placed as soon as possible after removal from the body in solutions of stabilizing or cross-linking compounds called
fixatives
To improve cell preservation in large organs, fixatives are often introduced via
blood vessels with vascular perfusion
One widely used fixative for light microscopy is
formalin, a buffered isotonic solution of 37% formaldehyde
a fixative used for electron microscopy
glutaraldehyde
Electron microscopy provides much greater magnification and resolution of very small cellular structures, and fixation must be done very carefully to preserve additional “ultrastructural” detail. Typically in such studies, glutaraldehyde-treated tissue is then immersed in buffered
osmium tetroxide
osmium tetroxide, preserves and stains what?
cellular lipids as well as proteins
To permit thin sectioning, fixed tissues are
infiltrated and embedded
Embedding materials include
paraffin, plastic resins
Before infiltration with such media, the fixed tissue must undergo
dehydration
dehydration is done by having the tissues’ water extracted gradually by transfers through
a series of increasing ethanol solutions, ending in 100% ethanol
The ethanol is then replaced by an organic solvent miscible with both alcohol and the embedding medium, a step referred to as
clearing
The fully cleared tissue is then placed in melted paraffin in an oven at
52°-60°C
The hardened block with tissue and surrounding embedding medium is trimmed and placed for sectioning in an instrument called a
microtome
Paraffin sections are typically cut at _______ thickness for light microscopy
3-10 μm
electron microscopy requires sections that are
less than 1 μm thick.
are tissue samples removed during surgery or routine medical procedures.
Biopsies
If results of such analyses are required before the medical procedure is completed, for example to know whether a growth is malignant before the patient is closed, a much more rapid processing method is used called a
Frozen section
What medium is used in frozen section
liquid nitrogen
What microtome is used in a frozen section
cryostat in a cabinet at subfreezing temperature
Freezing of tissues is also effective in
histochemical studies, study of lipids
Most cells and extracellular material are completely colorless, and to be studied microscopically tissue sections must be
stained
nucleic acids has what charge
net negative (anionic)
proteins with many ionized amino groups has what charge
net positive (cationic)
Cell components, such as nucleic acids with a net negative charge (anionic), have an affinity for basic dyes and are termed
basophilic
cationic components, such as proteins with many ionized amino groups, stain more readily with acidic dyes and are termed
acidophilic
Examples of basic dyes include
toluidine blue, alcian blue, and methylene blue.
behaves like a basic dye, staining basophilic tissue components
Hematoxylin
The main tissue components that ionize and react with basic dyes do so because of acids in their composition
DNA, RNA, and glycosaminoglycans
Acid dyes includes
eg, eosin, orange G, and acid fuchsin
Acid dyes (eg, eosin, orange G, and acid fuchsin) stain the acidophilic components of tissues such as
mitochondria, secretory granules, and collagen
Of all staining methods, the simple combination of is used most commonly
hematoxylin and eosin (H&E)
Hematoxylin stains
DNA in the cell nucleus, RNA-rich portions of the cytoplasm, and the matrix of cartilage
eosin stains
other cytoplasmic structures and collagen
Here eosin is considered a
counterstain
More complex procedures, such as__________, allow greater distinctions among various extracellular tissue components.
trichrome stains (eg, Masson’s trichrome)
utilizes the hexose rings of polysaccharides and other carbohydrate-rich tissue structures and stains such macromolecules distinctly purple or magenta
periodic acid–Schiff (PAS) reaction
The DNA of cell nuclei can be specifically stained using a modification of the PAS procedure called the
Feulgen reaction
Basophilic or PAS-positive material can be further identified by
enzyme digestion
Examining lipids are stained by?
lipid-soluble dyes such as Sudan black
Less common methods of staining can employ
metal impregnation
typically using solutions of silver salts to visual certain ECM fibers and specific cellular elements in nervous tissue.
metal impregnation techniques
Slide preparation, from tissue fixation to observation with a light microscope, may take from
12 hours to 2½ days
Small pieces of tissue are placed in solutions of chemicals that cross-link proteins and inactivate degradative enzymes, which preserve cell and tissue structure
Fixation
The tissue is transferred through a series of increasingly concentrated alcohol solutions, ending in 100%, which removes all water.
Dehydration
Alcohol is removed in organic solvents in which both alcohol and paraffin are miscible
Clearing
The tissue is then placed in melted paraffin until it becomes completely infiltrated with this substance.
Infiltration
The paraffin-infiltrated tissue is placed in a small mold with melted paraffin and allowed to harden
Embedding
The resulting paraffin block is trimmed to expose the tissue for sectioning (slicing) on a microtome.
