Histology & its methods of study

1. Histology &
Its Methods of Study
Department Of General Histology

2. Recommended Reading:
 Benefits of General and Clinical
Pathology Department
 Student Workbook
 Ross – “Histology”
 Holes “Human Anatomy and
Physiology”
 Any book on the physiology and
histology with a note: For Medical
Students.

3. Introduction
 Histology is the study of the tissues of
the body and how these tissues are
arranged to constitute organs. The
Greek root histo can be translated as
either "tissue" or "web" and both
translations are appropriate because
most tissues are webs of interwoven
filaments and fibers, both cellular and
noncellular, with membranous linings.
Histology involves all aspects of tissue
biology, with the focus on how cells'
structure and arrangement optimize
functions specific to each organ.

4.  Tissues are made of two interacting components:
cells and extracellular matrix. The extracellular
matrix consists of many kinds of molecules, most
of which are highly organized and form complex
structures, such as collagen fibrils and basement
membranes. The main functions once attributed
to the extracellular matrix were to furnish
mechanical support for the cells, to transport
nutrients to the cells, and to carry away
catabolites and secretory products. We now know
that, although the cells produce the extracellular
matrix, they are also influenced and sometimes
controlled by molecules of the matrix. There is,
thus, an intense interaction between cells and
matrix, with many components of the matrix
recognized by and attaching to receptors present
on cell surfaces. Most of these receptors are
molecules that cross the cell membranes and
connect to structural components of the
intracellular cytoplasm. Thus, cells and

5. Sponsored
Medical Lecture Notes – All Subjects
USMLE Exam (America) – Practice

6.  Each of the fundamental tissues is formed
by several types of cells and typically by
specific associations of cells and
extracellular matrix. These characteristic
associations facilitate the recognition of
the many subtypes of tissues by students.
Most organs are formed by an orderly
combination of several tissues, except the
central nervous system, which is formed
almost solely by nervous tissue. The
precise combination of these tissues
allows the functioning of each organ and
of the organism as a whole.

7.  The small size of cells and matrix
components makes histology dependent
on the use of microscopes. Advances in
chemistry, molecular biology, physiology,
immunology, and pathology—and the
interactions among these fields—are
essential for a better knowledge of
tissue biology. Familiarity with the tools
and methods of any branch of science is
essential for a proper understanding of
the subject. This chapter reviews
several of the more common methods
used to study cells and tissues and the
principles involved in these methods.

8. Overview of methods used
Histology
 The objective of a histology course is
to lead the student to understand the
microanatomy of cells, tissues and
organs and correlate structure with
function.
 Auxillary techniques include^
Histochemistry and biochemistry
Autoradiography
Organ and tissue culture

9.  Ross - Histology

10. Types of the Microscope

11. Components and light path of
a bright-field microscope

12. Fluorescent microscopy
Kidney cells stained with acridine
orange, which binds nucleic acids.
Under a fluorescence microscope,
nuclear DNA emits yellow light and the
RNA-rich cytoplasm appears reddish or
orange
The less dense culture of kidney
cells stained with DAPI (4',6-
diamino-2-phenylindole) which
binds DNA, and with phalloidin,
which binds actin filaments.

13. Phase-contrast & Bright-field
microscopy
Bright-field microscopy: without
fixation and staining, only the two
pigment cells can be seen.
Phase-contrast microscopy: cell
boundaries, nuclei, and cytoplasmic
structures with different refractive indices
affect in-phase light differently and
produce an image of these features in all
the cells.

14. Differential interference
microscopy
Cellular details are highlighted in a
different manner using Nomarski optics.
Phase-contrast microscopy, with or
without differential interference, is
widely used to observe live cells grown
in tissue culture.

15. Principle of confocal
microscopy
Although a very small spot of light
originating from one plane of the section
crosses the pinhole and reaches the
detector, rays originating from other
planes are blocked by the blind. Thus,
only one very thin plane of the specimen
is focused at a time. The diagram shows
the practical arrangement of a confocal
microscope. Light from a laser source
hits the specimen and is reflected. A
beam splitter directs the reflected light
to a pinhole and a detector. Light from
components of the specimen that are
above or below the focused plane is
blocked by the blind. The laser scans the
specimen so that a larger area of the
specimen can be observed.

