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OVERVIEW OF METHODS USED IN HISTOLOGY b. Allows it to be thinly sliced in the range of 5-15um
- Histology (histos = tissue; logia = science) aka VI. Microtome – designed slicing machine
Microscopic anatomy – scientific study of structures VII. Mounting – use water bath
- Virtual microscopy – method of viewing a digitized a. Mount on glass slides using mounting mediums
microscopic specimen on a computer screen or mobile (pinene or acrlic resins)
device VIII. Staining
- Electron Microscope – more detailed interpretation a. Paraffin must be dissolved with xylol or toluol
 Transmission electron microscope (TEM) b. Rehydrated with DESCENDING alcohol
 Scanning electron microscope (SEM) concentration
- Atomic force microscope – comparable or higher c. Then stained with hematoxylin in WATER
resolution to those obtained from TEM d. Specimen is dehydrated again
- EM and AFM – often last step in data acquisition e. Stained with eosin IN ALCOHOL
f. Passed through xylol or toluol to a nonaqueous
TISSUE PREPARATION
mounting medium
Consists mostly of formalin-fixed, paraffin-embedded,
 Other Staining Procedures:
hematoxylin-eosin (H&E) stained specimens
o Use of ORCEIN and RESORCIN-FUCHSIN –
 Most photomicrographs are taken from such slides
ELASTIC material
Steps in tissue preparation
o SILVER impregnation – reticular fibers
I. Fixation – permanently preserves tissue structure
a. Terminates cell metabolism HISTOCHEMISTRY AND CYTOCHEMISTRY
b. Prevents autolysis - Based on specific binding of a dye
c. Kills pathogenic microorganism - Use of fluorescent due-labeled antibody
d. Hardens tissue - Inherent enzymatic activity of a cell component
i. Cross-linking - Autoradiography – localizes large molecules
ii. Denaturing protein molecules Chemical composition of Histologic samples
 Formalin – most commonly used fixative (37% aqueous Components remain after fixation consist mostly of large
solution of formaldehyde) molecules that do not readily dissolve
o Preserves by reacting with amino group of  Nucleoproteins – nucleic acids bound to protein
proteins  Intracellular cytoskeletal proteins
o Proteins maintain ability to react with specific  Extracellular proteins – large insoluble aggregates
antibodies – important in immunocytochemistry  Membrane phospholipid-protein (or carbohydrate)
methods complexes
o Acts relatively slow but penetrates tissue well Structural element is also a functional unit
o Dissolves lipids - Muscle cells – filaments are visible and actual
o 20:1 participants of contractile process
 Other methods: - RNA of the cytoplasm – also participant in synthesis of
o Frozen sections and dye that dissolves fat protein
 Retains neutral fat Many tissue components are lost during routine
 Alcohol and organic solvents dissolve neutral preparation of H&E
fats Molecules lost:
o Other fixatives containing heavy metals bind to  Neutral lipids – dissolved by organic solvents
phospholipids  Small proteins and small nucleic acids (tRNA)
 Osmium tetroxide – fixative for EM  Glycogen
 Permanganate  Proteoglycans and glycosaminoglycans (Carbs found in
II. Dehydration extracellular CT)t pathol
a. Remove extractable water from fixed tissue  Metabolites, glucose, sodium, chloride
b. Series of ASCENDING concentrations of alcohol o Do not make up formed elements
III. Clearing o Can be studied in special preparation
a. Uses organic solvent (xylene, toluene) o Provide information about metabolism, active
b. Renders elements transparent or transluscent transport, and vital cellular processes
c. Miscible in both alcohol and paraffin CLINICAL CORRELATION: FROZEN SECTIONS
IV. Infiltration Frozen section requested when:
a. Supporting medium (paraffin) - Pathologic diagnosis may determine how surgery will
b. Fixed tissue not firm enough to allow sectioning proceed
without supporting medium - No preoperative diagnosis was available
V.Embedding - Unexpected intraoperative findings
