Microscopy And Histology Flashcards
What is cytology
Study of cells
Define histology
Study of tissues
What are the two major types of microscopes?
- Light microscopes
- Electron microscopes
The ability to make small objects seem larger
Magnification
Discrimination between two objects that are close together
Resolution
The ability to perceive the presence of an object
Detection
Magnification without revealing more detail (every optical system has a finite limit of resolution/resolving power)
Empty magnification
The type of tissue preparation that takes several days but has the highest resolution
Paraffin-Embedded Sections
In Paraffin Embedded tissue preparation, what is the water in tissue replaced with?
Paraffin wax
What are the 6 steps of Paraffin Embedded tissue preparation
- Fixation: using formaldehyde
- Dehydration: using alcohols
- Clearing: using xylene
- Infiltration and embedding: using paraffin wax
- Sectioning: use microtome
- Staining
What type of tissue preparation is often used during surgery?
Frozen sections
What type of tissue preparation only takes a few minutes but has lower resolution
Frozen sections
What are the 4 steps of frozen section tissue preparation?
- Fixation: using acetone
- Embedding and freezing: using glycols and resin (removes water)
- Sectioning: using cryostat
- Staining
If tissues dye and are not fixed fast enough, what artifact can occur?
Post mortem degeneration, where cells are pulled away from connective tissue leaving open spaces
When does shrinkage occur?
When a tissue sample is not fixed fast enough
Which tissue preparation uses formalin for fixation?
Paraffin-embedded Sections
What is required to visualize colorless tissue and cells under a microscope?
Staining
What is the most common type of tissue staining?
Hematoxylin and Eosin (H&E)
What charge and color is Hematoxylin?
What does it bind to?
A blue dye with a positive charge. Binds to RNA and DNA (negative charged phosphate group). Binds to sulfate groups
Define basophilic
Tissue components that stains with basic dyes. Example: DNA and RNA
What color will the Nucleus stain using H&E
Blue
What components in tissue are basophilic?
Nucleus (DNA) and basal ER (RNA)
What color and charge is Eosin dye?
Eosin is a red, negatively charged dye
Is Eosin basic or acidic?
Acidic
Define acidophilic
Tissue components that stain with acidic dyes
What color would tissue components containing a NH3+ stain when using H&E
Red
What color would proteins stain when using H&E
Red (NH3+ group)
What other components stain red in tissue?
Proteins, cytosol, and proteins compacted into different granules
Stain with hematoxylin in…
Water
Stain with eosin in…
Alcohol
Does a Pancreas stained with H&E have polarity, how can you tell?
Red top half, contains a lot of proteins. Blue bottom half contains nucleus and the rough ER
What does a brown color in stained skin cells represent?
Melanin
Would hemoglobin show a nucleus in the center when stained?
No, hemoglobin is a mature RBC and do not have a nucleus
Would blood vessels stain red or blue?
Red because RBC’s do not have a nucleus
What technique is used when staining connective tissue?
Trichrome stain. Blue color shows cartilage, red shows muscle
What staining technique is used for neurons?
Golgi (silver) stain
What is the Feulgen reaction?
Treat tissue with HCl acid, purine base on DNA is cleaved, forming a aldehyde group which reacts with Schiff’s reagent
What technique is used to stain Nucleic Acid?
Feulgen
What is Periodic Acid Schiff (PAS) stain specific for?
Sugars
What happens when glucose in a tissue sample is treated with periodic acid (HIO4)?
An aldehyde group is formed, which can react with schiff’s reagent
What color is produced with an aldehyde group reacts with Schiffs reagent?
Red or magenta
What staining technique would you use if you wanted to quantify the amount of sugar in the liver?
Periodic Acid Schiff (PAS) stain
What technique would you use to visualize the basement membrane and brush border of kidney tubules?
Periodic Acid Schiff (PAS)
Which staining technique is specific for fats?
Oil red O and Sudan Black
Can you use paraffin embedded sections to visualize fat?
No, you actually remove fat from tissue using xylene when using the paraffin embedded tissue preparation
Why do you need to use frozen sections to stain fats?
Fats are soluble in alcohol and xylene (paraffin embedded tissue). staining fat must be done in unfixed cells and tissue
Structures that stain with eosin are called…
Acidophilic
What is Immunocytochemistry/immunohistochemistry?
Antibody staining
The antibody must be tagged. You can use…
- Enzymes
Or - Fluorescent molecules
Or - Colloidal gold
Immunohistochemistry (IHC), uses what to tag antibodies?
Enzymes
Immunoelectron microscopy
Antibodies are tagged with colloidal gold and viewed with an electron microscope
Immunofluorescence
Antibodies are tagged with fluorescent molecules and viewed with a fluorescent microscope
Immunoperoxidase stain (type of IHC)
Produces a brownish/black precipitate that is viewed on the slides.
Example: incubate pituitary cells with an antibody to growth hormone, and then a second antibody with the peroxidase enzyme on it. Peroxidase enzyme will detect the growth hormone, than add the DAB substrate, the peroxidase reacts with the peroxide, produces the brown product
Which microscope has the higher resolution? Light or electron?
Electron microscope
A transmission electron microscope is used to view…
Uses electrons that pass through tissue, identify objects that are inside a cell
What would you use to visualize a 3D image of the surface of cells?
Scanning electron microscope (SEM), electrons are scattered
Myelinated nerve axons must be viewed using…
Transmission electron microscope (TEM), inside a cell
RBC in a fibrin clot must be viewed using a…
Scanning electron microscope (SEM)
Good tip to identify Osteoclasts under the microscope?
Multiple nuclei
What is the size of a Paraffin section diameter ?
Usually 5 to 7 micrometers
RBC are found in most tissue, how big are RBC?
About 7 micrometers
In paraffin embedded tissue preparation, which steps comes before dehydration with alcohols?
Fixing in a chemical such as formaldehyde
Which microscope has the lowest resolution: fluorescent, scanning electron, or transmission electron?
Fluorescent
In an H&E staining section, which dye stains basophilic structures and what feature of a cell would it stain?
Hematoxylin, it would stain the nucleus, RER, and mitochondria
Why is mitochondria basophilic?
It contains DNA, which is negatively charged and binds to the positively charged hematoxylin dye
Cytosol and secretory vesicles would stain [ ] due to positively charged proteins
Red, Eosin