Trimming
Similar steps are used in preparing tissue for transmission electron microscopy (TEM), except special fixatives and dehydrating solutions are used with smaller tissue samples and embedding involves _______ which become harder than paraffin to allow very thin sectioning.
epoxy resins
With _________ stained tissue is examined with ordinary light passing through the preparation
bright-field microscope
highly sensitive to low light levels with a camera and monitor used to view the microscope
charge-coupled device (CCD)
The critical factor in obtaining a crisp, detailed image with a light microscope is its
resolving power
defined as the smallest distance between two structures at which they can be seen as separate objects
resolving power
The maximal resolving power of the
light microscope is approximately
0.2 μm
Objects smaller or thinner than 0.2 μm such cannot be distinguished with light microscope
as a single ribosome or cytoplasmic
microfilament
typically used for study of brightfield
microscopic preparations, involves the conversion of a stained tissue preparation to high-resolution digital images and permits study of tissues using a computer or other digital device,
Virtual microscopy
In virtual microscopy regions of a glass-mounted specimen are captured digitally in a grid-like pattern at multiple magnifications using a
specialized slide-scanning microscope
When certain cellular substances are irradiated by light of a proper wavelength, they emit light with a longer wavelength—a phenomenon called
fluorescence
In _______ tissue sections are usually irradiated with ultraviolet (UV) light and the emission is in the visible portion of the spectrum before analyzing
fluorescence microscopy
Fluorescent compounds with affinity for specific cell macromolecules may be used as fluorescent stains. _____________ which binds both DNA and RNA, is an example
Acridine orange
Other compounds, such as _____________ stain specifically bind DNA and are used to stain cell nuclei, emitting a characteristic blue fluorescence under UV
DAPI and Hoechst
Another important application of fluorescence microscopy is achieved by coupling compounds such as __________________to molecules that will specifically bind to certain cellular components and thus allow the identification of these structures under the microscope
fluorescein
Unstained cells and tissue sections, which are usually transparent and colorless, can be studied with these modified light microscopes
Phase-Contrast Microscopy
A modification of phase-contrast microscopy is
differential interference contrast microscopy
differential interference contrast microscopy produces what images
image of living cells with a more apparent three-dimensional (3D) aspect
DAPI
4′,6-diamino-2-phenylindole
binds actin filaments green
fluorescein phalloidin
avoids problems found in bright field microscopes and achieves high resolution and sharp focus by using (1) a small point of high-intensity light, often from a laser and (2) a plate with a pinhole aperture in front of the image detector
Confocal microscopy
Confocal microscopy avoids problems found in bright field microscopes and achieves high resolution and sharp focus by using
(1) a small point of high-intensity light, often from a laser
(2) a plate with a pinhole aperture in front of the image detector
allows the recognition of stained or unstained structures made of highly organized subunits
Polarizing microscopy
The ability to rotate the direction of vibration of polarized light is called
birefringence
are based on the interaction of tissue components with beams of electrons
Transmission and scanning electron microscopes
is an imaging system that permits resolution around 3 nm
transmission electron microscope (TEM)
The transmission electron microscope (TEM) is an imaging system that permits resolution around 3 nm. This high resolution allows isolated particles magnified as much as
400,000 times to be viewed in detail
Specimens typically studied by TEM
Very thin (40-90 nm), resin-embedded tissue sections
parts of Transmission electron microscope
Cathode
Anode
Condensor lens
Objective lens
Intermediate lens
Projector lens
Image on viewing screen
Electron detector with CCD camera
Electron gun
Copper grid with 3 sections (3nm)
Specimen holder
Column
TEM image
Parts of Scanning electron microscope
Cathode
Anode
Lens
Lens #2
Scanner
Lens #3
Specimen
Electron gun
Column
Electron detector
SEM image
To improve contrast and resolution in TEM, compounds with _________ are often added to the fixative or dehydrating solutions used for tissue preparation.
heavy metal ions
Examples of heavy metal ions used in tissue preparation for TEM
osmium tetroxide, lead citrate, and uranyl compounds
are techniques that allow TEM study of cells without fixation or embedding and have been particularly useful in the study of membrane structure
Cryofracture and freeze etching
In cryofracture and freeze etching, a replica of the frozen exposed surface of the specimen is produced in a vacuum by applying thin coats of
vaporized platinum or other metal atoms
provides a high-resolution view of the surfaces of cells, tissues, and organs. Like the TEM, this microscope produces and focuses a very narrow beam of electrons, but in this instrument the beam does not pass through the specimen
Scanning electron microscopy (SEM)
Often used heavy metal in SEM
gold
is a method of localizing newly synthesized macromolecules in cells or tissue sections.
Microscopic autoradiography
act as microdetectors of the radiation in the same way that they respond to light in photographic film in samples with radiolabeled cells or tissue sections
silver bromide crystals
Much histological information becomes available by autoradiography. If a radioactive precursor of DNA such as_____________ is used, it is possible to know which cells in a tissue and how many are replicating DNA
tritium-labeled thymidine
Autoradiographs are tissue preparations in which particles called ________ indicate the cells or regions of cells in which specific macromolecules were synthesized just prior to fixation.
silver grains
Live cells and tissues can be maintained and studied outside the body in culture
in vitro
Live cells and tissues can be maintained and studied in the organism, cells are bathed in fluid derived from blood plasma and containing many different molecules required for survival and growth
in vivo
primary cell cultures steps
- cells and tissues are GROWN in complex solutions of known composition (salts, amino acids, vitamins)
- serum or specific growth factors are ADDED
- Cells to be cultured are DISPERSED mechanically or enzymatically from a tissue or organ
- PLACED with sterile procedures in a clear dish to which they adhere
Some cells can be maintained in vitro for long periods because they become immortalized and constitute a permanent
cell line
Most cells obtained from normal tissues have a finite, genetically programmed life span. However, certain changes can promote cell immortality, a process called
transformation
Cervical cancer cells from a patient later identified as Henrietta Lacks, who died from the disease in 1951, were used to establish one of the first cell lines, called
HeLa cells
HeLa cells are used to study the potential treatment benefits of a drug called ______ against certain blood cancers and sickle cell anemia.
