Microscopy

Classification of Microscopes

  • Microscopes are classified based on their energy source:

    • Continuous light (visible light) in a light microscope.

    • Polarized light in a polarizing microscope.

    • Ultraviolet light in an ultraviolet microscope and a fluorescence microscope.

    • Electrons in an electron microscope.

Microscopy Techniques

Tissues can be visualized using the following microscopy techniques:

  • Conventional Brightfield (Light) Microscopy

  • Fluorescence Microscopy

  • Phase-Contrast Microscopy

  • Differential Interference Microscopy

  • Confocal Microscopy

  • Polarizing Microscopy

  • All of the above microscopy types are based on the interaction of light and tissue components.

  • Electron Microscopy

    • Based on interactions of electrons and tissue components.

Light (Brightfield) Microscope

  • Key Features:

    • Light source: ordinary light with colors imparted by tissue staining, such as Hematoxylin and Eosin (H&E).

    • Composed of a series of lenses:

    • Condenser Lens: Focuses the beam of light at the level of the specimen.

    • Stage: Where the slide or specimen is placed.

    • Objective Lens: Gathers light that has passed through the specimen.

    • Ocular (Eyepiece) Lens.

    • Magnification ranges from x40 to x1000.

    • Application: Observing tissue to identify any pathology.

Resolution and Magnification

  • The resolution and magnification of cells in a light microscope largely depend on:

    • Objective Lens.

  • Resolving Power:

    • Definition: The ability of a microscope lens or optical system to produce separate images of closely positioned objects.

    • Distance Between Resolvable Points and Magnification:

    • Human eye: $0.2 ext{ mm}$

    • Light Microscope (LM): $0.2 ext{ μm}$ or $200 ext{ nm}$ (x1000 = “grand” view)

    • UV Microscope: $0.1 ext{ μm}$

    • Super-resolution Optical Microscope: $10-100 ext{ nm}$

    • Scanning Electron Microscope (SEM): $2.5 ext{ nm}$ (x1 million)

    • Transmission Electron Microscope (TEM): $0.1 ext{ nm}$ (x50 million)

    • Atomic Force Microscope (AFM): $0.05 ext{ nm}$ (x100 million)

Ultraviolet Microscope

  • Key Features:

    • Uses quartz lenses and an ultraviolet light source. Quartz does not absorb UV light as glass does, making these microscopes more expensive.

    • Uses light with a shorter wavelength (sub-300 nm) for better resolution than a normal optical microscope.

    • Image formation is dependent on the absorption of UV light by conventional fluorescent dyes in the specimen.

    • The image is recorded on a photographic plate or fluorescent screen since UV light is not visible.

  • Clinical Applications:

    • Useful in detecting nucleic acids (purine and pyrimidine bases) and amino acids.

    • Microscopy with Ultraviolet Surface Excitation (MUSE) does not require tissue sectioning/staining, allowing assessments of biopsies for quicker cancer reporting while analyzing blood cells accurately for hemoglobin content.

Confocal Scanning Microscopy

  • Key Features:

    • Provides 3D visualization of biological specimens.

    • Creates multiple images at varying depths within the specimen, layer-by-layer.

    • Better image quality than fluorescence microscopy, resulting in less blurring.

  • Clinical Applications:

    • Useful for visualizing living cells and tracking the movement and function of tagged molecules.

Electron Microscopy

  • Provides a much better resolution, as the wavelength of the electron beam is less than that of visible light, although sample preparation is complex, time-consuming, and requires expensive instrumentation.

  • Types of Electron Microscopes:

    1. Transmission Electron Microscope (TEM): Similar to a light microscope, showing 2-D structure.

    2. Scanning Electron Microscope (SEM): Bounces electrons off the surface to show 3-D structure.

  • Clinical Use:

    • Visualizing viruses, cellular components, cellular processes, and diseases not visible under a light microscope.

  • Details in Electron Microscopy:

    • TEM shows ultrastructural detail, e.g. in the Rough Endoplasmic Reticulum (RER).

    • Electron detectors, such as charge-coupled devices (CCD), convert images to real-time observations on a monitor.

  • Recent advancements include Three-Dimensional Electron Microscopy (3D EM) for reconstructing entire cells and their organelles, e.g. using Electron Tomography (ET), Cryo-EM, Serial Block-face Scanning EM.

