Module 1

• Tissues have two interacting components: cells and extracellular matrix (ECM)
• Most common procedure used in histologic research is the preparation of tissue slices or “sections” that can be examined visually with transmitted light.
• The ECM consists of many kinds of

  macromolecules, most of which form complex structures, such as collagen fibrils.
• PREPARATION OF TISSUES

  Fixation: Small pieces of tissue are placed in solutions of chemicals that cross-link proteins and inactivate degradative enzymes, which preserve cell and tissue structure.
• Dehydration: The tissue is transferred through a series of increasingly concentrated alcohol solutions, ending in 100%, which removes all water.
• Clearing: Alcohol is removed in organic solvents in which both alcohol and paraffin are miscible.
• Infiltration: The tissue is then placed in melted paraffin until it becomes completely infiltrated with this substance.
• Embedding: The paraffin-infiltrated tissue is placed in a small mold with melted paraffin and allowed to harden.
• Trimming: The resulting paraffin block is trimmed to expose the tissue for sectioning (slicing) on a microtome.
• A microtome is used for sectioning paraffin-embedded tissues for light microscopy. (1 to 10 μm.)
• fixatives are solutions of stabilizing or cross-linking compounds
• One widely used fixative for light microscopy is formalin, a buffered isotonic solution of 37% formaldehyde.
• Electron microscopy provides much greater magnification and resolution of very small cellular structures, and fixation must be done very carefully to preserve additional “ultrastructural” detail.
• The fully cleared tissue is then placed in melted paraffin in an oven at 52°C-60°C
• Paraffin sections are typically cut at 3-10 μm thickness for light microscopy, but electron microscopy requires sections less than 1 μm thick.
• Biopsies are tissue samples removed during surgery or routine medical procedures
• A microtome called a cryostat in a cabinet at subfreezing temperature is used to section the block with tissue
• Basophilic - nucleic acids with a net negative charge (anionic), have an affinity for basic dyes
• Acidophilic - cationic components, such as proteins with many ionized amino groups, stain more readily with acidic dyes
• Simple combination of hematoxylin and eosin (H&E) is used most commonly.
• Periodic acid–Schiff (PAS) reaction - utilizes the hexose rings of polysaccharides and other carbohydrate-rich tissue structures and stains such macromolecules distinctly purple or magenta
• Hematoxylin produces dark blue or purple color in nuclei. While, eosin counterstain the cytoplasmic structures pink.
• Sudan black - lipid-soluble dyes to reveal lipid-rich structures.
• Metal impregnation - using solutions of silver salts to visualize certain ECM fibers and specific cellular elements in nervous tissue
• Light microscopy - interaction of light with tissue components and are used to reveal and study tissue features.

  Examples are bright-field, fluorescence, phase-contrast, confocal, and polarizing microscopy
• Parts of Microscope

  • condenser - focusing light on the object
  • objective lens - enlarging and projecting the image
  • eyepiece - further magnifying this image and projecting it onto the viewer’s retina or a charge-coupled device (CCD)
• Resolving power - the critical factor in obtaining a crisp, detailed image with a light microscope
• Virtual microscopy - involves the conversion of a stained tissue preparation to high-resolution digital images and permits study of tissues using a computer
• Fluorescence microscopy - is irradiated with ultraviolet (UV) light and the emission is in the visible portion of the spectrum.

  • Fluorescence - irradiated by light of a proper wavelength, they emit light with a longer wavelength
  • fluorescent substances appear bright on dark background
• Phase-contrast microscopy - uses a lens system that produces visible images from transparent objects and, importantly, can be used with living, cultured cells
• Additional info about phase-contrast microscopy:

  • Phase-contrast microscopy is based on the principle that light changes its speed when passing through cellular and extracellular structures with different refractive indices
  • A modification of phase-contrast microscopy is differential interference contrast microscopy with Nomarski optics, which produces an image of living cells with a more apparent 3D aspect
• Confocal microscopy - Avoids Stray (excess) light reduces contrast in the image by using (1) a small point of high-intensity light, often from a laser and (2) a plate with a pinhole aperture in front of the image detector
• Additional info about Confocal microscopy:

  • all optically conjugated or aligned to each other in the focal plane (confocal), and unfocused light does not pass through the pinhole
  • include a computer-driven mirror system (the beam splitter) to move the point of illumination across the specimen automatically and rapidly
• Polarizing Microscopy - allows the recognition of stained or unstained structures made of highly organized subunits

  • When normal light passes through a polarizing filter, it exits vibrating in only one direction
  • No light passes through the second filter above the first one.
  • Appear as bright structures against dark background if tissue are between two polarizing filters.
• Additional info about Polarizing Microscopy:

  • birefringence - ability to rotate the direction of vibration of polarized light, a feature of crystalline substances
  • Also a feature of crystalline substances containing highly oriented molecules, such as cellulose, collagen, microtubules, and actin filaments.
• Transmission and scanning electron microscopes are based on the interaction of tissue components with beams of electrons.
• Transmission electron microscope (TEM) - is an imaging system that permits resolution around 3 nm.

  • an upside-down light microscope
  • 400,000x
  • Very thin (40-90 nm), resin-embedded tissue are 120,000x
  • Electrons passing through a hole in the anode form a beam that is focused electromagnetically by circular electric coils in a manner analogous to the effect of optical lenses on light
• Additional info about TEM:

  • specimen through electron passed appear bright (electron lucent)
  • bind heavy metal ions preparation absorb/deflect electron appear darker (electron dense)
  • Osmium tetroxide, lead citrate, and uranyl compounds
• Scanning electron microscope (SEM) -Like the TEM, but in this instrument, the beam does not pass through the specimen.

  • surface of specimen is first dried and spray-coated with a very thin layer of heavy metal (often gold) that reflects electrons in a beam scanning the specimen
  • reflected electrons are captured by a detector, producing signals that are processed to produce a black-and-white image.
• Autoradiography - particles called silver grains indicate the cells or regions of cells in which specific macromolecules were synthesized just prior to fixation