1. Topic 1: Introduction to Histology
Animal Histology BIOL 241
Topic 1:
Introduction to Histology
Dr. Issa Al-Amri
Department of Biological Sciences & Chemistry
College of Arts & Sciences

2. Introduction Histology: study of tissue using a microscope.
Body composed of systems which made of organs. Organs made of tissues which composed of cells.
Anatomy subdivided into:
Gross anatomy
Microscopic anatomy
Microscopic anatomy subdivided into:
Organology (study of organs)
Histology (study of tissues)
Cytology (study of cells)
Histology also related to study of cell biology, and organ systems.

3. Introduction
It forms the structural basis for understanding organ function (Physiology) and is the preparation for the study of abnormal structure and function (Pathology).
Structures of tissue and cell size measured by special units under the microscopes.
Units of measurements:
1 centimeter (cm) = 10 millimeters (mm)
1 millimeter (mm) = 1000 micrometers (µm)
1 micrometer (1µm) = 0.001mm
1 nanometer (1nm) = µm
1 Angstrom (1 Å) = 0.1nm = µm

4. Histological Techniques
Methodology (Histological Techniques):
1. Type of microscope used
2. Type of sample preparation methods
Type of Microscopes
Type of microscopes used depends on magnification and ability to resolve structural details (resolution).
Light microscope (LM) uses visible light as source of illumination. Used for studying general structures (microstructure).
Electron microscope (EM) uses electron beam as source of illumination. Used for studying fine structures (ultrastructure).

5. Histological Techniques
Light Microscope (LM) composed of:
Frame and mechanical parts
Optical (magnification) system
Illumination system
Frame supports different parts of the optical system, it consists of:
Head (eyepiece, nosepiece)
Arm (optical system: objective lenses, focusing knobs)
Stage (thin section, central hole for light to pass through (aperture))
Base (illumination system: lamp, condenser lens, Iris)

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LM with ocular eyepiece lens

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LM with binocular eyepiece lenses

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The magnifying system composed of:
Eyepiece (ocular lens or binocular lenses) with 8, 10, 12, or 20 X of magnification.
Objective lens: five lenses with different choices of magnifications: 2, 5, 10, 20, 40, 60, 100 X (oil immersion lens); they are carried on an objective nose piece.
Objective lens collect light transmitted through a section from the condenser, focus and magnify the image and direct it to ocular lens. The eyepiece further magnifies the image and directs it to the viewer eye or camera.

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Magnification: The property of making things to appear larger.
The total magnification = ocular power x objective power
Resolution (or resolving power): The property of disclosing the fine details. It is the smallest distance between two points that can be distinguished from each other.
Resolution power of:
Human naked eye: 0.1–0.2 mm
LM: 0.1–0.2 µm
EM: 0.1–0.2 nm

12. Histological Techniques
To improve resolution of a microscope is to reduce substantially the wavelength of light. This was achieved by the electromagnetic beam of electron microscope.
Maximum magnification by LM X 2000 using 1 µm section thickness.
Illumination system consists of:
Light source (tungsten filament)
Condenser lens (to focus the light beam)
Iris diaphragm (to regulate amount of light)

13. Histological Techniques
Electron Microscope (EM): detailed morphology revealed by EM called fine structures or ultrastructure. There are two types of the electron microscopes, transmission and scanning.
Transmission Electron Microscope (TEM) composed of:
Illumination system (electron beam, condenser lens)
magnifying system (objective, projector lenses)
Vacuum system (vacuum pumps)
Imaging system (fluorescent screen, camera)

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TEM

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TEM

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TEM:
High resolving power ( nm) of EM made possible to study details of the interior structures of tissues and cells with final magnification of X1,600,000 .
Image seen on screen as black and white and recorded on photographs.
Electron microscope requires vacuum-enclosed system, high voltage (60-120kV), and mechanical stability.
Use of high voltage ( kV) allowed the use of relatively thicker sections to be examined for ultrahigh resolution and 3-D imaging.

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JEOL - TEM 120 kV

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ZEISS -TEM 120 kV

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JEOL - FETEM 200 kV

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Modern FEI - FETEM

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HVFETEM

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Principle of TEM:
Beam of electrons emitted by heated tungsten gun or filament (cathode) in a vacuum controlled system.
There is a voltage difference between the cathode and anode which accelerates electron beam and attracts electrons to the anode where they pass through its central hole.
While passing in the microscope tube (or column), the electron beam is subjected to electric coils with magnetic field, which deflect the electrons and change their path, thus called electromagnetic lenses. Beam pass through a section in the tube and image produced by objective lens.
The image projected and viewed directly on a fluorescent screen because the human eye is not sensitive to electrons.

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Principle of TEM

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Scanning Electron Microscope (SEM):
Contains similar systems to TEM, Except:
Study details of surface structures of tissues and cells, thus uses scanner to scan sequentially the different surface points of the specimen and the resulting image viewed in a monitor in black and white.
Provides three dimensional image (TEM 2D). Uses Large chamber for samples up to 75 mm diameter, whereas in TEM sections only 3 mm diameter & thickness 70 nm.
Uses electron detectors (secondary and backscattered) for image production.
Final magnification X 300,000, with resolution range of 3.0–1.0 nm, and voltage range of 1.0–30.0 kV.

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SEM

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Principle of SEM

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FE SEM

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Modern FE SEM

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Desktop SEM

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Comparison: LM, TEM, SEM

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Comparison between light (LM) and electron microscopes (EM)
Histological Techniques
Comparison between light (LM) and electron (EM)microscopes
Light Microscope
Electron Microscope
Image
Presented directly to the eye. Image keeps the colors given by staining
Presented on a screen in shades of green. In photographs, image appears in grey scale or in black and white
Magnification
Up to X 2000 (times), wider field of sample view; good orientation
High, up to X 1,600,000 TEM (90,000,000 STEM)
Resolution
0.1 – 0.2 μm
High, 0.1nm
Processing
time
By frozen section sample can be prepared in 20 min
Tissue processing takes one day at least. Frozen tissue for cryofructure and immunogold labeling
Stain
Routine H&E
Heavy metals
Specimen size
Can be large and alive
Very small samples (TEM)
Can be large (SEM)
Section thickness
1 – 10 μm
Very thin 40 – 90 nm (TEM)
Artifacts
Many
Fewer
3-D image
Can be constructed by serial sections
Obtained by thicker section of high-voltage EM, freeze-fructured techniques, and SEM
Light source
Visible light (electric)
Beam of electron
Lenses
Glass
Electromagnetic