Microscopy and Light Microscopy Types

Study Notes

Microscopy is the examination of small structural details in biological samples using a microscope
It's the starting point for discussions related to microscopic anatomy and cell biology
Microscopy relies on the principle that radiation's wavelength must be smaller than the object being examined to probe its structural details
Microscopes are laboratory equipment used to magnify and examine small features not visible to the naked eye
Light microscopes and electron microscopes are the two fundamental types
Light Microscopy: a range of wavelengths for visible light is 0.4 µm ~ 0.7 µm (400-700 nm)
Objects smaller than 0.4 µm will produce a blurry image
A compound light microscope uses multiple lenses, a light source with a tungsten filament, a condenser to focus the light and a quality lens that approaches 0.25 µm resolution limit

Bright-field Microscopy: The most common type for histology students; Transmitted light through the sample and appears dark against a bright background
Dark-field Microscopy: Simple and good for observing live, unstained samples; It utilizes an opaque disk to illuminate the sample against a dark background, making the sample appear bright
Phase Contrast Microscopy: Used for translucent and colorless samples by modifying its condenser to include an annular ring, with tungsten-halogen as a light source. Used to detect differences in thickness and refractive index
Differential Interference Contrast (DIC) Microscopy (Nomarski): Similar to phase contrast, but produces a 3D effect using shadowed, oblique illumination that works with coherent beams of light

Electron microscopy shows ultrastructural intracellular details
Resolves higher magnifications than light microscopy
Uses electron beams instead of light, and magnetic fields instead of lenses
Two imaging systems: Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM)
- TEM: Uses transmitted electrons through very thin sections of tissue to produce 2D images
- SEM: Directly studies solid surfaces by scanning the surface with electron beams. This shows a 3D character

Histological studies involve preparing and examining tissues.
Tissue Processing: Involves paraffin-embedding or fresh tissue processing
- Paraffin-Embedded: Tissues are preserved, dehydrated, cleared, and embedded in wax
- Fresh Tissue: Tissues are frozen and used without paraffin
Fixatives: Chemical solutions preserve the tissue structure and prevent decay by cross-linking proteins. A common fixative is 10% neutral-buffered formalin.
Ethyl Alcohol: Used for tissue dehydration, crucial for making them compatible for embedding

Acid-Base Reaction: Hematoxylin stains acidic structures (like nuclei) and eosin stains basic structures (like cytoplasm)
Affinity-Based Reaction: Specialized stains (like PAS) target specific tissue components through chemical interactions

Thin sections are essential for adequate light penetration during microscopy, enabling clear visualization of tissue components. Thick sectioning obscures visual clarity
Types of microtomes include rotary (for paraffin embedding), cryostat (for fresh frozen), and ultramicrotome (ultra-thin sections for electron microscopy)

Hematoxylin and Eosin (H&E): The most common stain for highlighting various structures based on their distinct affinities to the tissue structures.
- Useful for general tissue analysis
Periodic Acid-Schiff (PAS): Useful for staining carbohydrates in tissues
Importance of Clearing: Clearing removes alcohol from the tissue to make it compatible with the embedding medium (e.g., paraffin). This step allows visualization of structures and prevents artifacts from incomplete embedding.