a. Tissue is placed in melted paraffin
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- To know whether all of pathologic mass within healthy - Used with mordant – causes the stain to resemble a
tissue limit has been removed basic dye
- If margin of surgical resection is free of diseased tissue - Hematoxylin does not dissociate from tissue when
- Done with other procedures: Endoscopy or thin-needle placed in water
biopsy - True basic dyes are not used in sequences because
Three main steps in frozen section preparation: they tend to dissociate
1. Freezing of the tissue sample – using compressed Metachromasia
Carbon dioxide or by immersion in a cold fluid Presence of polyanions within tissue underlie metachromasia
(isopentane) at -50oC Toluidine blue – dye molecules form dimeric and polymeric
2. Sectioning the frozen tissue – performed in a cryostat; aggregates
5-10um - Absorption properties of aggregation differ from
3. Staining the cut section – differentiate nuclei individual dye molecules
a. Methylene blue - Will appear PURPLE to RED
b. H&E Cells that exhibit metachromasia: Ionized sulfate and
c. PAS phosphate groups
CHEMICAL BASIS FOR STAINING - Ground substance of cartilage
Acid and Basic Dyes - Heparin-containing granules of mast cells
Acidic dye (Eosin) – net negative charge [Na+-dye-] - RER of plasma cells
- React with cationic groups Aldehyde Groups and Schiff Reagent
- Acidophilia – reaction of cationic groups to acidic dye Period acid-Schiff (PAS) reaction – stains carbohydrates and
- Electrostatic linkage – major factor in primary binding carbohydrate-rich molecules
but not the only one - Demonstrates: glycans
o used in combination to color different tissue o Glycogen
constituents o Mucus
o Mallory staining technique: o Basement membrane – proteoglycans
 Aniline blue - collagen o Reticular fibers – proteo
 Acidic fuchsin – cytoplasm and nuclei - Also used in Feulgen stain – stain DNA
 Orange G – red blood cells - Alternativee to silver-impregnation
- Selective staining attributable to: Based on following facts:
o size and degree of aggregation of the dye - Hexose rings contain adjacent carbons with OH group
o Permeability and compactness of tissue - Hexosamines of glycosaminoglycans bear OH groups and
- Substances stained: NH2 group
o Cytoplasmic filaments – esp. muscle cells - Periodc acid cleaves the bond and forms aldehyde groups
o Intracellular membranous components - Aldehyde groups react with Schiff reagent
o Most extracellular fibers (ionized amino groups) Feulgen based on cleavage of purine by mild acid hydrolysis
Basic dye – net positive charge [dye+-Cl-]  sugar ring opens with formation of aldehyde groups
- React with anionic components - React with chiff
o Phosphate groups of nucleic acids - MAGENTA color
(heterochromatin and nucleoli; ergastplasm; - Stoichiometric reaction – can be measured through
ribosomal RNA) spectrophotometry
o Sulfate groups of glycosaminoglycans - RNA does not stain because it does not have deoxyribose
(extracellular materials) Felugen microspectrophotometry – study DNA increases in
o Carboxyl groups of proteins developing cells and analyze ploidy
- Basophilia – ability of anionic groups to react with - Two techniques:
basic dye o Static cytometry – for tissue sections
- Reaction of anion groups vary with pH  560nm
o High pH – three groups are ionized and ready for  Uses microspectrophotometry
reaction o Flow cytometry – for isolated cells
o Slightly acidic to neutral pH (5-7) – sulfate and  Scan only single cells flowing past a sensor
phosphate groups - Used to quantify amount of nuclear DNA
o Low pH (below 4) – only sulfate groups Enzyme digestion
- Can also be used in combination but not widely used - PAS stained specimen identified as glycogen by adding
o Methyl green and pyronin to study protein AMYLASE OR DIASTASE
synthesis and secretion o Abolition of staining = glycogen
Hematoxylin – does not meet definition of a strict basic dye - Pretreatment with DNAse will abolish Feulgen staining