16. Tissue appearance with bright-
field and polarizing
microscopy.
Under routine bright-field microscopy
collagen fibers appear red, along with thin
dark elastic fibers and cell nuclei.
Under polarizing light microscopy, only
collagen fibers are visible and these exhibit
intense birefringence and appear bright red
or yellow; elastic fibers and nuclei lack
oriented macromolecular structure and are
not visible.

17. Transmission Electron
Microscopy
 Schematic view of a transmission electron
microscope (TEM) with its lenses and the
pathway of the electrons. With the
microscope's entire column in a vacuum,
electrons are released by heating a very thin
metallic (usually tungsten) filament (cathode).
The released electrons are then submitted to a
voltage difference of 60–120 kV between the
cathode and the anode, which is a metallic
plate with a hole in its center. Electrons are
thus attracted to the anode, accelerated to
high speeds, and form a beam of electrons as
they pass through the central opening in the
anode. Passing through electric coils the beam
is deflected in a way roughly analogous to the
effect of optical lenses on light because
electrons change their path when submitted to
electromagnetic fields.
 The configuration of the TEM is similar to that
of an upside-down light microscope. The first
lens is a condenser that focuses the beam of
electrons on the section. Some electrons
interact with atoms of the section and continue
their course, while others simply cross the
specimen without interacting. Most electrons
reach the objective lens, which forms a
magnified image that is then projected through
other magnifying lenses. Because the human
eye is not sensitive to electrons, the image is
finally projected on a fluorescent screen or is
registered by photographic plates or a CCD
camera.

18. Schematic view of a scanning electron
microscope (SEM) with many similarities to
a TEM. However, here the electron beam
focused by electromagnetic lenses does
not pass through the specimen, but rather
is moved sequentially (scanned) from point
to point across its surface similar to the
way an electron beam is scanned across a
television tube. The specimen was coated
previously with a very thin coating of metal
atoms and the beam interacts with these
atoms, and produces reflected electrons
and newly emitted secondary electrons. All
of these are captured by a detector and
transmitted to amplifiers and other devices
which produce a signal to a cathode ray
tube monitor, resulting in a black-and-white
image. The SEM shows only surface views
of the coated specimen but with a striking
three-dimensional quality. The inside of
organs or cells can be analyzed by
sectioning them to expose their internal
surfaces.

19. Preparation of Tissues for
Study

20. Hematoxylin & Eosin (H&E) and Periodic
acid-Schiff (PAS) staining.
Micrograph stained with hematoxylin and
eosin (H&E)
Micrograph stained by the periodic
acid-Schiff (PAS) reaction for
glycoproteins. With H&E, basophilic
cell nuclei are stained purple while
cytoplasm stains pink

24. Autoradiography.
Autoradiographs are tissue preparations in which particles called
silver grains indicate the regions of cells in which specific
macromolecules were synthesized just prior to fixation. Precursors
such as nucleotides, amino acids, or sugars with isotopes substituted
for specific atoms are provided to the tissues and after a period of
incorporation, tissues are fixed, sectioned, and mounted on slides or
TEM grids as usual.

27. Detection Methods Using
Specific Interactions between
Molecules

28. Labeling by specific, high-
affinity interactions.
Compounds or macromolecules that have
specific affinity toward certain cell or tissue
macromolecules can be tagged with a label and
used to identify that component and determine
its location in cells and tissues. (1) Molecule A
has a high and specific affinity toward a portion
of molecule B. Examples of such interacting
macromolecules are an antibody that recognizes
specific antigens, usually proteins, or a segment
of single-stranded DNA with sequence-specific
complementarity to RNA molecules in a cell.
Molecule A can also be a small compound like
phalloidin, which specifically binds actin
filaments, or a protein such as "protein A" which
binds all immunoglobulins. (2) When A and B are
mixed, A binds to the portion of B it recognizes.
(3) Molecule A may be tagged with a label that
can be visualized with a light or electron
microscope. The label can be a fluorescent
compound, an enzyme such as peroxidase, an
electron-dense particle, or a radioisotope. (4) If
molecule B is present in a cell or extracellular
matrix that is incubated with labeled molecule A,
molecule B can be detected and localized by
visualizing the labeled molecule A bound to it.

