Tools of Histology Flashcards
Histology
Microscopic study of cells, tissues, organs
Cell
Smallest Living Unit
Tissue
Organized group of cells and their products that function in a collective manner
Organ
Structure composed of 2 or more tissue types that performs and specific function
Organ system
2 or more organs that perform a common function
- Micrometer (microns)
- Nanometer
Resolution (definition)
The smallest distance at which 2 points can be distinguished as seperate entities
- Smaller resolution = stronger
Resolution of
- human eye
- light microscope
- transmission electron microscope
- scanning electron microscope
Resolution of human eye: 100 um
Resolution of light microscope: 0.2 um (200 nm)
Resolution of transmission electron microscope: 3 nm
Resolution of scanning electron microscope = 1 nm
Ultrastructure
Ultrastructure = cellular structures that can only been seen using an electron microscope
Light microscope
Light travels through a thin section of tissue.
The ocular lens in the eyepiece (10x) and the objective lenses (4x,10x, 40x, 100x) magnify the image.
Low = 4X
Medium = 10 X
High = 40 X
Oil immersion = 100 x
Virtual microscope (5 steps)
1) Slide collections
2) Slide Scanner
3) Servers
4) Virtual microscope software
5) Histology lab and mobile devices
Routine slife preparation for light microscopy (7)
1) Fixation
2) Dehydration
3) Clearing
4) Infiltratoin
5) Embedding
6) Sectioning
7) Mounting on slide, removal of paraffin,
hydration, staining.
Fixation
① Fixation = Preserve with formalin (so does not decompose)
- cross-linking of proteins and inactivation of enzymes (Formaldehyde polymerizes so we add something to it to to stop it. Now called formalin)
- Macromolecules (like glycogen) get taken out of tissue
Dehydration with alcohol
② Dehydration = use of alcohol to remove all water
- To later embed in block of wax (so we can cut it into thin slices) we need to remove water with alcohol because water does not mix well with wax
Clearing
③ Clearing = use of organic solvent (e.g. xylol) to remove alcohol. The tissue is now saturated with organic solvent, which can dissolve paraffin used in the next step
- called “clearing” because this step renders the tissue transparent
- Will also wash out lipids with clearing agent
Infiltration
④ Infiltration = melted paraffin penetrates the tissue
Embedding
⑤ Embedding = paraffin placed in a mold and hardens. Block is trimmed
Sectioning
⑥ Sectioning = tissue is sectioned into thin slices (5-10um)
Mounting on slide, removal of paraffin,
hydration, staining.
⑦ Tissue slice is mounted on a glass slide. Paraffin is removed and the tissue is rehydrated. Stain is applied to color the clear cellular components so that they may be seen.
Artefact
Artefact = structural abnormality not present in the living tissue as a result of the preparation process
- Something you see under microscopy that is not there in real life
- tear, fold, air bubble
Planes of cut (3)
Cross section = the specimen was sectioned along its short axis (cut in top/bottom halves)
Longitudinal section = the specimen was sectioned along its long axis (cut in left/right halves)
Oblique section = the specimen was sectioned on an angle
Hematoxylin:
- behaves like
- stains what (and why)
- color change
Hematoxylin behaves like a base
Stains nucleic acids
- charged in solution, attracted to - charge in cell (- phosphate in nucleic acids)
Anything that turns blue = contains nucleic acids
Will stain things that are basophilic
(Base = Blue = Basophilic stains)
Eosin
- what does it stain (and why)
- what does it behave like
- what color change
- Eosin stains proteins
- Acts like an acid
- charge = acidic in solution, attracted to the + structures in cells like proteins (with ionized amino groups +)
- Will stain things that are acidophilic
- Stains things pink
Basophilic vs. Acidophilic
Basophilic: a structure that attracts basic dyes and is therefore stained by basic dyes (stained by hematoxylin)
Acidophilic: a structure that attracts acidic dyes and is therefore stained by acidic dyes (stained by eosin)
Using H&E together
- Often the two stains are used one after the other (hematoxylin always first) on the same tissue.
- hematoxylin can look purple in the final specimen
Metachromatic
The property of staining a different color than the dye.
- Occurs when a basic dye reacts with a tissue component.
- Example: Purple cell shown is metachromatic because it shifted the dye’s color from blue to purple
Periodic acid-Schiff (PAS) stain
- what is it used for?
- color changes?
PAS used for carbohydrates
In routine slide preparation with H&E: you lose glycogen during fixation, so will look like white holes where glycogen used to be
- can use other slide prep techniques to keep sugars and then use PAS to stain
- PAS stains lipids pink
Basement membrane
- function
- stained in PAS with hematoxylin vs. H&E
The basement membrane provides an attachment site for certain cell types.
- white in H&E
- pink in PAS
Osmium tetroxide
- what is it used for
- what color does it stain
- used for lipids
- in routine slide preparation with H&E, lipids get washed out during clearing, so can prep cells in other ways that leave lipids in and then use Osmium tetroxide
- will stain lipids black
- lipids
- proteins
- nucleic acids
- carbohydrates
B= answer
(C = longitudinal because long axis)