Phase Contrast Microscopy

  • Allows observation of unstained intact and living cells:

    • Light through denser areas is deflected, creating an out-of-phase effect.

  • Clinical Applications:

    • Used to observe living cells without staining and visualize organelles.

Darkfield Microscopy

  • Only light that has been scattered or diffracted by structures in the specimen reaches the objective lens, resulting in a dark background.

    • Clinical Use: Useful for examining urine for unstained crystals (like uric acid and oxalate) and thin unstained bacteria (e.g., Treponema pallidum).

Fluorescent Microscopy

  • The phenomenon of fluorescence occurs when certain substances emit light with a longer wavelength upon irradiation with light of a certain wavelength:

    • Tissue sections are irradiated with ultraviolet (UV) light. The fluorescent substance (fluorophore) gets excited and emits light in the visible spectrum that appears brilliant against a dark background.

  • Clinical Applications:

    • Used for identifying and localizing specific cellular structures and pathogens.

    • Green Fluorescent Protein (GFP) is commonly used as a fluorophore tagged with antibodies in immunohistochemistry.

  • This is the only microscopy mode where the specimen is the source of emitted visible light.

Staining with Fluorescent Compounds

  • Fluorescent compounds with an affinity for specific cell macromolecules can be used to stain:

    • Hoechst Stain and DAPI: Specifically bind to DNA to stain cell nuclei.

    • Acridine Orange: Binds both DNA and RNA.

Immunohistochemistry

  • A method for identifying and localizing specific proteins within cells or extracellular matrix.

  • Requires understanding of various methodologies, including autoradiography and enzyme histochemistry.

Atomic Force Microscopy (AFM)

  • AFM is a scanning probe microscopy technique using a sharp tip on a cantilever to scan a sample surface, creating high-resolution 3D images at an atomic level by measuring forces between the tip and surface.

  • Clinical Uses:

    • In studying mechanical and surface properties of cells and tissues, aiding in cancer diagnosis and treatment monitoring.

Virtual Microscopy

  • Virtual microscopy digitizes physical glass slides for analysis on a computer screen, allowing zooming, panning, and annotation.

Use Cases of Various Microscope Types

  1. Brightfield: Standard microscope.

  2. Darkfield: Allows examination of live specimens with contrast enhancement without staining.

  3. Phase Contrast: Produces high-contrast images without staining for live specimens.

  4. Differential Interference Contrast (DIC): Enhances contrast with interference patterns for detailed cell structures.

Additional Microscopy Techniques

  1. Fluorescence Microscopy: Identifies pathogens and localizes molecules using fluorescent stains.

  2. Confocal Microscopy: Produces high-resolution images at various depths and constructs 3D images from scanned 2D images.

  3. Two-Photon Microscopy: Uses a scanning technique with fluorochromes and long wavelengths to penetrate thick specimens.

  4. Transmission Electron Microscopy (TEM): Visualizes small specimens and subcellular structures using electron beams.

  5. Scanning Electron Microscopy (SEM): Visualizes surfaces and their 3D details using electron beams.

  6. Scanning Tunneling Microscopy (STM): Maps surface structures at atomic levels.

  7. Atomic Force Microscopy (AFM): Observes specimens at the atomic level and can work with non-conducting samples.

Correlation of 3D Organ Structure to 2D Microscopic View

  • Correlating a 3D model from multiple 2D slices or using techniques like deep learning to infer 3D structures from images.

  • Understanding the 3D architecture of organs is essential due to inherent limitations in microscopy observing specimens in 2D.

Magnification Levels in Histology Slides

  • Different levels of magnification in histology:

    1. Naked eye observation.

    2. Low Power (LP) magnification (x100).

    3. High Power (HP) magnification (x400 to x1000).

Artifacts in Microscopy

  • Definition: Apparent defects or structural details due to errors in specimen processing.

  • Common artifacts include:

    • Poor sampling,

    • Shrinkage,

    • Folds,

    • Stain precipitation and dust,

    • Knife defects,

    • Poor fixation leading to autolysis.

  • Examples of artifacts affecting tissue sections:

    1. Shrinkage causing rupture of the epithelium in stratified squamous nonkeratinized epithelium.

    2. Folds caused during processing of the epididymis.