29. Immunocytochemistry.
Direct immunocytochemistry uses an antibody made against the tissue protein of interest and tagged
directly with a label such as a fluorescent compound or peroxidase. When placed with the tissue
section on a slide, these labeled antibodies bind specifically to the protein (antigen) against which they
were produced and can be visualized by the appropriate method. The more widely used technique of
indirect immunocytochemistry uses two different antibodies. A primary antibody is made against the
protein (antigen) of interest and applied to the tissue section first to bind its specific antigen. Then a
labeled secondary antibody is obtained that was (1) made in another vertebrate species against
immunoglobulin proteins (antibodies) from the species in which the primary antibodies were made and
then (2) labeled with a fluorescent compound or peroxidase. When this labeled secondary antibody is
applied to the tissue section it specifically binds the primary antibodies, indirectly labeling the protein
of interest on the slide. Since more than one labeled secondary antibody can bind each primary
antibody molecule, labeling of the protein of interest is amplified by the indirect method.

30. Cells and tissues stained by
immunohistochemistry
A decidual cell grown in vitro stained to reveal a mesh of intermediate
filaments throughout the cytoplasm. Primary antibodies against the protein
desmin, which forms these intermediate filaments, and FITC-labeled
secondary antibodies were used in an indirect immunofluorescence
technique. The nucleus is counterstained light blue with DAPI.

31. A section of small intestine stained with an antibody against the enzyme
lysozyme. The secondary antibody labeled with peroxidase was then
applied and the localized brown color produced histochemically with the
peroxidase substrate DAB. The method demonstrates lysozyme-
containing structures in scattered macrophages and in the clustered
Paneth cells. Nuclei were counterstained with hematoxylin.

32. A section of pancreatic acinar cells in a TEM preparation incubated with an
antibody against the enzyme amylase antibody and then with protein A
coupled with gold particles. Protein A has high affinity toward antibody
molecules and the resulting image reveals the presence of amylase with
the gold particles localized as very small black dots over dense secretory
granules and developing granules (left).

33. Enzyme histochemistry. Part 1
Micrograph of cross sections of
kidney tubules treated
histochemically by the Gomori
method for alkaline phosphatases
show strong activity of this enzyme
at the apical surfaces of the cells at
the lumen of the tubules (arrows).

34. Enzyme histochemistry. Part 2
TEM image of a kidney cell in which acid phosphatase has been localized
histochemically in three lysosomes (Ly) near the nucleus (N). The dark
material within these structures is lead phosphate that precipitated in places
with acid phosphatase activity. X25,000. (Figure 1–10b, with permission, from
Eduardo Katchburian, Department of Morphology, Federal University of Sao
Paulo, Brazil.)

36. Cells stained by in situ
hybridization.
In situ hybridization shows that
many of the epithelial cells in this
section of a genital wart contain the
human papillomavirus (HPV),
which causes this benign
proliferative condition. The section
was incubated with a solution
containing a digoxygenin-labeled
cDNA probe for the HPV DNA. The
probe was then visualized by direct
immunohistochemistry using
peroxidase-labeled antibodies
against digoxygenin.

38. Interpretation of 3-D structures
in 2-D tissue sections.
Sections through a hollow swelling on a
tube produce large and small circles,
oblique sections through bent regions of
the tube produce ovals of various
dimensions.

39. A single section through a highly coiled
tube shows many small, separate round or
oval sections. On first observation it may
be difficult to realize that these represent a
coiled tube, but it is important to develop
such interpretive skill in understanding
histological preparations.

