Transmission Electron Microscopy (TEM)

• Transmission Electron Microscopy (TEM) is an advanced technique used to examine the ultrastructure of cells and tissues with extremely high resolution, down to the nanometer scale.
• TEM can magnify specimens up to a million times, allowing for detailed visualization of histopathological changes in diseases.

Principle of TEM Operation

• TEM uses an electron beam instead of light, enabling visualization of minute cellular details.
• Electrons pass through the ultra-thin specimen, producing high-contrast images that reveal cellular and tissue structures.
• The image is captured on a fluorescent screen or digital sensor for further analysis.

Sample Preparation for TEM

• Fixation of the Specimen
• Glutaraldehyde stabilizes cellular organelles.
• Osmium tetroxide enhances contrast by binding to lipids and membranes.
• Washing and Buffering: Specimens are rinsed with phosphate-buffered saline (PBS) to remove excess fixatives.
• Dehydration: A graded series of ethanol (50% → 70% → 90% → 100%) is used to remove water from the tissue.
• Embedding in Resin: Specimens are embedded in epoxy resin to provide stability for ultra-thin sectioning.
• Ultramicrotomy (Thin Sectioning): The sample is cut into ultra-thin sections (50-100 nm thick) using a diamond or glass knife.
• Staining with Heavy Metals: Uranyl acetate and lead citrate are applied to enhance contrast and improve visualization of cellular structures.

Applications of TEM in Histopathology

• Used for Cancer Diagnosis, Viral Infections, Kidney Diseases, Neurological Disorders, and Rare Histopathological Conditions

Advantages and Limitations of TEM

• Advantages:
  • Provides extremely high resolution (nanometer scale).
  • Enables detailed ultrastructural analysis of cells and tissues.
  • Essential for diagnosing complex and rare diseases.
• Limitations:
  • Sample preparation is complex and time-consuming.
  • Requires expensive equipment and specialized expertise.
  • Does not provide 3D imaging, unlike Scanning Electron Microscopy (SEM).

Introduction to Molecular Pathology

• Molecular pathology combines traditional histopathology with molecular techniques to study genetic, proteomic, and functional changes in cells and tissues due to diseases.
• It uses advanced techniques to analyze DNA, RNA, and proteins for accurate disease diagnosis and characterization.

Techniques Used in Molecular Pathology

• Polymerase Chain Reaction (PCR): Amplifies and analyzes specific DNA or RNA sequences to detect genetic mutations.
• Real-Time PCR (qPCR): Quantifies DNA or RNA levels in a sample using fluorescent dyes.
• Fluorescence In Situ Hybridization (FISH): Detects chromosomal abnormalities using fluorescent probes that bind to specific DNA sequences.
• Next-Generation Sequencing (NGS): Simultaneously sequences multiple genes to detect mutations and genetic variations.
• Gene Expression Profiling (Microarrays & RNA Sequencing): Determines gene expression levels in diseased cells.
• Gel Electrophoresis: Analyzes DNA or protein by separating them based on size.

Clinical Applications of Molecular Pathology in Histopathology

• Used for Cancer Diagnosis, Genetic Disorders, Infectious Diseases, and Personalized Medicine.

Advantages and Limitations of Molecular Pathology

• Advantages:
  • High accuracy in disease diagnosis compared to traditional methods.
  • Early disease detection before symptoms appear.
  • Provides detailed molecular insights for targeted treatments.
• Limitations:
  • High cost of advanced techniques such as NGS and FISH.
  • Requires specialized laboratories and skilled personnel for data analysis.
  • Some tests have long processing times for results.