- RNAse – abolish staining of ergastoplasm with basic dyes

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ENZYME HISTOCHEMISTRY - Expensive, labor intensive, not really adapted to
- Mild aldehyde fixation – preferred method automated procedures
- Capture reagent – trap or bind the reaction product by IMMUNOPEROXIDASE METHOD – conjugate polychlonal or
precipitation monoclonal antibodies with enzymes
- Tissue section is placed in a solution containing substrate Colloidal gold or ferritin – attached to antibody molecule –
(AB) and trapping agent (T): electron dense; viewed in EM
AB + T (enzyme)  AT + B HYBDIRIZATION TECHNIQUES
 AT = trapped end product Hybridization – ability of single-stranded RNA or DNA to
- Has demonstrated: interact with complementary sequences
o Acid hydrolase and esterases in lysosomes - Requires isolation of DNA or RNA mixed with
o ALP NUCELOTIDE PROBE (complementary sequence)
o ATPases - Detected using radioactive labels
o Various esterases In situ hybridization – binding performed within cells or
o Many respiratory enzymes tissues (cultured cells/whole embryos)
- Horseradish peroxidase – most common - Localizes 10-20 copies of mRNA or DNA per cell
o Widely used substrate – 3,3’ – diaminobenzidine - Oligonucelotide probes – as small as 20-40 base pairs
o Labeled with and visualized by:
(DAB) – produces a brown insoluble product
 radioactive isotopes (32P, 35S, 3H) - autoradiography
IMMUNOCHEMISTRY  modified nucleotide (Digoxigenin) –
Antibodies/Immunoglobulins – produced by specific cells in Immunocytochemistry
response to foreign protein, Antigen  Biotin (commonly used) – cytochemistry
- Can be purified and conjugated to fluorescent dye Strongest bond formed = DNA probe and complementary
(fluorochromes) DNA
Fluorochromes – absorb light of different wavelengths then Weakest bond = RNA probe and complementary RNA strand
emit visible light POLYMERASE CHAIN REACTION (PCR) – amplification of DNA
- Fluorescein – most commonly used; GREEN LIGHT
REVERSE TRANSCRIPTASE-PCR (RT-PCR) – for RNA
Reactions can be examined and photographed with
Fluorescence in situ hybridization (FISH) – visualize probes
Fluorescence microscope or confocal microscope
using fluorescent dyes
TWO TYPES OF ANTIBODY USED:
- Now used in screening procedures for cervical cancer, or
1. Polyclonal Antibodies – mixture of different antibodies
detection of HIV infected cells
produced by many clones of B lymphocytes; each
- Examine chromosomes from lymphocytes of astronauts –
recognize different regions
estimate radiation dose by measuring frequency of
2. Monoclonal antibody – produced by antibody-
chromosome translocation
producing cell line;
a. Single group of identical B lymphocytes AUTORADIOGRAPHY – makes use of photographic
b. Obtained from an individual with MULTIPLE emulsion placed over a tissue section to localize radioactive
MYELOMA material
i. Produce identical, homogenous antibodies - Tagged by incorporating radioactive atoms
with an identical specificity against an - Radioactivity is then traced
antigen - Labeled precursor molecules can be injected
c. Mouse or rat is immunized  activated B - Slide is usually dipped in melted photographic emulsion
lymphocytes isolated and fused with myeloma o Produces thin photographic film
cell line  HYBRIDOMA o Emulsion developed by standard photographic
Direct immunofluorescence – oldest immunohistochemistry techniques
technique - Used to indicate location or counted to provide
- Uses fluorochrome-labeled PRIMARY ANTIBODY that semiquantitative information
reacts with antigen MICROSCOPY
- Visualization is not ideal – low intensity of signal LIGHT MICROSCOPY
emission Simplest microscope – magnifying glass or reading glasses
Indirect immynofluorescence Resolving power – ability of an optical system to produce
- Sandwich or Double-layer technique separate images of closely positioned objects
