histology

MICROSCOPY & MICROTECHNIQUES

Goals of the Unit

  • Provide essential knowledge on types of microscopes and tissue preparation techniques.

  • Learning objectives include:

    1. Recognize various types of microscopes (properties and uses).

    2. Compare light and electron microscopes.

    3. Compare transmission and scanning electron microscopes.

    4. Use the light microscope in practical classes.

    5. Recognize measuring units for histological structures.

    6. Describe methods for preparing fixed tissues for examination.

    7. List different types of stains used for histological sections.

    8. Identify steps for preparing fixed tissues for paraffin sections.

    9. Identify principles of hematoxylin and eosin staining during practicals.

Histology Overview

  • Histology: Science of microscopic anatomy of cells, tissues, and organs correlating structure with function.

  • Importance of using:

    • Different types of microscopes.

    • Microtechniques to prepare tissues for examination.

Types of Microscopes

  • Visible Structures: Smallest structures visible to the human eye are ~50-100 um.

  • Microscope Categories:

    1. Conventional Light Microscope (LM): Common and uses visible light.

    2. Fluorescence Microscope: Uses fluorescence to visualize structures.

    3. Electron Microscope (EM): Uses electrons for high-resolution images; includes:

      • Transmission Electron Microscope (TEM): For internal structures.

      • Scanning Electron Microscope (SEM): For surface details.

  • Components of Light Microscope:

    • Ocular Lens, Body Tube, Revolving Nosepiece, Arm, Objectives, Stage, Stage Clips, Course Adjustment Knob, Diaphragm, Fine Adjustment, Light Source, Base.

Measurement Units in Microscopy

  • Millimeter (mm): 10^-3 m

  • Micrometer (µm): 10^-6 m

  • Nanometer (nm): 10^-9 m

Microtechniques for Histological Examination

Methods of Preparation

  • Objective: Preserve structure and chemical composition of tissues as in vivo.

  • Popular Techniques:

    • Paraffin Method: Common technique involving several steps:

      • Fixation: Utilizes neutral buffered formalin to prevent post-mortem changes.

      • Dehydration, Clearing, Embedding: Use chemicals like ethyl alcohol and xylol.

      • Sectioning: Obtain tissue sections (5-9 µm thick) using a rotatory microtome.

      • Mounting & Staining: Sections are mounted and stained, followed by dehydration and glass coverslipping.

    • Freezing Method: Cuts frozen tissues for urgent diagnoses; used for enzyme histochemistry and fat demonstration.

Staining of Histological Sections

  • Staining methods are critical to visualize tissue structures:

    1. Hematoxylin and Eosin (H&E): Common stain; hematoxylin stains nucleus and eosin stains cytoplasm.

Understanding Stains

  • Types of Stains:

    • Hematoxylin: Basic dye, appears violet.

    • Eosin: Acidic dye, appears pink.

  • Metachromatic Stain: Some tissues change color with certain dyes (e.g., mast cell granules with toluidine blue).

  • Immunohistochemistry: Techniques to locate specific antigens using labeled antibodies.

Light vs. Electron Microscopes

Feature

Light Microscope (LM)

Electron Microscope (EM)

Source of Illumination

Daylight or electric light

Beam of electrons

Magnification

Up to x1500

Up to x100,000

Section Thickness

5-9 µm

90-50 nm

Staining

Any stain

Heavy metals (lead citrate, osmium tetroxide)

Photo Output

Colored

Black and white

The Cell

Goals

  • Knowledge on microscopes and techniques for tissue examination.

  • Key learning objectives involve understanding cell membrane structure, organelle functions, cell cycle, and types of cell populations.

Cell Membrane (Plasma Membrane)

  • Vital, dynamic barrier that surrounds the cell.

  • Appears as a trilaminar structure: outer and inner dense lines with a lucent middle zone.

  • Composed of:

    • Lipid Bilayer: Phospholipids form a bimolecular layer.

    • Proteins: Integral (transmembrane) and peripheral proteins.

    • Carbohydrates: Glycoproteins and glycolipids contributing to the glycocalyx.

  • Functional Importance:

    • Essential for survival and homeostasis, controls selective permeability, vital for signaling.

Cytoplasmic Organelles

  • Organelles:

    • Membranous: e.g., ER, Golgi apparatus, lysosomes, mitochondria.

    • Non-Membranous: e.g., ribosomes, centrosome.

Ribosomes

  • Composed of nucleoproteins, crucial for protein synthesis:

    • Free Ribosomes: Scatter in cytoplasm; synthesize cytosolic proteins.

    • Attached Ribosomes: Bound to rough ER; synthesize secretory proteins.

Endoplasmic Reticulum (ER)

  • Types:

    1. Rough ER: Studded with ribosomes; involved in protein synthesis and post-translational modification.

    2. Smooth ER: Lacks ribosomes; synthesizes lipids and detoxifies.

Golgi Apparatus

  • Membranous organelle involved in processing and packing proteins from rER.

  • Consists of 4-6 saccules; has a cis face (receiving) and trans face (shipping). Functions include:

    • Modifying proteins, packaging them into vesicles for secretion or delivery within the cell.

Mitochondria

  • Powerhouses of the cell; site of ATP production.

  • Composed of outer and inner membranes with cristae.

  • Contains mitochondrial DNA and matrix enzymes.

Lysosomes

  • Membrane-bounded organelles with hydrolytic enzymes for intracellular digestion and metabolism.

Cytoskeleton

  • Three components:

    1. Microfilaments: Actin; involved in movement and shape.

    2. Microtubules: Tubular structures; important for vesicle movement and mitosis.

    3. Intermediate Filaments: Provide structural integrity, characteristic of different cell types.

Centrosome

  • Non-membranous organelle, organizing center for microtubules, crucial in cell division and cilia formation.

Cytoplasmic Inclusions

  • Metabolically inert accumulations representing stored energy (glycogen, lipids) and pigments (hemoglobin, melanin).

The Nucleus

  • Largest organelle; stores genetic material. Components:

    • Nuclear Envelope: Double membrane with pores for communication.

    • Chromatin: DNA with histone proteins; exists as heterochromatin (inactive) and euchromatin (active).

    • Nucleolus: Site of ribosomal RNA synthesis.

Cell Cycle

  • Comprises interphase (prep) and cell division (mitosis).

Types of Cell Populations

  • Static Population: Non-dividing cells (e.g., neurons).

  • Stable Population: Can proliferate (e.g., liver cells).

  • Labile Population: Continuously dividing (e.g., blood cells).