Human Anatomy EVALS 1

Lecture 2: Introduction to Histology

Dr. Gonzales

Histology Objective Lens Eyepiece Total

 study of tissues (x10) Magnification
 (evolved term) the study of microscopic structure of the 4x Scanning Objective x10 40x
body- microscopic anatomy. 10x Low Power Objective x10 100x
 encompasses the study of the microscopic structure of 40x High Power Objective x10 400x
organs, tissues, and cells (cytology) 100x Oil Immersion x10 1000x
Cells Objective
 capable of independent existence
 functional unit of all living things II. TISSUE PREPARATION FOR LIGHT MICROSCOPY
 grouped into 200 cell types  Preparation of a histologic specimen (tissue specimen;
histologic section; tissue section) for light microscopy
 sub-types of cells perform more specialized function. entails processing of a small tissue block of anatomical
 each cell Is assigned a specific task part of the body so that it becomes firm enough to allow
 some cells are unicellular (e.g. Entamoeba histolytica) very thin slices to be made.
 Most are multicellular (Humans consist of trillions of  Slices of the specimen are stained to provide contrast
cells) because biological specimens are inherently of low
Tissues contrast.
 Cells with the same function are grouped together to
form tissues H & E stain (Hematoxylin and Eosin)
Organ  most commonly used staining procedure in histology.
 Tissues with the same function are grouped together to Hematoxylin
form an organ.  blue basic dye
Organ System  binds with acidic structures
 Organs with the same function are grouped together to  -parts of specimen that take up hematoxylin are
form an organ system. colored blue and referred to as basophilic (e.g. Nuclei,
Ribosomes).
I. TOOLS OF HISTOLOGY Eosin
 red acid dye
Compound Light Microscope/ Light Microscope  binds with basic structures in a specimen
 the most commonly used tool in the study of basic  parts that take up eosin are colored various shades of
histology red and referred to as acidophilic or eosinophilic. (e.g.
Electron Microscope Mitochondria, granules in the Cytoplasm such as those
 often a useful adjunct because knowledge of the found in the Paneth cells of the intestines, acidophils of
ultrastructure of cells and tissues is oftentimes the adenohypophysis).
necessary for a more thorough understanding of their
architecture. Examples of other stains used to distinguish various parts of the
Resolving Power cells and tissues:
1. Periodic acid-Schiff (PAS) stain- glycogen and
 -shortest distance at which 2 points can be seen as carbohydrate-rich molecules are colored magenta.
separate
2. Best’s Carmine stain- glycogen is stained red.
 -using it beyond its RP leads to empty magnification 3. Silver stain- reticular fibers are colored black.
(Image becomes bigger but no additional details may
be seen.)
Resolving Power of different microscopes: III. STEPS IN THE PREPARATION OF HISTOLOGIC
 Light Microscope = 0.2 um (2 millionth of a meter) SPECIMENS USING H&E STAINS
 Electron Microscope = 0.2 nm (2 billionth of a meter) 1. Obtaining the specimen
 Transmission Electron Microscope = 0.2 nm (2 billionth  Using a sharp knife, the specimen is obtained as close
of a meter) as possible to the time of death or the time of removal
of organ from the body.
 Scanning Electron Microscope = 10 nm
 Specimen should be small enough to ensure that the
Light Microscope fixative shall be able to adequately penetrate the
interior.
 Also known as compound light microscope because
it uses not only one, but several lenses that are  Orientation of the specimen in relation to the entire
specifically arranged to magnify an image. organ is also considered. It must contain parts that will
enable the microscopist to orient himself/herself
 In light microscopy, a light beam is focused and made properly and make an intelligent interpretation of the
to pass through the specimen to be studied that is
architecture of the organ.
placed on the stage.
2. Fixation
 The light passing through the specimen is then made  Uses protein coagulants or cross -linking agents to
to enter an objective lens (usually 3-4) that are on a
allow preservation, as close to life as possible, of the
moveable turret located immediately above the
structures in the specimen.
specimen to be studied
 The image from the objective lens is further magnified  Shrinkage may occur.
by an ocular lens or eyepiece.  10% formaldehyde- most commonly used fixative
 The LM is designed to have only one ocular lens at a  arrests chemical and anti-microbial properties
time, usually a 10x lens 3. Dehydration
 Magnification (Linear Magnification)- is the product  Removal of water content by running the tissue sample
of the magnification of the eyepiece and the objective through a series of increasing concentration of an
that is in use. organic solvent like ethanol.
 HPO and OIO- generally used only when studying very  Needed in the preparation for the impregnation of the
minute parts of a specimen like individual cells or parts tissue sample by paraffin (not miscible with water)
of cells. 4. Clearing
 Specimens or sections studied under and LM and  With the use of xylene- miscible with alcohol that it
TEM microscope are very thin slices of the cell tissue replaces in the specimen and it is also miscible with
or organ under consideration. They are thin enough to paraffin, the embedding medium.
allow the passage of light or beam of electrons. 5. Embedding
Because of this thinness, these specimens are virtually  Tissue sample is placed in melted paraffin for a period
2-dimensional structures. of time to allow impregnation of the interior by this

