HISTOLOGY (HHIS 221)

LECTURE NO. 1: Histologic Tissue Preparation and Microscopy

Prepared by: Roiland A. Baybayon, RMT
Edited by: John Velasco, RMT, MD
School Year 2020-2021
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
DISCLOSURE STEPS IN TISSUE PREPARATION
This handout/review material must be treated with *See figure 1.1
utmost confidentiality. It shall be the responsibility of the
person, whose name appears therein, that the
1. FIXATION
handout/review materials are not photocopied or in
• PURPOSE: to preserve tissue structure and
away reproduced. Shared or lent to any person or
dispose in any manner. Any handout/review material in prevent degradation by enzymes released
the possession of another person whose name does not from the cells or microorganisms
appear in the class list enrolled in school year 2020-2021 • Usually done by a chemical or mixture of
shall be prima facie evidence of violation of RA 8293. chemicals, permanently preserving the
This material is not superior to the recommended book tissue structure for subsequent treatments
for this course. • SPECIMENS SHOULD BE IMMERSED IN
FIXATIVE IMMEDIATELY AFTER THEY ARE
TABLE OF CONTENTS REMOVED FROM THE BODY
Histology …………………………………………………. 1 • Fixation is used to:
Tissue Preparation ……………………………………… 1 ✓ Terminate cell metabolism
Chemical Composition of Histologic Samples …… 2 ✓ Prevent enzymatic degradation of
Chemical Basis of Staining …………………………… 2 cells and tissues by autolysis (self-
Microscopy ……………………………………………… 2-3 digestion)
✓ Kill pathogenic microorganisms
References: such as bacteria, fungi, and viruses
Junqueira’s Basic Histology (14th Edition) by Anthony L. ✓ Harden the tissue as a result of
Mescher (2016) either cross-linking or denaturing
protein molecules
• Uses FIXATIVES: must fully diffuse through the
tissues to preserve all cells, tissues are normally
HISTOLOGY cut into small fragments before fixation to
facilitate penetration.
• ORIGIN: Greek words
✓ HISTO: tissue/web
FORMALIN
✓ LOGOS: study
• Most widely used fixative is
• Scientific study of microscopic structures of
FORMALDEHYDE/FORMALIN
tissues and organs of the body
• STOCK SOLUTION: 37%
• Study of the tissues of the body and how these • MOST COMMON DILUTION: 10%
organs are arranged to constitute organs • Preserves the general structure of the cell and
TISSUES extracellular components by reacting with the
amino group of proteins (most often cross-
• Group of cells that are similar in structure and linked lysine residues)
perform a common function • Standard commercial solution of
• Have two interacting components: formaldehyde buffered with phosphates (pH:
✓ CELLS 7) acts relatively slowly but penetrates the
✓ EXTRACELLULAR MATRIX (ECM) tissue well
CELLS
2. DEHYDRATION
• BASIC STRUCTURAL AND FUNCTIONAL UNIT OF • Tissue is transferred through a series of
LIFE increasing/ascending concentrated alcohol
• Produce the ECM locally; strongly influenced solutions (up to 100%) which removes all water
by matrix molecules
3. CLEARING
EXTRACELLULAR MATRIX • Ethanol is replaced by an organic solvent
(xylene or toluene) miscible with both alcohol
• Consists of many kinds of macromolecules; and the paraffin before infiltration of the
most of which form complex structures such as specimen with melted paraffin.
collagen fibrils
• Supports the cells and the fluid transporting 4. INFILTRATION
nutrients to the cells, carrying away their • The fully cleared tissue is then placed in melted
paraffin in an oven at 52°C-60°C, which
wastes and secretory products
evaporates the clearing solvent
TISSUE PREPARATION
5. EMBEDDING
• The most common procedure used in • The paraffin-embedded tissue is placed in a
small mold with melted paraffin and allowed
histologic research is the preparation of tissue
to harden at room temperature
slices or sections that can be examined
visually with transmitted light 6. TRIMMING
• The ideal microscopic preparation is preserved • Removal of excess wax to expose the tissue for
so that the tissue on the slide has the same sectioning on a microtome
structural features it had in the body • SHAPE: TRUNCATED PYRAMID
• MOST COMMONLY STUDIED: HEMATOXYLIN
AND EOSIN-STAINED SECTION 7. SECTIONING
• The hardened block is placed for sectioning in
an instrument called microtome