40. Round structures in sections may be
portions of either spheres or cylinders.
Additional sections or the appearance of
similar nearby structures help reveal a
more complete picture.

41. Light Microscopy Stains

42. Hematoxylin and Eosin (H&E)
 Hematoxylin stains cellular regions rich in
basophilic macromolecules (DNA or RNA) a
purplish blue or blue-black color. It is the
most common stain for demonstrating cell
nuclei and cytoplasm rich in rough ER.
Usually used as the contrasting
"counterstain" with hematoxylin, eosin is an
acidic stain that binds to basic
macromolecules such as collagen and most
cytoplasmic proteins, especially those of
mitochondria. Eosin stains regions rich in
such structures a pinkish red color. Tissue
sections showing only structures with shades
of purple and pink are stained with H&E.

43. Pararosaniline-Toluidine Blue
(PT)
 This dye combination stains chromatin
shades of purple and cytoplasm and
collagen a lighter violet. These stains
penetrate plastic sections more readily
than H&E and are used here primarily
with acrylic resin-embedded sections to
provide better detail of cell and tissue
structures. Toluidine blue is also
commonly used for differential staining of
cellular components, particularly
cytoplasmic granules.

44. Mallory Trichrome
 This procedure employs a combination
of stains applied in series which results
in nuclei staining purple; cytoplasm,
keratin, and erythrocytes staining bright
red or orange; and collagen bright or
light blue. Mallory trichrome is
particularly useful in demonstrating cells
and small blood vessels of connective
tissue. Similar stains, such as Masson
trichrome and Gomori trichrome, yield
comparable results except that collagen
stains blue-green or green.

45. Picro-Sirius-Hematoxylin
(PSH)
 The dye Sirius red in a solution of
picric acid stains collagen red and
cytoplasm a lighter violet or pink, with
nuclei purple if first stained with
hematoxylin. Under the polarizing
microscope, collagen stained with
picro-sirius red is birefringent and can
be detected specifically.

46. Periodic Acid–Schiff Reaction
(PAS)
 This histochemical procedure stains
complex carbohydrate-containing cell
components, which become magenta
(shades of purplish pink). PAS is
commonly used to demonstrate cells
filled with mucin granules, glycogen
deposits, or the glycocalyx.

47. Wright-Giemsa Stain
 These are two similar combinations of
stains that are widely used on fixed
cells of blood or bone marrow smears
to demonstrate types of blood cells.
Granules in leukocytes are seen to
have differential affinity for the stain
components. Nuclei stain purple and
erythrocytes stain uniformly pink or
pinkish orange.

48. Silver or Gold Stains
 Various procedures employing
solutions of silver or gold salts have
been developed to demonstrate
filamentous structures in neurons and
fibers of reticulin (type III collagen). By
these "metal impregnation" techniques
these filaments stain dark brown or
black. Such stains have been largely
replaced now by
immunohistochemical procedures.

49. Stains for Elastin
 Several staining methods have been
developed to distinguish elastic
structures from collagen, most of
which stain the elastin-rich structures
brown or shades of purple. Examples
of such stains are Weigert's resorcin
fuchsin, aldehyde fuchsin, and orcein
Van Gieson stains.

50. Stains for Lipid
 When special preparation techniques
are used to retain lipids of cells, such
as in frozen sections, lipophilic dyes
are used to demonstrate lipid droplets
and myelin. Oil red O and Sudan black
stain lipid-rich structures as their
names suggest. Osmium tetroxide
(osmic acid), which is used as a
fixative for TEM, is reduced to a black
substance by unsaturated fatty acids
and is also used to demonstrate lipids.

51. Other Common Stains
 Many basic aniline dyes, including
azures, cresyl violet, brilliant cresyl
blue, luxol fast blue, and light green,
are used because of the permanence
and brightness of the colors they
impart to cellular and extracellular
structures in paraffin sections. Many
such stains were initially developed for
use in the textile industry.