Lecture 2: Tools of Histology Flashcards
Histology:
Histology: microscopic study of cells, tissues, organs
Cell:
Cell: smallest Living Unit
Tissue:
Tissue: organized group of cells and their products that function in a collective manner
Organ:
Organ: structure composed of 2 or more tissue types that performs and specific function
Organ system:
Organ system: 2 or more organs that perform a common function
- Decimeter
- Centimeter
- Millimeter
- 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:
- ____ travels through ____
- The ____ lens in the ____ (_x) and the ____ lenses (_x, _x, _x, _x) magnify the image
- Low = _x
- Medium = _x
- High = _x
- Oil immersion = _x
- 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 = 10x
- High = 40x
- Oil immersion = 100x
Virtual microscope (5 steps)
1) Slide collections
2) Slide Scanner
3) Servers
4) Virtual microscope software
5) Histology lab and mobile devices
Routine slide 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
4 chemical building blocks of tissues
1) nucleic acids
2) proteins
3) carbohydrates
4) lipids
* Tissue becomes colorless during “clearing”, so we need to color it. You can color (stain) any of the basic macromolecules*
Hematoxylin:
- behaves like
- has what charge
- stains what (generally)
- most commonly stains what (and why)
- causes what color change
Hematoxylin:
- behaves like a base
- has positive charge
- stains things that are basophilic
- stains nucleic acids (most common example)
- why: + charged in solution, attracted to - charge in cell (- phosphate in nucleic acids)
- So, anything that turns blue = contains nucleic acids
- stains nucleic acids (most common example)
(Base = Blue = Basophilic stains)
Eosin:
- acts like:
- what charge:
- what does it stain (generally):
- most commonly stains what:
- why?
- most commonly stains what:
- what color change:
Eosin:
- Acts like an acid
- has a negative charge
- Stains things that are acidophilic
- Stains proteins (most common example)
- why: - charge = acidic in solution, attracted to the + structures in cells like proteins (with ionized amino groups +)
- Stains proteins (most common example)
- 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:
- which is used first?
- what colors?
- 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 (eosin is pink)
Metachromatic
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 does it stain?
- why is it used?
- color changes?
Periodic acid-Schiff (PAS): stain is 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
- if on a low carb diet, you want to PAS on the sugars
- can use other slide prep techniques to keep the sugars in the cell and then use PAS to stain
- PAS stains sugars magenta
Basement membrane:
- function
- what stain is best for basement membranes and why
- what does it look like when stained in PAS with hematoxylin vs. H&E
The basement membrane provides an attachment site for certain cell types.
- contains sugars so it’s best stained with PAS
- when stained in PAS with hematoxylin = pink
- when stain in H&E = white (hard to see)
Osmium tetroxide:
- what is it used to stain
- common example
- why is it used to stain this
- what color does it stain
Osmium tetroxide:
- used to stain lipids
- example: used to stain myelin sheath surrounding axons (because myelin sheath contains 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 to stain
- will stain lipids black
- Glycogen lost during:
- Lipids lost during:
- Glycogen lost during fixation
- Lipids lost during clearing
What stains each of the following macromolecules?
- lipids
- proteins
- nucleic acids
- carbohydrates
What stains each of the following macromolecules?
- lipids = Osmium Tetroxide (black)
- proteins = Eosin (pink)
- nucleic acids = Hematoxylin (blue)
- carbohydrates = PAS (magenta)
which macromolecule is lost during routine slide preparation?
A) Lipids
B) Nucleic Acids
C) Proteins
D) Carbohydrates
which macromolecule is lost during routine slide preparation?
A) Lipids
- The organic solvent used during clearing will dissolve and wash out the lipids
-
B = Oblique angle (correct answer)
- This plane lies neither along the short nor the long axis of the tube. It is at an oblique angle.
- A = a cross-section because the plane is along the short axis of the tube
- C = longitudinal cut because the plane is along the long axis of the tube.