1
 HISTOLOGY (HHIS 221)
LECTURE NO. 1: Histologic Tissue Preparation and Microscopy
Prepared by: Roiland A. Baybayon, RMT
Edited by: John Velasco, RMT, MD
School Year 2020-2021
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
• THICKNESS: 3-10um (light microscopy); less
than 1um (electron microscopy)
DYE EXAMPLE COLOR
8. STAINING BASIC METHYL GREEN GREEN
• Most cells and extracellular material are METHYLENE BLUE BLUE
completely colorless, and to be studied PYRONIN G RED
microscopically tissue sections must be stained TOLUIDINE BLUE BLUE
• NUCLEIC ACID: has a net negative charge ACIDIC ACID FUCHSIN RED
(anionic) have an affinity for basic dyes and ANILINE BLUE BLUE
are termed as BASOPHILIC EOSIN RED
• CYTOPLASM/ORGANELLES/PROTEINS: has a net ORANGE G ORANGE
positive charge (cationic) have an affinity for SUBSTANCES THAT DISPLAY BASOPHILIA
acidic dyes and are termed as ACIDOPHILIC
✓ HETEROCHROMATIN
✓ NUCLEOLI
✓ CYTOPLASMIC COMPONENTS
9. MOUNTING ✓ EXTRACELLULAR MATERIALS
• Stained tissue sections are mounted SUSBSTANCES THAT DISPLAY ACIDOPHILIA
permanently on a glass slide using a mounting
media ✓ CYTOPLASMIC FILAMENTS
✓ INTRACELLULAR MEMBRANOUS COMPONENTS
CHEMICAL COMPOSITION OF ✓ EXTRACELLULAR FIBERS
HISTOLOGIC SAMPLES
1. The chemical composition of a tissue ready for MICROSCOPY
routine staining differs from living tissue
• The components that remain after fixation • The aid in the study of the human body
consist mostly of large molecules that do not structure by the use of a magnifying instrument
readily dissolve, especially after treatment with called a microscope
the fixative
2. Many tissue components are lost during the routine MICROSCOPE
preparation of H&E-stained sections *see figure 2
• Small proteins and small nucleic acids are • an instrument that magnifies an image and
generally lost during the preparation of the allows visualization of greater detail than is
tissue. possible with the unaided eye
• Examples of large molecules lost during routine
fixation are: RESOLVING POWER is the ability of a microscope
✓ GLYCOGEN lens or optical system to produce separate images
of closely positioned objects
✓ PROTEOGLYCANS
✓ GLYCOSAMINOGLYCANS
3. Soluble components, ions, and small molecules are RESOLUTION depends not only on the optical
also lost during the preparation of paraffin sections system but also on the wavelength of the light
• Intermediary metabolites, glucose, sodium, source and other factors such as specimen
chloride, and similar substances are lost thickness, quality of fixation, and staining intensity.
during preparation of routine H&E paraffin
sections
COMMONLY USED LINEAR EQUIVALENTS
UNIT SYMBOL/DEFINITION
CHEMICAL BASIS OF STAINING 1 picometer 0.01 angstrom
1 angstrom 0.1 nanometer
1.ACIDIC DYE 10 angstroms 1.0 nanometer
Carries a net negative charge
• 1 nanometer 1,000 picometers
React with cationic groups in cells and tissue
• 1,000 nanometers 1.0 micrometer
The reaction of cationic groups with an
• 1,000 micrometers 1.0 millimeter
acidic dye is called acidophilia (acid-loving)
2. BASIC DYE
• Carries a net positive charge 1. BRIGHT FIELD MICROSCOPE
• React with anionic components of the cells • Stained tissue is examined with ordinary light
and tissue passing through the preparation
• The ability of such anionic groups to react • Essentially consists of:
with a basic dye is called basophilia (base- ✓ LIGHT SOURCE: for illumination of
loving) specimen
ANIONIC ✓ Phosphate groups ✓ CONDENSER: focus the beam of light
COMPONENTS of nucleic acids at the level of the specimen
✓ Sulfate groups of ✓ STAGE: on which the slide or other
glycosaminoglycans specimen is placed
✓ Carboxyl group of ✓ OBJECTIVE LENS: gather the light that
proteins has passed through the specimen
CATIONIC • Proteins and other ✓ OCULAR LENS: through which the
COMPONENTS components in the
image formed by the objective lens
cytoplasm
may be examined directly

