HUMAN HISTOLOGY

BSMLS 2- 2ND SEM - PRELIMS

INTRODUCTION

Histology Physical and chemical properties of individual cells within a

heterogeneous population can be analyzed, measured, and separated
- is the study of tissues. Tissues are usually described as a into subpopulation through its fluorescence and light scatter properties
network of both cellular and noncellular materials, by means of flow cytometry.
intermingled filaments and fibers provided with membranous
linings. Techniques such as cell cloning, protein sequencing, and other
advancements in molecular genetics have substantially revolutionized
Human Histology our understanding of the cell.
- is defined as the study of the microscopic structures of
Visualization and interpretation of microscopic sections derived from
biological materials derived from humans and the means by
cells and tissues have improved with the introduction of enzyme
which their individual components are structurally and
(special stains) and immunohistochemistry. These methods allow
functionally related. In fact, the science of histology stands at
localization of specific protein and reveal subcellular components at the
the crossroad, critically linking the disciplines of
light microscopic level.
biochemistry, molecular biology, physiology, gross anatomy,
and embryology on one side, and the disease processes
Fundamental insights into the molecular mechanisms of cells are now
(pathology) and their effects on the other.
possible through in situ hybridization technique that demonstrates
specific DNA or mRNA sequences as well as localization of genes
Collection of Specimen for Histologic Study within a specific chromosome or messenger RNA.

Histologic examination requires obtaining small samples of cells and

Light or electron microscopic observation of cellular processes such as
tissues from different areas of the body. Samples of human biologic
tissue growth and the pathways involved in macromolecular synthesis
materials can be collected by means of quick, safe techniques using
are some of the dynamic activities which can be analyzed using
instruments such as:
radioactive precursors through the technique referred to as
autoradiography.
A. scalpels used directly on accessible tissues such as the
skin, mouth, nose, and other similar specimens;
B. needles which are used to gently pierce into solid organs Basic Histopathologic Techniques for the Preparation of
such as the brain, lymph nodes, breast, liver, kidney, bone
and bone marrow, eye, skeletal muscle, and other similar
Specimen Glass Slides for Histologic Examination
tissues; 1. Fixation:
C. endoscopic tubes which are inserted into the alimentary - In order to study tissues with a microscope they
tract; or must be first preserved (fixation). This is the
D. special flexible cannula which are passed along blood MOST important step in the preparation of tissue
vessels. specimens for histopathologic techniques. In
principle, this consists basically of a chemical or
Microscopic examination of cells and tissues is an increasingly physical method of killing the tissue and
important and direct way of diagnosing diseases. In instances where a inactivating its enzymatic actions and yet retaining
blood test or radiology fails to indicate the exact etiology of the its characteristic peculiarities of shape, structure,
disease, pieces of biologic specimens may be obtained and diagnosis and morphologic features. The prosector (more
through histologic examination of such specimens can be done for an often the pathologist who performs the dissection)
accurate basis of diagnosis to ensure proper clinical management of selects the appropriate sample and slices a thin
the patients' condition. representative section of the specimen to be
submitted for processing.
2. Dehydration:
Histologic Techniques - After a fixation time of approximately 24 hours on
The development of a wide range of investigative methods have average for most specimens, the tissue is
considerably broadened our knowledge of the structure and function of transferred into ascending grades of ethanol or
cells and tissues as well as their inter. relationship. isopropyl alcohol in order to remove excess
intracellular and extracellular water. This step
Valuable information on cell behavior is obtained through in vitro starts with a 70% alcohol serially increasing in
culture systems. concentration to the absolute concentration of
100% pure alcohol in order to be miscible with the
The technique of cell fractionation not only gives us the relative weight next chemical step, which is clearing
of cell constituents but has also been useful in the study of individual
organelles.
HUMAN HISTOLOGY
BSMLS 2- 2ND SEM - PRELIMS

