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ANA 205 General Histology
Objectives
At the end of this lecture, students should able to:
• identify a light microscope and its parts
• identify other types of light microscopy and how they work
• identify the two types of electron microscopy
• know methods of tissue preparation
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• know methods of tissue preparation
• identify sectioning equipments
• get acquainted with artifacts
Class activities
• Students will go to histology to observe histological activities
• Histology: study of the normal structure of tissues
of the body and of how they are arranged to
constitute organs.
• Histopathology: study of tissue malfunction in
disease states, changes in the microscopic structure
of the tissues. of the tissues.
• Four fundamental tissues are recognized: epithelial
tissue, connective tissue, muscular tissue, and
nervous tissue.
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Tissues
• Made of cells and extracellular matrix
• Extracellular matrix (ECM) consists of collagen
fibrils and basement membranes.
Functions of ECM
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- Furnish mechanical support for the cells
- Transport nutrients to the cells
- Carry away catabolites and secretory products.
• Cells produce ECM components and are also
influenced by them.
• The fundamental tissues are formed by cells
and by associations of cells and extracellular
matrix. matrix.
• Most organs are formed by several tissues,
except the central nervous system, which is
formed almost solely by nervous tissue.
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Preparation of Tissues for Microscopic Examination
• Cells are studied with the light microscope whereby
tissues are examined using a light beam that is
transmitted through the tissue.
• Tissues and organs are thick for light to pass
through them, they are sectioned to obtain thin,
translucent sections on glass slides before they are translucent sections on glass slides before they are
examined.
• Living cells, thin layers of tissues, or transparent
membranes of living animals (eg, the mesentery,
the tail of a tadpole, the wall of a hamster's cheek
pouch) can be observed directly in the microscope
without first being sectioned. 6
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Microtome
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These sections are cut from tissues for sectioning using microtomes or vibratomes.
Fixation (to preserve)
• For permanent sections to be made tissues must be fixed
Reasons
- To avoid tissue digestion by enzymes present within the cells
(autolysis) or by bacteria
- To preserve the structure of the tissue
Methods Methods
• Chemical
• Physical
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Chemical method
• Tissues are immersed in stabilizing or cross linking agents -fixatives.
• Intravascular perfusion greatly improves fixation.
• Fixative for routine light microscopy is a buffered
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• Fixative for routine light microscopy is a buffered isotonic solution of 4% formaldehyde andglutaraldehyde.
• For high resolution in electron microscopy, a double fixation procedure, using a buffered glutaraldehydesolution and buffered osmium tetroxide is used.
Physical Fixation
• Tissues are submitted to rapid freezing.
• Tissues are fixed by freezing (physically) and at the
same time become hard and can be sectioned.
• A freezing microtome—the cryostat has been
devised to section the frozen tissues.
• For histochemical study of enzymes or small • For histochemical study of enzymes or small
molecules, since freezing does not inactivate most
enzymes.
• Solvents such as xylene dissolves the tissue lipids,
the use of frozen sections is advised when these
compounds are to be studied.10
Dehydration
• Water is first extracted from the fragments to be embedded by bathing them in a graded series of mixtures of ethanol and water (from 70% to 100% ethanol).
Clearing
• Ethanol is replaced with a solvent miscible with the embedding medium. In paraffin embedding, the solvent used is usually xylene, benzene, chloroform. solvent used is usually xylene, benzene, chloroform.
Embedding
• Once the tissue is impregnated with the solvent, it is placed in melted paraffin in the oven, at 58–60°C to facilitate sectioning and to impart a rigid consistency to the tissue.
• Embedding materials include paraffin and plastic resins.
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Staining
• Tissue components are conspicuous and
permit distinctions to be made between them.
• Tissue components that stain with basic dyes
are basophilic while those with an affinity for
acid dyes are acidophilic.
• Basic dyes are toluidine blue and methylene blue,
hematoxylin, acid dyes (orange G, eosin, acid fuchsin)
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hematoxylin, acid dyes (orange G, eosin, acid fuchsin)
• Hematoxylin and eosin (H&E) is the most commonly used.
Hematoxylin stains the cell nucleus and other acidic
structures blue while eosin stains the cytoplasm and
collagen pink.
• When cells and cell limits are often not visible, a
counterstain is used to allow the recognition of nuclei or
cytoplasm.12
Types of Light Microscopes
• Conventional light
• Phase-contrast
• Differential interference
• Polarizing
• Confocal• Confocal
• Fluorescence microscopy
• They are all based on the interaction of light
and tissue components. 13
Microscope
• Composed of mechanical and optical parts.
Optical components consist of three lenses:
- Condenser collects and focuses light, producing a cone
of light that illuminates the object to be observed.
- Objective lenses enlarge and project the illuminated
image of the object in the direction of the eyepiece.
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image of the object in the direction of the eyepiece.
- Eyepiece further magnifies this image and projects it
onto the viewer's retina, a photographic plate, or a
detector such as a charged coupled device camera.
• Total magnification: multiply the magnifying power of
the objective and eyepiece.
Mechanical components
• Mechanical stage
• Fine and coarse adjustment knobs
• Body
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• Body
Resolution (resolving power)
• Critical factor in obtaining a crisp, detailed
image with the microscope
• Smallest distance between two particles at
which they can be seen as separate objects.
• Maximal resolving power of the light • Maximal resolving power of the light
microscope is approximately 0.2 m; this
power permits good images magnified 1000–
1500 times.
• Depends on the quality of its objective lens.
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Light
Microscope
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Light microscope
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Phase-Contrast Microscopy & Differential Interference Microscopy
• Unstained biological specimens are transparent and difficult to view, all parts of the specimen have the same optical density.