TRANSCRIBERS: CHA CRUZ, RJ BELTRAN, LEI MERCADO, GLY LAGASON 1 of 2

SUBTRANSHEAD: CARL ABELLAR
HUMAN ANATOMY: LECTURE # -- TOPIC TITLE

 substance-> place in a small plastic, metal or (2) Scanning Electron Microscope (SEM)
cardboard box where melted paraffin is poured o Used to give a highly magnified view of the
6. Sectioning surface of a solid specimen
o Affords a 3-D view of a structure or cell that is
 Paraffin is allowed to set-> Trim-> Placed on the under study unlike that of LM and TEM (2-D
microtome for sectioning
view)
 Sectioning of specimen must be oriented according to o A beam of electrons scans the surface of an
the needs of the histologist. object that has been previously coated with a
 The thin sections include both a part of the specimen heavy metal (gold or palladium)-> Electrons
and a part of the paraffin ejected from the heavy metal coat are
 These are floated on a water bath set at just below the captured by electron detectors that collate
melting point of the paraffin being used. At this temp, and interpret the behavior of the electrons to
the paraffin and tissue sample should flatten out and create a 3-D image
have no wrinkles.
7. Mounting
 Egg albumin is wiped on the glass slide on which the REFERENCES:
specimen is going to be mounted and then air-dried. 1. Lecture Notes
 Glass placed below the slice of paraffin and the tissue
and then slowly raised.
 The section is positioned on the glass slide with help of
dissecting needle
 To facilitate drying and make sure that the specimen
remains flat on the slide it is placed on a slide warmer.

8. Deparaffinization and Staining

- Done with H&E which are both water-soluble dyes,
therefore the tissue section impregnated with paraffin
will not, as is take up these stains
- Paraffin must be removed by passing it through xylol
(not miscible with water)
- Xylol must be removed by passing through decreasing
concentrations of alcohol. This will also hydrate the
specimen/.
- Only after this can the specimen be stained with H&E
and then covered with a cover slip

ELECTRON MICROSCOPE
 Electromagnets focus a beam of electrons on a
specimen.
 Has a greater resolving power than a light optical
microscope because electrons have much shorter
wavelength than visible light
 Present-day electron microscopes are good enough to
visualize macromolecules
 The 2 most common types of electron microscope are:
(1) Transmission Electron Microscope (TEM)
o Specimen preparation follows same basic
steps as in light microscopy but tissue blocks
are much thinner
o Image produced is not viewed directly with the
eye. Image is captured by an electron-
sensitive film producing a negative from which
a black and white photograph (positive) can
be printed.
o Electron micrograph- black and white
photograph

TRANSCRIBERS: CHA CRUZ, RJ BELTRAN, LEI MERCADO, GLY LAGASON 2 of 2

SUBTRANSHEAD: CARL ABELLAR