1.Intro to Histology Flashcards
Describe the microscopy methods to reveal cellular, tissues, and organ structures
bright field microscopy
fluorescence
immunohistochemistry
1) bright field microscopy:
examination by ordinary light to pass through specimen
H&E
2) fluorescence microscopy:
subtype of light microscopy that uses histochemical reagents
exam by proper wavelength and specimen will emit wavelength of visible spectrum
3) immunohistochemistry
extremely important research technique
• can be used with LM or TEM
• goal is to visualize some component (antigen) in a tissue section by means of a labeled antibody
• label for LM is usually a fluorescent molecule or enzyme such as peroxidase or alkaline phophatase bound to antibody
• label for TEM can be colloidal gold or ferritin iron bound to antibody
direct: 1:1 ration bc use 1 antibody to bind to 1 thing
indirect: 1:2 increases signal by using secondary antibody that is labelled
may be direct or indirect, with label directly attached to the primary antibody (which binds the molecule to be localized), or with the primary antibody being unlabeled and visualized by a labeled secondary antibody that binds specifically to the primary antibody.
4) Autoradiography and TEM
autoradiography is tissue prep where you can see where particles were synthesized prior to fixation by silver grains
Research method involving LM or TEM
• Goal: Localize in a tissue section a radioactive substance (drug, enzyme, etc.) that the living cells metabolized.
• After fixation and sectioning, the specimen is covered with a thin coat of photographic emulsion.
• Radiation from the isotope exposes and produces elemental silver after photographic development.
• The specific points in the emulsion (silver grains) indicate exactly where the radiolabeled metabolite is located in the cell.
• Isotopes that are low energy ß-emitters are usually best. H3 (tritium) and C14 are commonly used.
Explain common histologic methods
why do we have to section the tissue?
objectives vocab:
fixative
infiltration
embedding
Prep tissues to be studied under light microscope
- must be sectioned to allow light to pass through
1) place in fixative solution: crosslinks proteins and inactivates degredative enzymes
2) dehydration so that 100% of water is removed
3) clearing solution: removes alcohol
4) infiltration: tissue placed on melted paraffin at 58 degrees celsius to be infiltrated with the substance
As the tissues are infiltrated with the solvent, they generally become transparent (clearing)
5) embedding: tissue allowed to harden on mold containing melted paraffin
6) sectioning
explain strengths and limitation of different histological methods
light microscopy: maximum resolution is 0.2 microm limitations- 3D limited to the thickness of specimens which can only be 5-10 micrommillometers thick mm-microm limitation non-living if fixed and stained
Electron microscopy:
maximum resolution is 0.2 nano for TEM and 2nm for SEM (scanning electron microscope)
limitation: 2D image only because specimen thickness must be 0.1 - 0.5 microm
appreciate that structure and function are inseparable
use micro villa of enterocytes vs cilia of respiratory epithelium as example
microvilli:
show brush border which is important for absorption
cilia are motile and project from cell for movement
what are the levels of histological organization
- subcellular: cell organelles
ex: nucleus - cellular: specialized cells with surface modification
ex: micro villa for absorption - four basic types of tissues : epithelium, connective tissue, muscle, nervous tissue
- organs
- organ systems
what is the tenant of histology?
all tissues and organs are made by cells and the extracellular matrix they produce
describe mounting for each technique
use glass slide for light microscopy
use copper grid for TEM (transmission electron microscopy) bc glass is not transparent for electrons
describe the types of staining and colors
light microscopy:
eosin- acidic dye that bonds to (+) charged structures; pink
hematoxylin- basic dye that bonds to (-) charges; blue
Transmission electron microscopy:
heavy metal salt
scanning EM:
not stained but sprayed with heavy metal particles
what color will these things stain?
cytoplasm
nuclei
cytoplasm- pink
nuclei- purple
what are some problems in the study of tissues sections
1) shrinkage of artificial space between components
2) loss of molecules like mucus
3) wrinkles and dye precipitates shouldn’t be confused with small vessels or cytoplasmic granules
4) impossible staining differential for all tissues for LM procedure
5) TEM resolves organelles and ECMatrix but only in very small samples
describe CLARITY
1) hydrogel infusion
2) polymerization to hydrogel mesh
3) clearing of lipids by electrophoresis
4) imaging by confocal microscope
- CLARITY transforms intact tissue into optically transparent macromolecules with no slicing and keeps native molecule and structure
- confocal microscope increases optical resolution and enables reconstruction of 3d structures
resolution
“Resolution” is the ability to distinguish two close but distinct points. The best “resolving power” of various instruments is:
• human eye ~200 μm (0.2 mm)
• light microscope ~0.2 μm
• transmission electron microscope ~1 nm (0.001 μm) in tissue section.
• scanning electron microscope ~2 nm on a biological sample
PAS
Enzyme Histochemistry
• uses enzymes in specific cell organelles to localize those components specifically
• reaction of enzyme + an added substrate = metal salt (colored or dark for light microscopy; electron dense product for EM.
• The periodic acid-Schiff (PAS) or Feulgen reaction is a common example of histo- chemistry. This stains glycogen & various carbohydrate-containing molecules (e.g., glycoproteins on cell surfaces or in ECM).
what is the resolution of the human eye
0.1 mm
