Fundamentals of Microscopy Techniques and Cell Theory

Posted on Oct 4, 2025 in Biology

History of Optical Microscopy

Key Milestones

  • 1611: Johannes Kepler suggests how to construct a compound microscope.
  • 1655: Robert Hooke publishes Micrographia, introducing the concept of the cell.
  • In parallel, Antonie van Leeuwenhoek built more than 200 microscopes (simple magnifying glasses).

Early Optical Microscopy Pioneers

Robert Hooke (1655)

In 1655, Robert Hooke observed a piece of cork under his self-built microscope. He used the word cell to describe the small, closed compartments he noted.

Antonie van Leeuwenhoek

Antonie van Leeuwenhoek built more than 200 microscopes (simple magnifying glasses, achieving up to 270x magnification). These consisted of a copper plate holding a glass bead (lens) through which objects were observed, often mounted on a fixed needle.

19th Century Advancements

Giovanni Battista Amici corrected spherical and chromatic aberration. Later, Ernst Abbe built the first apochromatic objective.

The late 19th century saw the description of many different types of tissue, the cell nucleus, mitochondria of muscle cells, and the behavior of chromosomes during mitosis of animal cells. Bacteria causing tuberculosis and cholera were identified, and the Golgi apparatus was described.

1835: Matthias Schleiden and Theodor Schwann proposed the Cell Theory.

Sample Preparation for Optical Microscopy

  1. Staining: Staining the material with specific dyes.
  2. Fixation: Often, before staining, the sample must be fixed to prevent deterioration and facilitate dye impregnation.
  3. Freezing/Cryostat: To avoid cellular distortions and bypass fixation and inclusion steps, the sample can sometimes be frozen. This requires a special microtome (a Cryostat).
  4. Mounting: The final step. It may be temporary or permanent. Depending on the material, the sample is mounted in a non-soluble or water-soluble medium.

Sample Preparation for Electron Microscopy

The preparation is more complex, and living material cannot be observed.

  1. Fixation: Fix the sample with a stabilizing agent for proteins and lipids (e.g., glutaraldehyde or osmium tetroxide).
  2. Sectioning: Creating ultra-thin sections (between 50 and 100 nm thick) after infiltrating the sample with resin.
  3. Support: Placing the sections on a special support (a copper grid).
  4. Staining/Coating: Coating the object with a heavy metal (e.g., lead or uranium) to increase electron contrast.
  5. Viewing: The image is not seen directly, but through a fluorescent screen or captured photographically.

Types of Microscopy

Optical Microscopes (Visible Light)

  • Traditional optical microscope
  • Polarization microscope
  • Phase contrast microscope
  • Interference contrast microscope
  • Fluorescence microscope
  • Confocal microscope

Electron Microscopes

  • Transmission Electron Microscope (TEM)
  • Scanning Electron Microscope (SEM)

Microscopes Using Other Radiation or Methods

  • Infrared radiation microscope
  • Ultraviolet or X-ray microscope (Röntgen microscope)
  • Scanning Tunneling Microscope (STM)
  • Atomic Force Microscope (AFM)

The Cell Theory

The current cell theory argues that the cell is the structural, functional, and genetic unit of all living organisms.

  • Structural Unit: It is the smallest unit of all living organisms.
  • Functional Unit: It can nourish itself, reproduce, and interact with its environment.
  • Genetic Unit: From an evolutionary point of view, it originates from the first cells that appeared on Earth approximately 3.5 billion years ago.

Classification of Cells

Prokaryotic Cells

  • DNA molecules are distributed throughout the cytoplasm (lacking a nuclear membrane or typical chromosomes).
  • The cytoplasm lacks membrane-bound compartments (organelles).
  • All organisms in the Kingdom Monera possess this type of cell.

Eukaryotic Cells

  • Possess a membranous envelope that separates the genetic material from the rest of the cytoplasm (the cell nucleus).
  • The cytoplasm contains membrane-bound compartments (cellular organelles with specific functions).
  • Protists, fungi, animals, and plants are composed of these cell types, though they exhibit unique characteristics.