Histology and Microscopy Overview

Histology

• Histology is the study of the microscopic structure of normal tissues.
• The goal of histology is to understand microanatomy of cells, tissues, and organs and to connect structure with function.

Microscopy

• Light microscopes (LM) and Electron microscopes (EM) differ in resolution and magnification.
• Optical resolution is the minimum distance at which two points can be seen as separate.
• Resolution for the eye is 0.2 millimeters, for LM is 0.2 micrometers, and for EM is 0.2 nanometers.
• The maximum magnification for LM is about 1000x while EM can magnify over 100,000x.

Cell Staining

• Cells are colorless and can be indistinguishable under LM unless stained
• Acidic stains, like eosin, stain basic structures (acidophilic)
• Basic stains, like hematoxylin, stain acidic structures (basophilic)
• Neutral stains, like Leishman’s stain, combine acidic and basic stains to stain blood cells
• Vital stains, like trypan blue, stain living structures inside living animals
• Supravital stains, like brilliant cresyl blue, stain living cells outside a living person
• Metachromatic stains change color after staining, like toluidine blue staining mast cell granules violet.
• Orcein stain gives elastic fibers a brown color.
• Silver stain can stain reticular fibers brown or black, and can also demonstrate the Golgi apparatus.
• Osmic acid stains myelin sheath black
• Histochemical and cytochemical stains localize and demonstrate specific substances within a tissue or cell through biochemical reactions.

The Cell

• The cell is the functional and structural unit of all living tissues.
• It is the smallest living structure capable of growth, secretion, excretion, digestion, and reproduction.
• Cells vary in shape, size, and function, but have a similar composition.

Cytoplasm

• Cytoplasm is formed of:
  • Cytoplasmic matrix: a colloidal solution containing proteins, carbohydrates, lipids, minerals, and enzymes.
  • Cytoplasmic organelles: permanent, minute living structures essential for vital processes.
  • Cytoplasmic inclusions: non-living temporary structures, stored substances like glycogen, fat, and pigments.

Cytoplasmic Organelles

• Cytoplasmic organelles are classified by the presence or absence of membranes:
  • Membranous cell organelles:
    • Cell membrane
    • Mitochondria
    • Endoplasmic Reticulum (Rough and Smooth ER)
    • Golgi Apparatus
    • Lysosomes
  • Non-membranous cell organelles:
    • Ribosomes
    • Cytoskeleton:
      • Microtubules
      • Filaments: (Thin, Intermediate, and Thick)

Membranous Cell Organelles

Cell Membrane

• The cell membrane is an ultra-thin membrane that surrounds the cell.
• It is invisible under LM, but can be stained with silver or PAS.
• Under EM, it appears as three parallel lines: two dark layers separated by a light layer (trilamellar membrane).
• The cell membrane has an outer covering rich in carbohydrates called the cell coat.
• Molecular structure: Composed of lipids, proteins, and carbohydrates.
• Functions:
  • Encapsulation: forms an envelope around the cell.
  • Regulation of substance exchange: Controls what enters and leaves the cell.
  • Selective permeability: Possesses receptors in the cell coat that allow only certain substances to enter (receptor-mediated endocytosis).
  • Bulk transport (vesicular transport): Macromolecules enter and leave the cell through changes in plasma membrane and formation/fusion of vesicles.
    • Exocytosis: Substances leave the cell.
    • Endocytosis: Substances enter the cell.
      • Phagocytosis: Solid substances enter.
      • Pinocytosis: Fluid substances enter.

Mitochondria

• It is a membranous cell organelle, considered the “powerhouse” of the cell.
• Responsible for cellular respiration and energy production.
• Number varies based on activity (liver cells have 1000-2000 mitochondria).
• Absent in red blood cells.
• Site: Often found where activity is highest (like at the apical end of ciliated cells).
• Under LM: Appear as granules, rods, or filaments. Requires special stains to be visualized (iron hematoxylin stains black, Janus green stains green).
• Under EM:
  • Each mitochondrion is a rounded or oval vesicle.
  • Covered by double membranes, separated by a narrow intermembranous space.
  • Outer membrane is smooth, while inner membrane is folded into incomplete folds called cristae.
  • Mitochondrial matrix fills the internal cavity and contains lipids, proteins, carbohydrates, calcium, magnesium, DNA, RNA, and oxidative enzymes.
• Functions:
  • Produce energy (ATP) through oxidative enzymes.
  • Can form their own proteins and divide because they contain DNA and RNA.

