Histology Lec E

List the 3 components of the body

1. cells (between 270-300 dif types but not universally agreed upon)

2. intercellular substances

3. body fluids - including blood, lymph, etc

Important histological events of the 1600s

Janssen - developed the microscope

Leeuwenhoek - father of microscopy

Hooke - further developed the microscop, coined the term “cell” NOT WITH EM

Malpighi - father of histology

Important histological events of the 1800s

Early - compound microscope with multiple objectives → better magnification

Middle - cell theory by Schleiden and Schwann (with later addendum by Virchow)

Late - microtome which could cut tissue into very small pieces

Important histological events of the 1900s

General - explosion of new information bc of better microscopes, especially in field of developmental biology

1930s - invention of the electron microscope EM

1940s through 1950s - EM being used for more biological work thanks to think sectioning with the ultramicrotome

List the common linear equivalents used in histology (smallest → biggest)

• Angstrom Å

• Nanometer nm

• Micrometer μm

• Millimeter mm

List the steps of preparing dead tissue

1. rapid removal to prevent degradation by autolysis

2. fixation by way of freezing or chemicals (such as aldehydes, acid, and alcohols)

3. embedding in paraffin (for LM) or plastic (LM or EM)

4. sectioning on cryostat, microtome, or ultramicrotome

5. mounting specimens on slides and staining

6. protecting specimens with cover slips

what kind of compounds are used for tissue staining?

complex organic compounds; some require even more specific processing steps

State the 2 possible staining techniques for Microdissection

1. Vital dyes - injected into the living organism (ex. trypan blue for studying phagocytic activity in macrophages)

2. Supra-vital dyes - add to cells previously removed (ex. mitochondria with janus green, lysosome w neutral red, nerve fibers w methylene blue)

What is used in histochemistry & cytochemistry?

1 of the 3 to locate organic and inorganic compounds

• specific binding of a dye

• fluorescent-dye labeled antibody

• inherent enzymatic activity

What plays a role in how tissue responds to the various staining techniques?

• Chemical composition of the tissue

What is the most common stain used in histology?

H & E: hematoxylin & eosin

• Hematoxylin stains nuclei blue

• Eosin tains cytoplasm pink

What color does the nuclei stain if using Iron hematoxylin?

• Black

Describe the main differences between acidic and basic stains?

• Basic dyes possess a “+” charge that then reacts with anionic components of tissue like:

  • phosphate groups on nucleic acids

  • sulfate groups on GAGs

  • carboxyl groups on proteins

  • BASOPHILIA

• Acidic dyes possess a “-” charge that reacts with cationic components of tissue like:

  • cytoplasmic filaments

  • intracellular membranous components

  • extracellular components

  • ACIDOPHILIA

List 3 examples of basic aniline dyes

1. Methyl green

2. methylene blue

3. Janus green

List 3 examples of acidic dyes

1. eosin

2. aniline blue

3. acid fuchsin

How are elastic fibers stained?

• orcein

• resorcin fuchsin

• OR Weigert’s elastic stain

How are reticular fibers stained?

silver precipitate from an alkaline solution

• the structures that are stained with silver are called argyrophilic

What stain is used for collagen?

NO SPECIFIC STAIN FOR COLLAGEN

• can use acid aniline of a trichrome method such as Gomori’s, Masson’s, or Mallory’s

Describe what a trichrome staining method is?

One procedure gives three distinct different colors

• ex. Masson’s stain w. “CT” (stained green), nuclei (stained blue), and cytoplasm (stained red)

How are lipids stained?

• Sudan black B (sudanophilia) for enzyme localization and glycogen stains

• Periodic Acid-Schiff (PAS) reaction

What aspects of enzyme research have become important tools for method research?

• enzyme digestion

• enzyme histochemistry

Describe immunocytochemistry staining methods

• direct methods of staining with Ag/Ab rxn OR

• indirect method with Ag/Ab w/ a 2nd Ab rxn to the combined Ag/Ab complex

Describe autoradiography in staining methods

• uses radioisotopes (i.e., C-14, H-3, I-125) with photographic emulsions for LM or EM study of chemicals and metabolism of the cell

How does sectioning affect how tissue is viewed?

• different orientations of a tubular structure results in different overall shapes of the different histological sections

Compare the LM, TEM, and SEM in terms of resolving power (weakest → strongest)

• LM = 0.2 mm OR 200 um

• SEM = 0.2 um OR 200 nm

• TEM = 0.1 nm OR 1 A

What is the relationship between wavelength and energy of visible light

increased wavelength = decreased energy

VICE VERSA

Describe what the phase contrast microscope does?

• takes advantage of the contrast created by optical means due to differential refractive indices

  • ex. air has a lower refractive index than glass (1.0 to 1.3, respectively)

• the light that travels through equal distances of these 2 will emerge out of phase when leaving the various materials

• you use optical plates in the condenser and objectives to change phase differences into amplitude differences which makes transparent objects visible

What is a phase contrast microscope used to observe?

• living cells and tissues (ex. blood cells, membranes, tissue culture, etc.)

• unstained tissues

• plastic-embedded thick sections

Describe the darkfield microscopes

• uses a strong oblique light that does NOT enter the objective lens

  • special darkfield condenser allows no light to pass through the center of the lens

• Light enters specimen @ oblique angle → reflection of light off particles in the specimen → results in glistening spots so that objects BELOW normal levels of resolution are visible

  • think dust in a sunbeam of a dark room

  • helps us look at unstained tissue

Describe fluorescent microscopy

• Happens bc many substances emit light when exposed to UV light (aka the specimen glows)

• the specimens may be naturally occurring or chemically-induced fluorescent structures

Describe confocal scanning microscopy

• is a type of light microscopy

• provides a 3-D view of histological specimens when a detecter aperture (aka a pinhole) is conjugated with the focal point of a lens

• using a computer provides images similar to that of a CAT scan

Describe a UV microscope

• uses a quartz lens not glass bc glass is opaque to UV light which increases the resolution 2-fold to 0.1 um

• good for detecting proteins with certain amino acids and nucleic acids

Describe how polarizing microscope is used

• takes advantage of the property birefringence

• a polarizer (located below the condenser) converts normal, diffuse light into plane polarized light and an analyzer rotates perpendicular to plane polarized light waves results in production of darkfield effects

Isotropic vs Anisotropic

• Isotropic - singly refractice structures (light waves bent only once) which remain dark in the polarizing microscope

  • EX striated muscle I band

• Anisotropic - refers to doubly refractive (light waves bent twice) resulting in birefringent strucures which appear light in the polarizing microscope

  • EX striated muscle A bands, “CT” fibers, lipid droplets

Describe interference microscopy

• depends on objects’ abilities to refract light using two beams of light that combine on the image plane

• after recombination, the different refracted light beams interfere w each other and can detect and measure the refractive index of an object

Describe electron microscopy

• Plain EM - depicts high velocity electrons w wavelength = 0.05A

• transmission EM provides 2-D images on a phosphorescent screen of an electron photomicrograph

  • fix with glutaraldehyde, embed in plastic, section w glass or diamond

• scanning EM provides 3-D images on TV screen or electron photomicrograph

  • fix as freeze fracturing, dehydrate and coat evenly w/ gold or platinum for 3-D study

Describe freeze fracturing and how to prepare specimens

• freeze fix in liquid N2, fracture in a specific plane w/ a sharp knife, warm slightly to etch and make a replica of the surface with heavy metal shadowing

• then remove specimen and put replica on grid and view surface at the macromolecular level

ALSO HOW TO FIX SCANNING EM

Define atomic force microscopy

• powerful tool to study surface topography at the molecular and atomic levels