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

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.

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) = 0.001 µm 1 Angstrom (1 Å) = 0.1nm = 0.0001 µm

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).

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)

Histological Techniques LM with ocular eyepiece lens

Histological Techniques LM with binocular eyepiece lenses

Histological Techniques 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.

Histological Techniques

Histological Techniques

Histological Techniques 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

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)

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)

Histological Techniques TEM

Histological Techniques TEM

Histological Techniques TEM: High resolving power (0.2-0.1 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 (500-3000kV) allowed the use of relatively thicker sections to be examined for ultrahigh resolution and 3-D imaging.

Histological Techniques JEOL - TEM 120 kV

Histological Techniques ZEISS -TEM 120 kV

Histological Techniques JEOL - FETEM 200 kV

Histological Techniques Modern FEI - FETEM

Histological Techniques HVFETEM

Histological Techniques 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.

Histological Techniques Principle of TEM

Histological Techniques 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.

Histological Techniques SEM

Histological Techniques Principle of SEM

Histological Techniques FE SEM

Histological Techniques Modern FE SEM

Histological Techniques Desktop SEM

Histological Techniques Comparison: LM, TEM, SEM

Histological Techniques 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