Preparation of samples for histological examination by light and electron microscopy

Sampling

 * Purpose: obtain specimen of cells or tissue
 * Size: 0.5-1 cm3 for light microscopy, ~1 mm3 for electron microscopy
 * Methods: biopsy (excision, puncture, curettage, ascites, and aspiration) or necropsy

Fixation

 * Purpose: stabilize cell and tissue structures by denaturing them . This is necessary, since freshly removed tissues are
 * chemically unstable
 * will dry up and shrink
 * suffer from hypoxia and bacteria
 * will autolyse (degrade via own enzymes )
 * A good fixative must
 * preserve the structure well
 * quickly penetrate into the tissue block
 * not interact negatively with the staining
 * Most commonly used substances include Formaldehyde for LM (12-24h); Glutaraldehyde for EM (1-3h).
 * Ethanol, organic acids, inorganic acids, heavy metal salts, or compounds are also used
 * Excess fixative is rinsed off with water
 * Water is removed via ascending series of ethanol
 * To allow embedding medium to enter, ethanol is cleared with a solvent miscible in both (ex: xylen)

Embedding

 * Purpose: give firm texture to the sample to allow thin cutting
 * Hard tissues (like dental or bone) require softening by acid (decalcification), or grinding to thin specimens
 * Infiltration: tissue is placed into molten embedding medium (typically paraffin for LM, epoxide for EM)
 * Hardening at room temperature
 * Stable method: Formalin-Fixed-Paraffin-Embedded (FFPE) . Many applications.

Cutting

 * Microtomes are used to precisely control thickness
 * Types: sliding, rotary, cryotomes, ultramicrotomes
 * Cryostat: rotary microtome in freezing box; used to cut frozen tissue without embedding
 * Thickness: 5-10 μm (light microscopy), 70-100 nm (electron microscopy)

Staining

 * Before starting, one must affix sections to microscopic glass using albumin or gelatin
 * Purpose: cells and their contents are usually colorless and thus invisible without staining for light microscopy. Electron microscopy uses heavy metals instead of staining for visualization
 * Chromophilic compounds have high affinity for dyes, chormophobic ones do not.
 * Basophilic components take up basic dyes (ex: nucleic acids); acidophilic components take up acidic dyes (cytoplasm, ionized proteins). Eosinophilic compounds take up eosin (ex: collagen)
 * Routine staining visualizes all tissue components ; special staining visualizes a particular structure.
 * Mounting: attach cover slip with transparent adhesive over stained sample on slide