Principles of Microscopy and Tissue Processing

purpose for tissue processing

• tissue begins to decay; autolysis of cells leaks enzymes into the tissue, causing breakdown of proteins, fats, and other substances

• pathogens (bacteria) can also be destructive

• the tissue may not be visible; must be cut into thin slices (paraffin sections are usually 3-5 micrometers thick); difficult to cut thin sections if the tissue is not fixed with a more sturdy architexture

fixation

• heating (more common with smears of microorganisms)

• microwaving

• cryopreservation (freeze drying)

• chemical fixation is more commonly used for diagnostic tissue preservation

  • tissue is placed in a container of fixative for a specific amount of time based on the tissue size (ex: in the video, the rat kidney is fixed for 24 hours)

  • type of fixative depends on the tissue and purpose (what you are looking for in the tissue)

  • some fixatives work to denature; replace free water and destabilize hydrophobic bonds, ultimately making water soluble proteins insoluble so that they are not lost in further treatment of the tissue

  • some fixatives work to cross-link proteins, stabilizing them

  • combinations of different chemicals are used to achieve the desired effect including lack of shrinkage of the tissue, which occurs when it is exposed to alcohols

dehydration

removes the water from tissue so that the paraffin (wax) can penetrate the tissue effectively; this provides a stable architecture so the tissue can be thinly sliced

• tissue is exposed to increasing concentrations of alcohols over time to avoid shrinkage

• fixation process removes lipids so they appear as white spaces in the cell

clearing

• reagents remove the alcohols to make the tissue more homogenous for the paraffin; also makes the tissue more translucent for light microscopy

• most common reagent: xylene

• toluene, chlorform are also reagents (chlorform is not as common, health hazard)

embedding

tissue is placed in molten wax/paraffin

sectioning

• the solidified embedded tissue is then sectioned using a microtome

• sections are placed on a slide and then dried

staining

• wax is removed and tissue is rehydrated through a series of washes

• staining is accomplished by placing the slide in different dyes

  • type of dye depends on what is being stained; most common at Hematoxylin and Eosin

mounting

• coverslips are added to protect the tissue

• reagent used to mount the cover slips is specifically designed so it does not change the light refraction index from the glass slide, preventing distortion when viewing the tissue

light microscopy (LM)

• uses a light source and different levels of objectives to magnify the field of vision

  • different types of microscopes and set-ups allow you to view both fixed cells/tissue and live cells

  • objective used is often noted in light micrograph images (20x, 40x, 100x)

  • different stains allow you to view different components of the cell

  • antibodies that recognize specific proteins allow you to stain those proteins; you can also label DNA and RNA

  • immunofluorescence is commonly used to visualize localization of proteins

electron microscopy (EM)

higher resolution, can view details of individual organelles

transmission electron microscope (TEM)

tissue is stained with heavy metals using antibodies that recognize specific proteins or other molecules

scanning electron microscope (SEM)

scans the surface of the tissue

hematoxylin

Basic dye; binds to acidic components which are basophilic (attracted to basic); Stains DNA and RNA blue (nucleus). Used on all tissues

eosin

Acidic dye; binds to basic components that are acidophilic (attracted to acidic); Stains cytoplasmic elements pink

Wright and Giemsa stains

Contain eosin and methylene blue; protein stains pink, nuclei stain bluish purple; Used in blood smears and bone marrow

Luxol fast blue + cresyl violet

Stains nuclei and Nissl substances violet-purple; Used to stain nerve tissue

osmic acid

Stain and fixative used to preserve lipids; Commonly used for cell membranes in EM

Gomori aldehyde fuchsin (GAF)

Stains elastic fibers dark purple-black

Trichrome - uses 3+ stains (masson ttrichrome is common)

Collagen is blue/green, nuclei are blue-black, muscle fibers are red; Used to look at the collagen in muscle

Periodic acid-Schiff (PAS)

Carbohydrate groups stain purple-magenta, hematoxylin stains the nuclei blue; Used commonly to look at cartilage