Histology: Tissue Preparation and Fixation

• Histology is the study of the microscopic structure of tissues.
• It bridges the gap between molecular biology, cell biology, and physiology.
• Histological analysis is essential for understanding tissue organization, function, and pathology.

Tissue Preparation

• Tissue preparation is crucial for accurate histological analysis.
• The main steps include fixation, processing, embedding, sectioning, and staining.

Fixation

• Fixation aims to preserve tissue structure and prevent autolysis (self-digestion).
• Common fixatives include formaldehyde (formalin) and glutaraldehyde.
• Formalin cross-links proteins, preserving overall tissue architecture.
• Glutaraldehyde provides better ultrastructural preservation for electron microscopy.
• Fixation stabilizes the tissue, preventing degradation.

Processing

• Tissue processing involves dehydration, clearing, and infiltration.
• Dehydration removes water from the tissue using increasing concentrations of ethanol.
• Clearing replaces the ethanol with a solvent miscible with both ethanol and paraffin wax, such as xylene.
• Infiltration replaces the clearing agent with a support medium, usually molten paraffin wax.
• Processing prepares the tissue for embedding.

Embedding

• Embedding involves encasing the tissue in a solid medium, usually paraffin wax.
• The embedded tissue provides support during sectioning.
• The tissue is placed in a mold, and molten paraffin wax is poured around it.
• After cooling and solidifying, the paraffin block is ready for sectioning.

Sectioning

• Sectioning involves cutting the paraffin block into thin slices using a microtome.
• Microtomes use a sharp blade to cut sections typically 5-10 micrometers thick.
• The thin sections are then floated on a water bath to remove wrinkles.

Staining

• Staining enhances contrast and allows visualization of tissue components.
• Hematoxylin and eosin (H&E) staining is the most common histological stain.
• Hematoxylin stains acidic structures (e.g., DNA, RNA) blue or purple.
• Eosin stains basic structures (e.g., proteins) pink or red.
• Other stains, such as trichrome stains, periodic acid-Schiff (PAS), and silver stains, are used for specific tissue components.
• Immunohistochemistry uses antibodies to detect specific proteins.

Microscopy

• Microscopy is used to view stained tissue sections.
• Light microscopy uses visible light to magnify the image.
• Electron microscopy uses electrons to achieve higher magnification and resolution.

Light Microscopy

• Bright-field microscopy is the most common type of light microscopy.
• The sample is illuminated from below, and the image is viewed through an objective lens and eyepiece.
• Phase-contrast microscopy enhances contrast in unstained samples by exploiting differences in refractive index.
• Fluorescence microscopy uses fluorescent dyes to label specific molecules.

Electron Microscopy

• Transmission electron microscopy (TEM) uses a beam of electrons to create an image.
• TEM provides high-resolution images of cellular ultrastructure.
• Scanning electron microscopy (SEM) scans the surface of a sample with a focused beam of electrons.
• SEM provides detailed images of the sample's surface topography.

Basic Tissue Types

• There are four basic tissue types: epithelial, connective, muscle, and nervous.
• These tissues are defined by their structure, function, and the characteristics of their cells and extracellular matrix.

Epithelial Tissue

• Epithelial tissue covers surfaces, lines cavities, and forms glands.
• Epithelial cells are tightly packed and attached to a basement membrane.
• Epithelial tissue functions in protection, absorption, secretion, and excretion.
• Types of epithelium include:
  • Simple squamous epithelium: single layer of flattened cells (e.g., lining of blood vessels).
  • Simple cuboidal epithelium: single layer of cube-shaped cells (e.g., kidney tubules).
  • Simple columnar epithelium: single layer of column-shaped cells (e.g., lining of the stomach).
  • Stratified squamous epithelium: multiple layers of flattened cells (e.g., epidermis of the skin).
  • Pseudostratified columnar epithelium: single layer of cells with varying heights (e.g., trachea).
  • Transitional epithelium: capable of stretching (e.g., urinary bladder).

Connective Tissue

• Connective tissue supports, connects, and separates different tissues and organs.
• Connective tissue consists of cells and an extracellular matrix composed of fibers and ground substance.
• Types of connective tissue include:
  • Connective tissue proper: loose and dense connective tissue.
  • Cartilage: hyaline, elastic, and fibrocartilage.
  • Bone: compact and spongy bone.
  • Blood: red blood cells, white blood cells, and platelets.
• Fibers in the extracellular matrix include collagen fibers (strength), elastic fibers (elasticity), and reticular fibers (support).
• Cells in connective tissue include fibroblasts, adipocytes, mast cells, and macrophages.

Muscle Tissue

• Muscle tissue is responsible for movement.
• There are three types of muscle tissue: skeletal, smooth, and cardiac.
• Skeletal muscle is striated and voluntary (e.g., biceps muscle).
• Smooth muscle is non-striated and involuntary (e.g., walls of blood vessels).
• Cardiac muscle is striated and involuntary (e.g., heart).

Nervous Tissue

• Nervous tissue is responsible for communication and control.
• Nervous tissue consists of neurons and glial cells.
• Neurons transmit electrical signals.
• Glial cells support and protect neurons.
• Nervous tissue is found in the brain, spinal cord, and nerves.

Glands

• Glands are specialized epithelial tissues that secrete substances.
• Exocrine glands secrete substances onto a surface through ducts.
• Endocrine glands secrete hormones into the bloodstream.
• Examples of exocrine glands include sweat glands, salivary glands, and digestive glands.
• Examples of endocrine glands include the thyroid gland, adrenal gland, and pituitary gland.

Specialized Tissues and Organs

• Histology is essential for understanding the structure and function of specialized tissues and organs.
• Examples include:
  • Skin: epidermis, dermis, and hypodermis.
  • Respiratory system: trachea, bronchi, and alveoli.
  • Digestive system: esophagus, stomach, small intestine, and large intestine.
  • Cardiovascular system: heart and blood vessels.
  • Urinary system: kidneys, ureters, bladder, and urethra.
  • Nervous system: brain, spinal cord, and nerves.
  • Endocrine system: glands that secrete hormones.
  • Reproductive system: male and female reproductive organs.
  • Lymphatic system: lymphatic vessels and lymphoid organs.

Pathological Histology

• Pathological histology, also known as histopathology, involves the microscopic examination of abnormal tissues.
• It is used to diagnose diseases, evaluate the extent of disease, and monitor the response to treatment.
• Biopsies and surgical specimens are commonly examined in histopathology.
• Pathologists analyze tissue sections to identify abnormalities in cell structure, tissue organization, and the presence of pathogens.
• Histopathology is essential for the diagnosis of cancer, infectious diseases, and other conditions.