Endoplasmic Reticulum

• A membranous organelle formed by flattened communicating tubules that create a network within the cytoplasm.
• Classified based on the presence or absence of ribosomes.
  • Rough endoplasmic reticulum (rER): has ribosomes attached to its surface.
  • Smooth endoplasmic reticulum (sER): lacks ribosomes.

Non-Membranous Cell Organelles

Ribosomes

• Appear as small electron-dense particles under EM.
• Formed of two subunits, one large and one small, connected by messenger RNA (mRNA).
• Large subunit has a central groove where the newly formed polypeptide chain is situated.
• Three types of ribosomes:
  • Free ribosomes: Scattered diffusely in the cytoplasm (common in immature cells like embryonic and stem cells).
  • Polyribosomes: Ribosomes are connected by mRNA in a spiral or rosette form.
  • Attached ribosomes: Ribosomes are attached to the rER through the large subunit.
• Function: Provide the site for protein synthesis, where amino acids are linked together to form polypeptide chains.
  • Proteins formed by free ribosomes are used within the cell.
  • Proteins formed by rER are secreted and used outside the cell via transfer vesicles to the Golgi apparatus, then secretory vesicles, then the cell membrane.

Nucleus

• Shape: Can be rounded, oval, flat, horseshoe, kidney shaped, segmented, or tabulated.
• Site: Can be central, eccentric, peripheral, or basal.
• Number:
  • Single: Mononucleated.
  • Two nuclei: Binucleated.
  • Many nuclei: Multinucleated.
• Under LM: Appears as a prominent basophilic structure within the cell due to its nucleic acids (DNA and RNA).
  • Vesicular nucleus: Pale stained with extended chromatin.
  • Condensed nucleus: Darkly stained with condensed chromatin.
• Under EM: Composed of:
  • Nuclear membrane
  • Nuclear chromatin
  • Nuclear sap
  • Nucleolus

Nuclear Membrane

• Under LM: Appears as a blue line due to the presence of chromatin on its inner surface and ribosomes on its outer surface.
• Under EM: Consists of a double-walled membrane with two layers separated by a perinuclear space.
• Nuclear pores: present at intervals where the two layers fuse together. Covered by diaphragms. Allow substances to pass between the nucleus and cytoplasm in both directions.

Nuclear Chromatin

• Definition: Nucleoprotein (DNA + protein) that forms the chromosomes of the cell.
• Under LM: Appears as a basophilic substance filling the nucleus.
• Under EM: Classified according to cell activity:
  • Euchromatin: Extended form of chromatin, invisible under LM, pale-stained, carries active genes (present in active cells).
  • Heterochromatin: Condensed form of chromatin, large masses, darkly stained, carries inactive genes (present in inactive cells).
  • Classification of chromatin by site:
    • Peripheral: Adherent to the nuclear membrane.
    • Nucleolus-associated: Present around the nucleolus.
    • Chromatin islands: Scattered between the nucleolus and the nuclear membrane.

Nucleolus

• Under LM: Appears as a dark basophilic structure within the nucleus.
• Can have one, two, or no nucleoli depending on activity.
• Under EM: Consists of:
  • Light area: Nuclear sap.
  • Dark area:
    • Granular part: Ribosomal RNA (rRNA).
    • Fibrillary part: New filaments of rRNA around DNA filaments.
    • Nucleolus-associated chromatin.
• Function: Formation of rRNA, which is essential for protein synthesis. Cells that produce a lot of protein have well-defined nucleoli.

Nuclear Sap

• Fluid that fills the areas between the nucleolus and chromatin islands.
• Acts as a transport medium for ribosomes from the nucleus to the cytoplasm.

Functions of the Nucleus

• Responsible for cell division because it contains chromosomes carrying genetic information.
• Controls all vital processes within the cell, such as protein formation.
• Forms the different types of RNA (mRNA, tRNA, rRNA).

This quiz covers key concepts in histology, including the microscopic structure of tissues, various microscopy techniques, and the importance of cell staining. Understand the differences between light and electron microscopes, as well as the different staining methods used to visualize cells.