INTRODUCTION

3. Clearing: certain tissue components or organelles. The

- This step involves removal of the dehydrating stained section is again dehydrated in ascending
agent and its replacement with a hydrocarbon grades of alcohol up to the absolute concentration,
chemical (e.g., xylene, toluene, benzene) which is then cleared with xylene to retain its translucency.
miscible with the alcohol and embedding medium 8. Mounting and Labeling:
such as paraffin wax. - The stained section mounted on a glass slide is
4. Wax Impregnation or Infiltration: covered with a glass coverslip and cemented with
- It is the process by which a tissue is permeated a resinous mounting medium. This syrupy liquid
with an embedding medium, usually liquid paraffin, will harden after some time and maintains the
at its given melting point temperature of 56-58 refractive index similar to that of glass in order to
degrees centigrade. minimize optical distortion for a crisp, clear view
5. Wax or Tissue Embedding: during microscopic examination. The slide is finally
- The tissue impregnated with wax is surrounded labeled for identification and permanent archival
further in liquid paraffin wax in a mold made of purposes.
paper, plastic, or metal. The wax, when cooled,
solidifies to provide sufficient external support in
the form of a paraffin block. This will be REVIEW OF MICROSCOPY
subsequently subjected to sectioning using a The thorough examination of the small structural details comprising
microtome cutting instrument. each biologic sample is facilitated by observing the specimen under the
6. Sectioning: microscope Thus, microscopy is the starting point of any discussion
- The paraffin block is sliced into micro-thin sections related to microscopic anatomy. The birth of the science of cell biology
(or serial sections called ribbons), on an average commences with the discovery of the light microscope (LM).
of about 4-6 microns in thickness, using steel
knives mounted in a precision cutting instrument
called a microtome. The sections or ribbons are Microscopy
prepared in a technique in order to mount this with - basically operates on the principle that any given type of
the correct orientation on a glass slide. For radiation cannot be used to probe on the structural details of
electron microscopy, the sections are considerably an object with a size smaller than its wavelength. An
less than one ten-thousandth of a millimeter (0.1 essential part of this study is the microscope-an equipment
micron, um) thick. This is accomplished by used in the laboratory to magnify and examine structural
embedding the tissue in a plastic resin such as details of specimens that are too small to be visualized by
epon or araldite (epoxy resins) and sectioning it on the naked eye. This is the common instrument utilized in
special ultramicrotomes equipped with a fine order to observe histologic sections.
mechanical or thermal advance. Sections are cut
with glass or diamond knives and mounted on There are two fundamental types of microscopes:
copper mesh grids.
● light microscope and
7. Staining:
● electron microscope.
- The sections are deparaffinized, rehydrated (using
descending grades of alcohol), and routinely
stained with hematoxylin and eosin (often referred
LIGHT MICROSCOPY
to as H&E) stain, which is a form of "differential
staining" used in the study of basic
Histology.Hematoxylin is a basic natural dye that - For light microscopy, the range of wavelength for visible light
stains the nucleus in shades of blue to blue-violet is 0.4 pm - 0.7 pm (400 nm - 700 m). Thus, any object with a
to black (hence "basophilic"), while Eosin stain is size smaller than 0.4 pm would demonstrate an obscure
an acid dye that has an affinity for staining the image when viewed under a light microscope.The classic
cytoplasm in shades of pink to orange to red compound light microscope, as it is known, utilizes more
(hence "eosinophilic" or "orangeophilic"). In than just a single lens. Its light source is an electric bulb with
working with light microscopic examination of tungsten filament, and light rays are gathered into a focused
tissue sections, it is difficult to recognize the beam by means of a condenser lens. The quality of an
various components of cells and tissues without image does not solely depend on the capability of the lens to
differential staining. The stains may react magnify the image but also on its resolution. The quality of a
chemically or physically and a wide variation of lens depends on how close its resolution approaches the
color is possible. The staining method can be theoretical limit of 025 pm, a restriction that is determined by
altered using other "special stains" to suit the the wavelength of visible light.
needs of the histologist in order to accentuate
HUMAN HISTOLOGY
BSMLS 2- 2ND SEM - PRELIMS

INTRODUCTION

Light microscope can be made to perform special functions by using 4. Differential Interference Contrast (DIC) Microscope
different light sources, by modifying its condenser, or by adding special - is also branded as Nomarski Interference Contrast (NIC)
types of prism or file such as in a phase contrast microscope, microscope. Similar to a phase contrast microscope, DIC is
interference microscope, or fluorescence microscope. However, the also used to provide contrast in unstained and transparent
details that can be resolved using these types of microscope remain samples. The image formed in DIC is nearly
limited. three-dimensional (3D) shadowed under very oblique
illumination. DIC works using two coherent beams of light
Types of Light Microscope coming from the same light source and prism.

