Microscopy techniques

Microscopy techniques

Microscopy techniques

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Light Microscopy - The Basic Toolkit

Labeled Diagram of a Compound Light Microscope

  • Principle: Uses visible light passed through a specimen and glass lenses. Total magnification = (Objective lens) x (Eyepiece lens).
  • Resolution: The ability to distinguish between two close points. Limited by the wavelength of light. Max resolution ≈ 0.2 µm.
    • Formula: $R = 0.61λ / NA$ (where λ = wavelength, NA = numerical aperture).
  • Common Stains (H&E):
    • Hematoxylin (H): Basophilic. Stains acidic, negatively charged structures (e.g., DNA, RNA, ribosomes) a purplish-blue.
    • Eosin (E): Acidophilic/Eosinophilic. Stains basic, positively charged structures (e.g., cytoplasm, collagen) a reddish-pink.
    • 📌 Mnemonic: BASophilic = Blue; Acidophilic = piNK.

⭐ The absolute limit of resolution for light microscopy is ~200 nm. Therefore, structures smaller than this (e.g., individual ribosomes, viruses, macromolecules) cannot be visualized.

Electron Microscopy - Ultimate Zoom Lens

  • Principle: Utilizes a focused beam of electrons instead of light, achieving significantly higher resolution (down to 0.1 nm) to visualize cellular ultrastructure.
  • Types:
    • Transmission EM (TEM): Provides high-magnification, 2D images of a specimen's internal composition. Electrons are transmitted through an ultra-thin slice. Essential for viewing organelles, viruses, and molecular complexes.
      • 📌 Mnemonic: TEM = Transmits Through.
    • Scanning EM (SEM): Generates lower-magnification, 3D images of a specimen's surface topography. An electron beam scans the surface, causing secondary electron emission.
      • 📌 Mnemonic: SEM = Surface Scanning.

⭐ EM is the gold standard for diagnosing diseases involving ultrastructural changes, such as Minimal Change Disease, by visualizing the effacement of podocyte foot processes in the glomerulus.

TEM vs. SEM: Principles and Components

Special Techniques - Seeing the Unseen

  • Immunofluorescence (IF): Visualizes antigens using fluorescently-tagged antibodies.

    • Direct IF: Labeled primary antibody.
    • Indirect IF: Labeled secondary antibody; offers ↑ signal amplification. Immunofluorescence Methods Comparison
  • Immunohistochemistry (IHC): Uses an enzyme-linked antibody to create a visible colored product (e.g., brown). Routinely used to classify tumors.

  • Electron Microscopy (EM): High-resolution view of cellular ultrastructure.

    • Transmission (TEM): 2D view of internal structures (organelles).
    • Scanning (SEM): 3D surface view.
  • Flow Cytometry: Analyzes properties of single cells in suspension using laser optics. Identifies cell surface markers (e.g., CD markers).

  • In Situ Hybridization (ISH): Detects specific DNA/RNA sequences in tissues using a labeled probe. Used for viral detection (CMV, HPV) or gene localization.

⭐ Flow cytometry is essential for diagnosing and classifying leukemias/lymphomas by detecting specific lymphocyte CD markers.

Artifacts - When Glitches Happen

  • Shrinkage: Fixation/dehydration causes artificial spaces between cells and stroma.
  • Folds/Wrinkles: Tissue folding during mounting creates dense, dark lines obscuring details.
  • Knife Marks: Parallel lines/tears from a nicked microtome blade.
  • Air Bubbles: Round, clear spaces under the coverslip that refract light.
  • Ice Crystal Artifact: "Swiss cheese" holes in frozen sections from ice formation.

⭐ Formalin pigment, a brown/black granular deposit, can mimic hemosiderin. It's an acid hematin artifact, preventable with buffered formalin.

Histology artifacts: knife marks and shrinkage

High‑Yield Points - ⚡ Biggest Takeaways

  • Hematoxylin (blue) stains basophilic structures (e.g., DNA); Eosin (pink) stains acidophilic proteins.
  • Electron microscopy offers supreme resolution: TEM for internal ultrastructure, SEM for 3D surface views.
  • Immunofluorescence uses fluorophore-tagged antibodies to localize specific proteins.
  • Flow cytometry counts and sorts cells, often using CD markers for identification.
  • In Situ Hybridization (ISH) detects specific DNA or RNA sequences directly in tissue.
  • Darkfield microscopy is vital for visualizing unstained live organisms like spirochetes.

Practice Questions: Microscopy techniques

Test your understanding with these related questions

A medical technician is trying to isolate a pathogen from the sputum sample of a patient. The sample is heat fixed to a slide then covered with carbol fuchsin stain and heated again. After washing off the stain with clean water, the slide is covered with 1% sulfuric acid for decolorization. The sample is rinsed again and stained with methylene blue. Microscopic examination shows numerous red, branching filamentous organisms. Which of the following is the most likely isolated pathogen?

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Flashcards: Microscopy techniques

1/10

The base of a cilium below the cell membrane, called the _____, consists of 9 microtubule triplets with no central microtubules

TAP TO REVEAL ANSWER

The base of a cilium below the cell membrane, called the _____, consists of 9 microtubule triplets with no central microtubules

basal body

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