Histology Methods

Histology: Definition and Importance

• Histology is the study of the structure of biological material and the ways in which individual components are structurally and functionally related.
• It is equally important as gross anatomy, biochemistry, molecular physiology, and study of disease processes as they are all inter-related.
• Knowledge of normal histological appearances is essential as it is a prerequisite in studying abnormal or pathological structures.

History of Histology

• Histology began with the discovery and development of the first microscopes and techniques for preparing microscopic specimens.
• French gross anatomist Marie F.X.Bichat introduced the word "tissue" in medical sciences in the 18th century.
• Bichat classified tissues into four types: epithelial, muscular, nervous, and connective tissues.

Methods of Studying Histology

Cell Culture Technique

• Involves growing cells in a controlled environment.

Microscopic Examination of Tissue Sections

• There are two types: paraffin technique and frozen section technique.
• Paraffin technique involves steps such as fixation, washing, dehydration, clearing, embedding, sectioning, and staining.
• Frozen section technique involves rapid freezing and sectioning of tissue samples.

Other Techniques

• Stained smears
• Transmission Electron Microscopy (TEM)
• Scanning Electron Microscopy (SEM)
• Freeze-Fracturing Technique
• Autoradiography
• Immunocytochemistry
• Histochemistry

Paraffin Technique

Fixation

• Stops postmortem autolysis (self-digestion) of tissue samples.
• Minimizes changes in the in vivo morphology of tissues.

Washing

• Removes excess fixative from tissue samples.

Dehydration

• Removes water from tissue samples and introduces ethanol.

Clearing

• Removes ethanol from tissue samples and introduces a clearing agent.

Embedding

• Infiltrates tissue samples with a solution that later solidifies to allow thin sectioning.

Sectioning

• Cuts tissue blocks into thin sections using a microtome.

Staining

• Uses dyes to distinguish different components of tissue sections.

Electron Microscopy

Transmission Electron Microscopy (TEM)

• Uses an electron beam to provide contrast and resolution.

Scanning Electron Microscopy (SEM)

• Uses an electron beam to provide depth of focus and three-dimensionality.

Artifacts

• Structures or features in tissue resulting from poor handling or improper processing.
• Examples: shrinkage, dark precipitates, folds, wrinkles, scratches, chatter, and bubbles.

Cell Theory

• Enunciated by Theodore Schwann in 1838 and 1839.
• States that all living things are composed of cells, and that cells can only arise from pre-existing cells.

Protoplasm

• The colorless material comprising the living part of a cell.
• Functions: metabolism, irritability, contractility, secretion, conductivity, endocytosis, exocytosis, growth, maintenance, and reproduction.### Cellular Structure and Function
• Cell is the basic structural and functional unit of living organisms.
• Cellular structure consists of:
  • Protoplasm (cytoplasm and nucleoplasm)
  • Nucleus (control center of the cell)
  • Cell membrane (plasma membrane)
• Cellular function includes:
  • Growth (increase in number or size of cells)
  • Maintenance (renewal and replacement of cells)
  • Reproduction (increase in number of cells through mitosis)
  • Excretion (removal of waste products)

Nucleus

• Nucleus is the command and control center of the cell.
• Parts of the nucleus:
  • Nuclear membrane (envelope)
  • Nucleoplasm (ground substance of the nucleus)
  • Chromatin (DNA-containing granules)
  • Nucleolus (site of mRNA synthesis)
• Nucleus is broken down and reconstructed during each cell cycle.

Cell Morphology

• Cell shape is influenced by functional specialization.
• Cells have organized structures with predictable positions.
• Cell size varies among different species and within the same species.

Organelles

• Organelles are living components of the cell.
• Classified as active and passive organelles.
• Active organelles:
  • Participate in metabolic processes
  • Classified as membranous (e.g., cell membrane, ER, Golgi apparatus) and non-membranous (e.g., ribosomes)
• Passive organelles:
  • Do not participate in metabolic processes
  • Include inclusions (e.g., glycogen, lipids, pigments)

Cell Membrane

• Cell membrane is a selective barrier that controls the movement of materials in and out of the cell.
• Structure of the cell membrane:
  • Trilaminar (three-layered) structure with inner and outer dark (osmiophilic) leaflets of lipids separated by a middle light (osmiophobic) leaflet of fatty acids
  • Cell coat (glycocalyx) is a carbohydrate-rich covering of the cell membrane

Cytoplasm

• Cytoplasm is the medium in which the various formed elements are suspended.
• Parts of the cytoplasm:
  • Cytosol (matrix or ground substance)
  • Formed elements (organelles and inclusions)