1. Bright-field Microscope
- is the most commonly used type of microscope by students
of histology. The source of illumination is a beam of tungsten
light that is transmitted to the sample after it passes through
a condenser. In this type of microscope, the image appears
dark against a bright contrast/background.
2. Dark-field Microscope
- is simple yet very effective and appropriately used in
observing live and unstained biological samples. It utilizes an
opaque disc which is located underneath the condenser. The 5. Fluorescence Microscope
specimen appears brightly lit against a dark background. - This type of an optical microscope uses fluorophore
Treponema pallidum, the causative agent of syphilis, can be (fluorescent dyes) to generate fluorescence in a biological
identified using dark- field microscopy. sample. Fluorescence is a type of short-lived luminescence,
which is produced when light is emitted by a material that
has absorbed excitation light or electromagnetic radiation.
The components of a fluorescence microscope are xenon
arc or mercury vapor lamps, excitation filter, dichroic mirror,
and emission filter.

3. Phase Contrast Microscope

- is the type of light microscope preferred when the specimen
that will be observed is translucent and colorless. The
modification in the design of the phase contrast microscope
includes an annular ring positioned in the substage
condenser front focal plane. The light source is a 6. Confocal Microscope
tungsten-halogen lamp. In a phase contrast microscope, the - Similar to the fluorescence microscope, the confocal
differences in the thickness and refractive index of the microscope also uses fluorophore dyes to visualize the
different components of the specimen can be converted into sample. A typical fluorescence microscope is wide field. In a
differences in brightness or contrast developed in the image. confocal microscope, pinhole is provided (inside the optical
As a result, more detailed intracellular parts of living cells pathway) to focus the light onto a definite spot in the sample
can be observed with relatively high resolution. Phase and rejecting out-of-focus fluorescent glare.
contrast microscopy was conventionally the first accurate
method used in manual counting of platelets.
HUMAN HISTOLOGY
BSMLS 2- 2ND SEM - PRELIMS

INTRODUCTION

specimen, thereby producing a highly magnified image. SEM images of

ELECTRON MICROSCOPY surfaces appear to be three-dimensional (3D); however, there is no
The ultrastructural intracellular details of individual cells can be more measurable depth information provided.
defined using the electron microscope. Electron Microscopy is a
system analogous to that of visible light microscope but its resolving
power and magnification is higher. It has been a valuable instrument
for modern morphological research. An electron microscope utilizes
electron beams instead of light rays and magnetic fields instead of
lenses.

There are two imaging systems-the transmission electron

microscope (TEM) and the scanning electron microscope (SEM).
1. Transmission Electron Microscope (TEM)
- The transmission electron microscope makes use of a beam
of electrons that is transmitted through a specimen or in the
case of histology, through very thin slices of tissue to
produce a two-dimensional image of the specimen. The
specimen is most often an ultrathin section less than 100-nm
thick.

TEM has the following essential systems:

A. an electron gun and the condenser, which produces the
electron beam, and which focuses the beam onto the object,
respectively;
B. the image-producing system, which consists of the objective
lens, movable specimen stage, and intermediate and
projector lenses, which focus the electrons passing through
the specimen to form a real, highly magnified image; and
C. the image-recording system, which converts the electron
image into some form perceptible to the human eye. It
consists of a fluorescent screen for viewing and focusing the
image and a digital camera to produce permanent records of
the images. TEM provides a higher resolution of the image
as compared to SEM.

2. Scanning Electron Microscope (SEM)

- The scanning electron microscope (SEM) is designed for
directly studying surfaces of solid objects. SEM utilizes a
beam of electrons to scan the specimen.
- In SEM, the narrow beams of electrons are scattered or
emitted from the surface of the specimen thus producing a
three-dimensional character of the surface.

The essential parts of a scanning electron microscope include the

following:
a. source of electrons
b. column down which electrons travel with electromagnetic
lenses
c. electron detector
d. sample chamber
e. computer and display to view the images

The electrons are produced at the top of the column, accelerated

down, and passed through a combination of lenses and apertures to
produce a focused beam of electrons, which hits the surface of the