Introduction to Microscopic Techniques

  • Microscopes: Optical devices for observing microscopic objects (<70µm).
  • Historical Context:
    • First microscope by Zacharias Jansen (1590);
    • Robert Hooke's Micrographia (1665): introduced biological term "cell".
    • Antonie van Leeuwenhoek (1676): designed a simple microscope.
    • Carl Zeiss initiated mass production of microscopes (1847).
    • First electron microscope constructed in 1933.
  • Importance: Standard equipment in biomedicine.

Types of Microscopes

1. Light Microscopes

  • Use white or ultraviolet light.
  • Light Sources: Sunlight, bulb or vapour lamp.
  • Optical Parts: Cut glass lenses; provides low resolving power (0.2 µm), max theoretical magnification 2,000x (usually up to 1,000x in practice).
    • Observation Methods: Native samples in passing light; fluorescence/inverted for specific methods.

2. Electron Microscopes

  • Use electron beams from cathodes, electromagnetic lenses.
  • Higher resolving power (0.2 nm), max magnification up to 1,000,000x.
  • Sample Preparation: Fixation, staining, contrasting required.
    • Types:
    • Transmission Electron Microscope (TEM): Electron beam through sample.
    • Scanning Electron Microscope (SEM): Electron beam scans surface for details.

Specific Microscope Types

1.2.1 Stereomicroscope

  • Also called dissecting microscopes.
  • Focus from two angles for 3D view; low magnification (<100x).
  • Used in diagnostics and surgeries (gynaecology, neurosurgery).

1.2.2 Inverted Microscope

  • Light source above, optics below; designed for cell culture observation.

1.2.3 Fluorescence Microscope

  • Uses UV light to excite fluorescent dyes; important for molecular cytogenetics (e.g., FISH).

1.2.4 Polarized Microscope

  • Uses polarized light to view structures (chitin, cell fibers).

Construction of Light Microscope

  • Comprised of optical, lighting, and mechanical parts.
    • Optical Part: Two lens types (objective and eyepiece). Magnification = Objective x Eyepiece (40x10 = 400x).
    • Lighting System: Includes light source, filters, and condenser.

Recommended Procedure for Microscopic Observation

  • Steps for preparing and viewing the slide:
    1. Place slide on stage, secure with clips.
    2. Start with low power objective.
    3. Adjust for focus using coarse and fine knobs.
    4. Rotate objectives for increased magnification.

Errors in Microscopy

  • Common errors include:
    • Low condenser position: raise it.
    • Dirty slides: clean them.

Types of Slide Preparations

  • Impression Preparations: Clean slide pressed onto tissue surface (
    e.g., liver cells).
  • Smear Preparations: Suspension spread across the slide (e.g., blood smear).
  • Covered Slides: Suspensions covered by cover slip.

The Cell

  • Definition: Basic unit of life (Latin: "cellula").
  • Functions: Metabolism, growth, reproduction.
  • Observations led to the cell theory via Hooke (1665), Leeuwenhoek (1715-1722), and the reformulation by Schleiden, Schwann, and Virchow.

Prokaryotic Cell

  • Composed of cytoplasm, nucleoid, membrane, and cell wall.
  • Genetic Material: Single circular DNA, smaller ribosomes (70S).
  • Reproduction: Asexual (binary fission, conjugation).

Eukaryotic Cell

  • Contains a nucleus and multiple organelles; organized into tissues.
  • More complex in structure than prokaryotes.

Molecular Structure of Cell Membranes

  • Composition: Phospholipid bilayer with proteins.
  • Functions: Selective permeability, signaling, transport structures.

Transport Mechanisms

  • Passive Transport: No energy required, occurs down concentration gradient (e.g., osmosis, diffusion).
  • Active Transport: Requires energy, moves substances against the gradient (e.g., Na+-K+ pump).

Endocytosis and Exocytosis

  • Endocytosis: Invagination of the membrane to bring substances into the cell (e.g., phagocytosis, pinocytosis).
  • Exocytosis: Vesicular transportation out of the cell.

Intercellular Communication

  • Mechanisms include nerve and humoral signaling.
  • Types: Endocrine, paracrine, and autocrine interactions.

Cell Organelles

  • Key organelles include ribosomes, mitochondria, Golgi apparatus, endoplasmic reticulum, and lysosomes.
  • Functionality: Energy production (mitochondria), biosynthesis (ER), protein processing (Golgi).

DNA and Replication

  • Nucleic Acids: DNA as the carrier of genetic information.
  • Replication Process: Semiconservative, involving DNA polymerase.

Transcription and Translation

  • Transcription creates mRNA from DNA; translation creates proteins from mRNA.
  • Modifications and Mechanisms: splicing involves removing introns from hnRNA to produce functional mRNA.

Laboratory Techniques in Molecular Genetics

1. Isolation Techniques

  • Various methods for obtaining DNA from biological specimens.
  • PCR: Amplification method for DNA sequences.

2. Electrophoresis

  • Technique for separating nucleic acid fragments.

3. Enzymatic Modifications

  • Restriction enzymes used for cutting DNA.

Viruses

  • Basic characteristics: acellular, requiring host cells for replication.
  • Classification: By nucleic acid type (DNA/RNA), shape, and infection cycle (lytic vs lysogenic).
  • Human Impact: Pathogenicity varies, resulting in various diseases.