• Uses a lens system that produces visible images from transparent objects.
- Structures appear lighter or darker relative to each - Structures appear lighter or darker relative to each other
- Used to observe living cells.
• Another way to observe unstained cells or tissue sections is Nomarski differential interference microscopy, which produces a three-dimensional image 19
The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again. Phase Contrast
MicroscopeThe image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again.
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Polarizing Microscopy
• Allows structures made of highly
organized molecules (cellulose,
collagen, microtubules,
microfilaments) to be recognized.
• Possession of two polarizing filters.
• Appear as bright structures against a dark
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• Appear as bright structures against a dark
background.
• Ability to rotate the direction of vibration of
polarized light is called birefringence
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Polarizing
Microscope
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Confocal Microscopy
• A very thin plane of the specimen is seen in focus at
one time.
• There are two principles on which this is based:
- specimen is illuminated by a very small beam of light
- image collected from the specimen passes through a
small pinhole.
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small pinhole.
• Only the image originating from the focused plane
reaches the detector whereas the images in front of
and behind this plane are blocked.
• Focused object becomes better and allows the
localization of any specimen component with much
greater precision than in the common light microscope.
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Fluorescence Microscopy
• Tissue sections are irradiated with either UV light or laser
and the emission is in the visible portion of the specimen
• The fluorescent substances appear brilliant or colored on a
dark background
• Examples of fluorescent stain is acridine orange which
combines with DNA and RNA. It is possible to identify and
localize nucleic acids in the cells
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localize nucleic acids in the cellsThe image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again.
Electron Microscopy
• Transmission and scanning electron microscopes are
based on the interaction between electrons and tissue
components.
Transmission Electron Microscopy
• Theoretically permits very high resolution (0.1 nm-
3nm).
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3nm).
• High resolution allows magnifications of up to 400,000
times to be viewed with detail.
• Electrons are released by heating a very thin metallic
(tungsten) filament (the cathode) in a vacuum and are
attracted to the anode
• Beam of electrons are deflected by electromagnetic
fields
• Electrons released are submitted to a voltage difference of 60–120 kV between the cathode and the anode.
• Resulting image is black and white.
• Dark areas of an electron micrograph are usually called electron dense, whereas light areas are called electron lucent.
• Electron microscopy requires very thin sections (40–
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• Electron microscopy requires very thin sections (40–90 nm)
- Embedding is performed with a resin that becomes very hard.
• Blocks obtained are hard that glass or diamond knives are usually necessary to section them.
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Transmission electron microscope and micrograph
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Freezing techniques
• Allow the examination of tissues by electron
microscopy without the need for fixation and
embedding.
• There are fewer artifacts
• Technique is usually arduous.
• Frozen tissues may be sectioned or fractured
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• Frozen tissues may be sectioned or fractured
(cryofracture, freeze fracture) to reveal details of
the internal structure of the membranes.
Scanning Electron Microscopy
• Permits pseudo-three-dimensional views of the
surfaces of cells, tissues, and organs.
• Produces a narrow electron beam that is scanned
from point to point across the specimen which do
not pass through the specimen.
• Resulting in a black-and-white image.
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• Resulting in a black-and-white image.
• Shows only surface views.
• Inside of organs can be analyzed by freezing the
organs and fracturing them to expose their internal
surfaces.
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Scanning electron microscope and micrograph
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Autoradiography of Tissue Sections
• Study of biological events in tissue sections using radioactivity.
• Permits the localization of radioactive substances in tissues by means of emitted radiation.
• A radioactive compound is delivered to the cells such as radioactive amino acids, radioactive nucleotides, and radioactive sugars.
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radioactive sugars.
• When the silver bromide crystals present in the photographic emulsion are hit by radiation they are transformed into small black granules of metallic silver-- revealing the existence of radioactivity in the tissue.
• Used in both light and electron microscopy
• Determines where new cells are produced in an organ and where they divide or migrate
Cell & Tissue Culture
• Live cells and tissues are maintained and studied outside
the body.
• Direct observation of the behavior of living cells under a
microscope.
• Experiments that cannot be performed in the living animal
(in vivo) can be reproduced in vitro.
• Cells and tissues are grown in complex solutions of known
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• Cells and tissues are grown in complex solutions of known
composition to which serum components are added.
• Cells are isolated, dispersed and cultivated in a suspension
or spread out on a Petri dish or glass slide where they
adhere as a single layer of cells.
• Cultures of cells that are isolated in this way are called
primary cell cultures.
• Cell types that have been maintained in vitro and immortalized constitute a permanent cell line.
• Changes can promote cell immortality -transformation, which may be a first step in transforming a normal cell into a cancer cell.
MEDICAL APPLICATION
• Used for the study of the metabolism of normal and cancerous cells
• Used for the development of new drugs.
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• Used for the development of new drugs.
• Useful in the study of parasites that grow only within cells, such as viruses, mycoplasma, and some protozoa.
• Determination of human karyotypes
- Detect anomalies in the number and morphology of the chromosomes
• Central to contemporary techniques of molecular biology and recombinant DNA technology.
Distortions & Artifacts Caused by Tissue Processing
• Shrinkage produced by the fixative and ethanol, these decrease when specimens are embedded in resin.
• Heat needed for paraffin embedding.
• A consequence of shrinkage is the appearance of artificial spaces between cells and other tissue components.
• Another source of artificial spaces is the loss of
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• Another source of artificial spaces is the loss of molecules (glycogen, lipids) that were removed by the fixatives, dehydrating and clearing fluids.
• All these artificial spaces and other distortions are called artifacts.
• Other artifacts may include wrinkles of the section, precipitates of stain, and many more.