Hydroxyurea
is a method for localizing cellular structures using a specific enzymatic activity present in those structures
Enzyme histochemistry (or cytochemistry)
To preserve the endogenous enzymes, histochemical procedures usually use
unfixed or mildly fixed tissue
For enzyme histochemistry tissue preparation is done by:
(1) tissue sections are
immersed in a solution containing the substrate of the enzyme
to be localized
(2) the enzyme is allowed to act on its substrate
(3) the section is then put in contact with a marker compound that reacts with a product of the enzymatic action on the substrate
(4) the final product from the marker, which must be insoluble and visible by light or electron microscopy, precipitates over the site of the enzymes, identifying their location.
Examples of enzymes that can be detected histochemically include the following:
Phosphatases
Dehydrogenases
Peroxidase
used to diagnose the iron storage diseases, hemochromatosis and hemosiderosis
Perls’ Prussian blue reaction for iron
to detect glycogenosis and mucopolysaccharidosis
PAS-amylase and alcian blue
reactions for polysaccharides
to detect sphingolipidosis
reactions for lipids and sphingolipids
The most commonly used labels are
fluorescent compounds
radioactive atoms
peroxidase or other enzymes (histochemistry)
metal (usually gold)
Examples of molecules that interact specifically with other molecules include the following (visualizing specific molecules)
Phalloidin
Protein A
Lectins
(visualizing specific molecules) glycoproteins derived mainly from plant seeds, bind to carbohydrates with high affinity and specificity
Lectins
(visualizing specific molecules) purified from Staphylococcus aureus bacteria, binds to the Fc region of antibody molecules, and can therefore be used to localize naturally occurring or applied antibodies bound to cell structures
Protein A
(visualizing specific molecules) a compound extracted from mushroom, Amanita phalloides, interacts strongly with the actin protein of microfilaments.
Phalloidin
labeled antibodies are routinely used in _____________ to identify and localize many specific proteins, not just those with enzymatic activity that can be demonstrated by histochemistry.
immunohistochemistry
Widely applied for both research and diagnostic purposes, every immunohistochemical technique requires an antibody against the protein that is to be detected. This means that the protein must have been previously purified using
biochemical or molecular methods
Growth and activity of activated lymphocytes can be prolonged indefinitely by fusing them with ____________ to produce _________
lymphocytic tumor cells
hybridoma cells
Antibodies are commonly tagged
with fluorescent compounds, with __________ for histochemical detection
peroxidase or alkaline
phosphatase
There are other indirect methods that involve the use of other intermediate molecules, such as the__________ technique, which are also used to amplify detection signals.
biotin-avidin
usually implies the specific binding between
two single strands of nucleic acid, which occurs under appropriate conditions if the strands are complementary
Hybridization
A single cultured uterine cell stained fluorescently to reveal a meshwork of intermediate filaments (green) throughout
the cytoplasm. Primary antibodies against the filament protein__________ were used in the indirect staining technique, with the nucleus counterstained blue with DAPI. (X650)
desmin and fluorescein isothiocyanate (FITC)-labeled secondary antibodies
DAB
3,3′-diamino-azobenzidine
Examples of specific antigens with diagnostic importance.
Specific cytokeratins
Tumors of epithelial origin
Examples of specific antigens with diagnostic importance.
Protein and polypeptide hormones
Certain endocrine tumors
Examples of specific antigens with diagnostic importance.
Carcinoembryonic antigen (CEA)
Glandular tumors, mainly of the digestive tract and breast
Examples of specific antigens with diagnostic importance.
Steroid hormone receptors
Breast duct cell tumors
Examples of specific antigens with diagnostic importance.
Antigens produced by viruses
Specific virus infections
Hybridization at stringent conditions allows the specific identification of sequences in genes or RNA. This can occur with cellular DNA or RNA when nucleic acid sequences
in solution are applied directly to prepared cells and tissue sections, a procedure called
in situ hybridization (ISH)
in situ hybridization (ISH) is ideal for?
determining if a cell has
a specific sequence of DNA
identifying the cells containing specific messenger RNAs (mRNAs)
determining the localization of a gene
in a specific chromosome
in ISH the nucleotide sequences of interest are detected with
probes
probes consists of?
single-stranded complementary
DNA (cDNA)
The probe may be obtained by
cloning by polymerase chain reaction (PCR) amplification of the target sequence
or by chemical synthesis
The probe is tagged with nucleotides containing a radioactive isotope (localized by autoradiography) or modified with
a small compound such as
digoxigenin
Certain steps in tissue procedure may distort the tissues slightly, producing minor structural abnormalities called
artifacts