- Fluorochrome is conjugated with a SECONDARY - Determined by spacing of photoreceptors in the retina
ANTIBODY directed against primary antibody - Depends on:
- Enhances fluorescence signal o Wavelength of light source
- Single secondary antibody used to localize several o Specimen thickness
different primary antibodies o Quality of fixation
o Staining intensity
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0.2um – greatest attainable resolving power of light - Resembles spectrophotometers
microscope - Specimen cannot be inspected directly because UV is
Ocular or eyepiece magnifies but it cannot increase resolution injurious to the eye
Parts of a bright field microscope: - Useful for detecting:
 Light source – illumination o Purine and pyrimidine bases
 Condenser lens – focus beam of light o Proteins that contain certain amino acids
 Stage – in which slide is placed o Determine quantitavely DNA and RNA (Feulgen
 Objective lens – gather light microspectrophotometry)
 Ocular lens – through w/c the image is examined directly Confocal scanning microscope – allows visualization in three
Does not produce useful level of contrast in unstained dimensions
specimen - Two lenses (objective and phototube lens) – perfectly
aligned to focus light
EXAMINATION OF HISTOLOGIC SLIDE PREPARATION - Major difference from conventional microscope –
Artifacts – an error in preparation process; caused by: addition of DETECTOR APERTURE (pinhole)
- Inferior purity of chemicals and reagents o Conjugate with the focal point of the lens (confocal)
- Imperfections in execution o Allows only “in-focus” light to pass into
- Improper equipment photomultiplier (detector) device
OTHER OPTICAL SYSTEMS - 0.2-0.5um
Phase contrast microscope – takes advantage of small - Uses laser light system
differences in refractive index Polarizing Microscope – modification of light microscopy
- High refractive index – light is deflected; becomes out - polarizing filter (POLARIZER) is located between LIGHT
of phase SOURCE and SPECIMEN
- Dark portions correspond to dense portions - Second polarizer (ANALYZER) located between objective
- Light portions – less dense portions lens and the viewer
- Examine living cells and tissue - Difference between angles of polarizer and analyzer is
- Examine unstained semithin (0.5um) sections of used to determine degree by which a structure affects
plastic-embedded tissue the beam of polarized light
- Two modifications: - BIREFRINGENCE – ability of a crystal to rotate the plane
o INTERFERENCE MICROSCOPE – allows of polarized light
quantification o Striated muscle
o DIFFERENTIAL INTERFERENCE MICROSCOPE – o Leydig cells
NOMARSKI OPTICS; assessing surface properties ELECTRON MICROSCOPY
of cells Transmission EM - uses beam of electrons; principle is as
Dark-field microscopy – only light that has been scattered or follows
diffracted reaches the objective  Electron source (cathode, electron gun) emits electron
- Field of view = dark background  Electrons are attracted toward an ANODE
- Small particles that reflect some light = appear bright  An electrical difference b/n cathode and anode imparts
- Useful in examining: accelerating voltage creating the ELECTRON BEAM
o Autoradiographs – silver grains appear white  Beam passes through ELECTROMAGNETIC LENSES which
o Urine crystals (uric acid and oxalate) serve the same function as the glass lens of light
o Specific bacteria – spirochetes Treponema microscope
pallidum; syphilis Condenser lens – shapes and changes the diameter of
Fluorescence microscope – used to display naturally occurring electron beam
fluorescent molecules such as VITAMIN A AND Objective lens – focuses and magnifies beam
NEUROTRANSMITTERS Fluorescent screen/Photographic plate – where final image is
- Useful in studying: viewed
o Gap junctions Charge-coupled device (CCD) – placed above or below viewing
o Tracing pathway of nerve fibers screen to observe image in the real time