Endoplasmic Reticulum (ER)

• ER is a system of hollow membranous structures (tubules or flattened vesicles) extending throughout the cytoplasm.
• There are two types of ER:
  • Rough ER (RER) with attached ribosomes that synthesize proteins for extracellular use
  • Smooth ER (SER) with enzymes associated with various specialized functions

Golgi Apparatus

• Golgi apparatus is a complex of flattened membranous saccules and associated vesicles.
• Functions of the Golgi apparatus:
  • Concentrates and packages secretory products
  • Synthesizes polysaccharides
  • Produces lysosomes

Lysosomes

• Lysosomes are membrane-bound vesicles containing enzymes called acid hydrolases.
• Functions of lysosomes:
  • Degrade material taken up from outside the cell
  • Digest obsolete components of the cell itself
• Types of lysosomes:
  • Phagolysosome (heterophagic vacuole)
  • Autophagic vacuole
  • Multivesicular body

Mitochondria

• Mitochondria are the "powerhouse of the cell" as they generate ATP.
• Structure of mitochondria:
  • Double membrane structure with outer and inner walls
  • Inner wall forms platelike or tubular folds (cristae)
  • Cristae contain matrix rich in granules (calcium ions)
• Functions of mitochondria:
  • Generate ATP
  • Participate in Kreb's citric acid cycle
  • Contain DNA and RNA

Cytoskeleton

• Cytoskeleton is the internal skeleton of a cell.
• Components of the cytoskeleton:
  • Microfilaments (actin)
  • Myosin
  • Microtubules
  • Intermediate filaments
• Functions of the cytoskeleton:
  • Maintains cell shape
  • Participates in cell division
  • Involved in cell migration and muscle contraction### Intermediate Filaments
• Diameter: 8-10 nm
• Types: neurofilaments, glial filaments, keratin filaments, and vimentin filaments
• Function: maintain cell shape, important in epithelial cell adhesion

Passive Organelles

• Do not participate in metabolic processes
• Provide anchorage or support to various cellular components
• Examples: centrioles, basal bodies, microbodies (peroxisomes), microtubules, and filaments & fibrils

Microfilaments

• Consist of actin protein
• Diameter: 5-8 nm
• Length: 1.0 um in muscle cells, microvilli, and stereocilia
• Function: involved in cell movement, changes in cell shape

Microtubules

• Composed of tubulin protein
• Diameter: 20-50 nm
• Function: form cytoplasmic support of cilia, flagella, and spindle fibers
• Involved in cell movement, separation of chromosomes during mitosis

Centrioles

• Contained within centrospheres
• Present in muscle cells
• Length: 1.5 um
• Diameter: 15 nm
• Function: form microtubules during mitosis

Basal Bodies

• Similar in structure to centrioles
• Form the base of cilia in ciliated cells

Microbodies or Peroxisomes

• Similar to mitochondria but smaller
• Function: not known

Microfilaments and Microfibrils

• Made up of tubulin
• Diameter: 6-10 nm
• Function: involved in cell adhesion, formation of terminal web

Inclusions

• Non-living, non-moving, membrane-bound materials
• Examples: secretory inclusions, nutritive inclusions, and miscellaneous inclusions

Secretory Inclusions

• Secretory products of cells
• Examples: enzymes, acids, proteins, mucus

Nutritive Inclusions

• Nutritive materials produced by or stored in the cell
• Examples: glycogen granules, lipid droplets

Miscellaneous Inclusions

• Examples: lipofuscin, melanin, and hemosiderin pigments

Tissues

• A group of similar cells and their extracellular products
• Perform a specific function or a spectrum of related functions

Connective Tissue

• Cells perform a specific function or a spectrum of related functions
• Composed of cells, intercellular substance, and fluid
• Intercellular substance: composed of collagen fibers, fibroblasts, chondroblasts, and osteoblasts

Collagen Fibers

• Predominant fiber in most connective tissue
• Composed of tropocollagen molecules
• Types: Type I, Type II, Type III, and Type IV

Elastic Fibers

• Provide "springiness" and stretchability to the tissue
• Stain with Verhoeff's stain

Reticular Fibers

• Composed of Type III collagen
• Stain with silver stain
• Form the framework of lymphoid organs

Nervous Tissue

• Makes up the organs of the nervous system
• Composed of cells, intercellular substance, and fluid

Epithelial Tissue

• Covers external surfaces of the body and forms exocrine glands
• Characterized by: polarity, avascularity, high turnover rate, and embryonic origin
• Types: simple squamous epithelium, simple cuboidal epithelium, simple columnar epithelium, and pseudostratified epithelium