Fixation: Preserves structural organization of cells, tissues, and organs. Prevents bacterial and enzymatic digestion, insolubilizes components to prevent diffusion, and reduces damage in later steps in tissue processing.
Chemical Fixation: Common approach. Fixatives are used individually or in mixtures (Table 1–2). Best results achieved by rapid penetration of living tissue with fixative. Small tissue pieces may be fixed by immersion. Entire organs better fixed by perfusion (fixative pumped through blood vessels).
Fixative-induced changes in chemical composition and fine structure may produce staining artifacts. Structural changes include denaturing and cross-linking proteins.
Freezing (Physical Fixation): Used for light or electron microscopy. Tissue embedded in cryoprotectant (glycerin). Rapid freezing reduces ice crystal formation and associated artifacts. Allows tissue to be sectioned (or fractured [Table 1–4]) without dehydration or clearing (Fig. 1–1). Faster. Avoids dissolving lipids and denaturing proteins (e.g., enzymes, antigens).
Less permanent. Sections are thicker. Resolution is poor.
Dehydration (substitution): Eases tissue penetration by clearing agent. Prepares fixed tissue for infiltration with embedding medium.
Replaces tissue water with organic solvent; commonly, ethanol. Fixed tissue immersed in series of alcohol–water mixtures with increasing alcohol concentration, to 100% alcohol.
Alcohol may denature proteins. Water loss causes uneven shrinkage of components with different water content. May create unnatural spaces between cells and tissue layers.
Clearing: Prepares fixed tissue for infiltration. Dehydrating agent replaced by clearing agent.
Dehydrated tissue immersed in series of clearing agent–alcohol mixtures with increasing clearing agent concentration, or placed directly in clearing agent. Xylene (paraffin solvent) commonly used for light microscopy. Propylene oxide (plastic solvent) commonly used for EM.
Clearing agents may denature proteins. Some components shrink unevenly as their proteins denature.
Infiltration: Prepares cleared tissue for embedding.
Cleared tissue immersed in series of clearing agent–embedding medium mixtures with increasing embedding medium concentrations, at medium-high temperature. Evaporating clearing agent replaced by embedding medium.
Heat denatures proteins. Bubbles form during poor infiltration.
Embedding: Prepares infiltrated tissue for sectioning. Makes tissue firm and prevents crushing during sectioning. Permits thin, uniform sectioning.
Infiltrated tissue positioned in a mold filled with embedding medium, which hardens into a block. Block attached to a chuck that holds it in microtome for sectioning. For light microscopy, paraffin commonly used; other media are celloidin, plastics, and polyethylene glycol (water soluble) wax. For EM, plastics, epoxy resins (e.g., Epon and Araldite). Require catalyst to harden (polymerize) after infiltration. Harder embedding media allow thinner sectioning, requirement for EM.
Improved sectioning allowed by embedding has limitations associated with dehydration, clearing, and infiltration.
Sectioning: Most tissues too thick and opaque for microscopic analysis. Thin slices allow light or electrons to penetrate specimen and form image.
For light microscopy, standard rotary microtome with steel blade cuts 3–8-μm sections of specimens embedded in paraffin, celloidin, or polyethylene glycol. Glass or diamond knives cut 1–5-μm sections of plastic-embedded tissue. Frozen sections, 5–25 μm, cut in a cryostat (standard microtome in a refrigerated chamber). For EM, ultramicrotome with a glass or diamond knife cuts very thin sections (0.08–0.1 μm or up to 0.5 μm for high-voltage EM). Ultramicrotomes include stereomicroscope to observe cutting.
Sections provide two-dimensional image of three-dimensional structure. Dull knife can crush or pinch tissue. Chatter (wavelike variations in section thickness) results from knife vibration during sectioning. Burr on knife can tear tissue.
Mounting: Eases handling and decreases damage to specimen during examination.
For light microscopy, sections placed on glass slides, often precoated with thin layer of albumin, gelatin, or polylysine to improve attachment. After staining, sections covered by glass coverslips to preserve them for repeated examination. For EM, specimens mounted on copper grids. Electron beam cannot penetrate glass.
Tissue sections may develop folds, making some regions appear to have more cells and stain darker. For grid-mounted specimens, only portions lying between crossbars are visible.
Staining: Most tissue sub-structure is indistinguishable. Stains, ligands with specific binding affinities and optical properties, and radiolabels help localize and distinguish cell and tissue components. Knowledge of specificities of such substances (Table 1–3) provides additional information about structure and composition.
For light microscopy: Once sections are on slide, paraffin is dissolved. Tissue may be rehydrated before staining. Plastic sections stained without removing plastic. Most stain affinities based on reciprocal acid–base characteristics of stain and tissue components. Acidic stains (e.g., eosin) bind basic (i.e., acidophilic) structures and compounds (e.g., cytoplasmic proteins). Basic stains (e.g., hematoxylin) bind acidic (i.e., basophilic) tissue components (e.g., nucleic acids in ribosomes). Stain mixtures reveal multiple cell components. Hematoxylin and eosin (H & E), most common stain mixture for light microscopy, distinguishes nucleus from cytoplasm.
Acid–base boundaries may not correspond to boundaries between structures. Multiple staining procedures may be needed to characterize a particular component. Colors are controlled artifacts, best to focus more on component structure than color.
For TEM: Most stains (contrast agents) for TEM chosen for electron-absorbing or -scattering ability and affinity for particular cell components. Heavy metal salts, e.g., lead citrate and uranyl acetate, are common. Osmium tetroxide interacts with lipids to form electron-dense precipitate, doubles as stain for cell membranes.
TEM stains stop electrons from penetrating. TEM images are shadows of heavy metal deposits. Actual tissue structures not seen.
For SEM: Specimens not stained. First subjected to critical-point drying (prevents surface-tension artifacts). Dehydrated, specimens soaked in liquid miscible with CO2 or Freon and put in critical-point chamber. Chamber heated to a critical temperature (31°C), raising pressure to 73 atm, at which the gas and liquid phases exist without surface tension, liquid escapes specimen without altering structure. Specimen then mounted on a stub and sputter-coated (sprayed) with fine mist of heavy metal particles (e.g., gold) before viewing (Fig. 1–1).
SEM reveals surface architecture in detail, but heavy metal coating prevents electrons from penetrating to reveal internal structure.