o Fluorescent growth markers in mineralized Routine preparation for TEM
tissues GLUTARALDEHYDE – preserves protein
- Uses a second filter between specimen and objective OSMIUM TETROXIDE – reacts with phospholipids; imparts
lens electron density to cell and tissue structures
Ultraviolet (UV) microscope – depends on absorption of UV Tissue pieces no more than 1mm3 are fixed
light by molecules in the specimen Expoxy resin – infiltrating agent
- 200nm wavelength SECTIONS are 50nm-150nm
- 0.1um resolution
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- Cantilever – where sharp tip is mounted; highly flexible

Diamond knives with nearly perfect cutting edge are used o Tip detects cantilever as it encounters the
Plastic-coated copper grids are used with 50-400 holes per ‘atomic force’
inch o Upper surface is reflective
Stained by adding materials of great density o Laser beam is deflected off the cantilever to a
- Bound during fixation or dehydration, or by soaking, or diode
after sectioning - Can work in two ways:
- Uranyl nitrate – added to alcohol solutions to increase o Contact mode
density of components of cell junctions o Tapping mode
- Sequential soaking of URANYL ACETATE and LEAD CITATE - Z-axis – movements are recorded on the diode
– routinely used to stain o A piezoelectric device activated to move the
EM autoradiography – have been refined for use with TEM specimen up and down so that the laser beam is
Fluorescent dye instead of heavy metal-containing centered on the diode
compounds can be used with TEM for immunocytochemical  Current through piezoelectrice device is
methods interpreted as z-axis
Freeze Fracture - Major advantage: Specimen does not have to be in
Cryoprotectant (glycerol) allowed to infiltrate tissue vacuum
 Tissue rapidly frozen to -160oC VIRTUAL MICROSCOPY
o Ice crystal formation is prevented - Integrates light microscopy with digital technologies
 Frozen tissue then placed in vacuum - Glass slides are scanned to create 2-D digital files
 Struck with knife edge or razor blade - Virtual slide – digital representation of a glass slide
Resulting fracture produces two new surfaces: - Allows for:
 E-face – backed by extracellular space o Remote viewing
 P – face – backed by protoplasm o Seamless progressive zooming in and out
Evaporated platinum is then used to coat specimen o Switching with ease b/n HPO and LPO
 Surface replica is picked up on grids to be examined o Orientation (navigation) thumbnail
SCANNING ELECTRON MICROSCOPE o Magnified glass thumbnail
- Portray surface appearance and has three-dimensional o Drag, rotate, measuring tools, color
appearance adjustment
- Sample is dehydrated by critical point drying coated - Also utilized in pathology education and pathology
with evaporated gold-carbon film mounted on practice (telepathology)
aluminum stub
Backscattered electrons – electrons reflected from the surface Some basic and acidic Dyes
Secondary electrons – electrons forced out of the surface Dye Color
Electrons (Backscattered and secondary) collected and Basic Dyes
reprocessed to form 3-D image Methyl Green Green
High resolution cathode ray tube (CRT) or photographic plate Methylene Blue Blue
– image capturing devices in earlier models Pyronin G Red
Sensitive detectors and CCD – image capturing devices in
Toluidine Blue Blue
modern instruments
Acidic Dyes
Other detectors can be used to measure:
Acid fuchsin Red
- Cathodoluminscence – tissue below surface
Aniline Blue Blue
- Auger electrons – emitted at surface
SCANNING TRANSMISSION EM (STEM) Eosin Red
SEM can produce transmission image by INSERTING GRID Orange G Orange
HOLDER
STEM facilitates use of instrument for electron probe x-ray
microanalysis
ATOMIC FORCE MICROSCOPY
- One of the most powerful tools
- Nonoptical microscope
o Works in the same way as a fingertip
- Ultra-sharp, pointed probe approaches the size of a
single atom at the tip
o Scans following parallel lines along x-axis
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