2
 HISTOLOGY (HHIS 221)
LECTURE NO. 1: Histologic Tissue Preparation and Microscopy
Prepared by: Roiland A. Baybayon, RMT
Edited by: John Velasco, RMT, MD
School Year 2020-2021
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
• A specimen to be examined with the bright- • Allows visualization of a biologic specimen in
field microscope must be sufficiently thin for three dimensions
light to pass through it • The two lenses in the confocal microscope
• Does not produce a useful level of contrast in (objective and phototube lens) are perfectly
an unstained specimen aligned to focus light from the focal point of
one lens to the focal point of the other lens
2. PHASE-CONTRAST MICROSCOPY • Combines components of light optical
• Uses a lens system that produces visible microscope with a scanning system to
images from transparent objects and can be dissect a specimen optically
used with living, cultured cells • Achieves high resolution and sharp focus by
• Based on the principle that light changes its using:
speed when passing through cellular and ✓ Small point of high-intensity light
extracellular structures with different refractive (LASER)
indices ✓ Plate with a pinhole aperture in front
• Used to examine living cells and tissues and is of the image detector
used extensively to examine unstained ✓
semithin (approximately 0.5um) sections of 6. POLARIZING MICROSCOPE
plastic-embedded tissue • Simple modification of the light microscope
• 2 MODIFICATIONS: in which a polarizing filter (polarizer) is
✓ INTERFERENCE MICROSCOPE locate between the light source and the
- Allows quantification of tissue mass specimen, and a second polarizer
✓ DIFFERENTIAL INTERFERENCE MICROSCOPE (analyzer) is located between the objective
- Uses Nomarski optics, which lens and the viewer
produces an image of living cells • Allows the recognition of stained or
with a more apparent 3D aspect, unstained structures made of highly
which is especially useful for organized subunits such as birefringent
assessing surface properties of crystals
cells and other biologic objects
7. ELECTRON MICROSCOPE
• Primary improvement versus the light
3. DARK FIELD MICROSCOPE microscope is that the wavelength of the EM
• Only light that has been scattered or beam is approximately 1/2000 that of the
diffracted by structures in the specimen light microscope beam, thereby increasing
reaches the objective resolution by a factor of 103
• Equipped with a special condenser that
illuminates the specimen with strong, oblique a. TRANSMISSION ELECTRON MICROSCOPE (TEM)
light • Imaging system that permits resolution
• Field of view appears as a dark background around 3nm; allowing isolated particles
on which small particles in the specimen that magnified as much as 400,000 times
reflect some light into the objective appear • a beam of electrons focused using
bright electromagnetic lenses passes through the
• Useful in examining urine for crystals (uric acid tissue section to produce an image with
and oxalate), and in demonstrating specific black, white, and intermediate shades of
bacteria such as spirochetes gray regions.
• Uses a beam of electrons rather than a
4. FLUORESENCE MICROSCOPE beam of light
• Tissue sections are usually irradiated with • Specimens for TEM begins with fixation using
ultraviolet (UV) light and the emission is in the glutaraldehyde
visible portion of the spectrum • Cryofracture and freeze etching are
• The fluorescent substances appear bright on techniques that allows TEM study of cells
a dark background without fixation or embedding; particularly
• Fluorescent compounds with affinity for useful in the study of membrane structures
specific cell macromolecules may be used • The principle of the microscope is as follows:
as fluorescent stains (ex. Acridine orange) ✓ An electron source (cathode,
• Makes use of the ability of certain molecules electron gun), such as a heated
to fluoresce under ultraviolet light tungsten filament, emits electrons
• Uses quartz lenses with an ultraviolet light ✓ The electrons are attracted toward
source an anode
• Resembles the workings of a ✓ The beam the passes through a
spectrophotometer series of electromagnetic lenses that
• The specimen cannot be inspected directly serve the same function as the glass
through an ocular lens because UV light is lenses of a light microscope
not visible and is injurious to the eye
• UV microscopy is useful in detecting nucleic
acids (purine and pyrimidine bases); also for b. SCANNING ELECTRON MICROSCOPE (SEM)
proteins that contain certain amino acids • Provides a high-resolution view of the
surfaces of the cells, tissues, and organs
• Produces and focuses a very narrow beam
5. CONFOCAL MICROSCOPE of electrons

3
 HISTOLOGY (HHIS 221)
LECTURE NO. 1: Histologic Tissue Preparation and Microscopy
Prepared by: Roiland A. Baybayon, RMT
Edited by: John Velasco, RMT, MD
School Year 2020-2021
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
• Surface of the specimen is first dried and
spray-coated with a very thin layer of heavy
metal (often gold) which reflects electrons 9. VIRTUAL MICROSCOPY
and produces a black-and-white image • It is the digital procedure that is an
• Easy to interpret since they present a three- alternative to the examination of glass slides
dimensional image using a light microscope
• Integrates conventional light microscopy
8. ATOMIC FORCE MICROSCOPE with digital technologies
• Most useful for biological studies
• Non-optical microscope
• One of the most powerful tools for studying
the surface topography at molecular and
atomic resolution

FIGURE 1.1 SECTIONING FIXED AND EMBEDDED TISSUE (HTTPS://MEDICALLABSCIENTIST.ORG/TISSUE-PROCESSING-FACTORS-STEPS-OF-TISSUE-PROCESSING-TYPES/)

FIGURE 2 PARTS OF COMPOUND MICROSCOPE (HTTPS://MICROBENOTES.COM/COMPOUND-MICROSCOPE-PRINCIPLE-INSTRUMENTATION-AND-APPLICATIONS/)