Examining Cells Flashcards
Define tisssue
Woven
Name four tissue classifications
Muscle, nerves, epithelial, connective
How does epithelial tissue communicate?
Through junctions at their lateral and basal surfaces
Four properties of epithelial tissue
- on the edge of other tissue or surrounding them
- form glands when in clusters
- polarised when at surfaces
- held together by anchoring proteins
What type of membrane does epithelial tissue have on the basal?
Basement
What are connective tissues made from?
Cells, extra cellular proteins, glycoproteins and gel
Name three mains cells in connective tissue
Fibroblasts, chondrocytes, osteocytes
Main products of connective tissue
- fibres
- wax and gel materials
What are the three muscle cells muscle tissue is made from
- skeletal
- cardiac
- smooth
Three functions of muscle tissue
- movement
- stability
- movement of tissue
(Made function to contract)
Minor functions of hormones
- natriuretic factors (for the heart)
- myostatin (regulates the heart muscle)
What do nerve cells congregate into?
Nerve fibres
What is the main role of nerve tissue?
Communication
What is the standard measurement for a cell
Um
What is used to measure a cell?
A measurement graticule
What is the limit of resolution
The smallest distance by which two objects can be separated and still be distinguishable as two separate objects
Tissue procurement methods
- endometrial sample (upper uterus)
- venepuncture (intravenously)
- bone marrow aspiration
Why do you use a buffered formalin solution during fixation?
To prevent the cell from swelling
In basic terms what is fixation?
To preserve cells and tissue components
What is the step after fixation?
Washing and dehydration
What is the most common fixation chemical and why is it isotonic with the intracellular fluid?
Formalin
Allows better penetration of the formaldehyde
Name two properties of the sample during fixation and why
Small - fixation needs to be rapid
24-48 hours - no longer or they will shrink
X
Why use paraffin wax?
It is fluid when heated but hardens when solid
What happens in washing?
The tissue is washed in a series of alcohol solutions
Then in solvents
What is the key characteristic of the solvents
Miscible with both alcohol and hot paraffin wax (completely dissolved in each other)
What happens to tissue after being washed with the solvent?
It is immersed in hot paraffin wax overnight
Then placed in a mold
More hot wax over to completely cover
Set aside to harden fully (can now be stored indefinitely)
What is used to cut the hardened specimen?
Microtome
What blade can a microtome use?
Steel
Glass
Diamond knife
Where is the cut specimen then put and why?
A warm water bath (using a fine paintbrush)
The surface tension causes the section to stretch slightly - cutting artefacts are reduced
What can you cover the microscope slid with and why?
A sticky substance
Specimen adheres when dry
What must you do to the paraffin section before you can look at it through a microscope?
Dissolve
Rehydrate
With different percentage of alcohols
Stain it
What are common stains used?
Haematoxylin
Eosin
How do you stain using haematoxylin?
And what does it bind to?
Immerse in an aqueous solution of haematoxylin
It is basic, so will bind to acidic structures (DNA, RNA)
How do you then stain with eosin?
And what does it bind to?
Wash it in water and alcohol - eosin is more soluble in alcohol
Immerse in eosin
Binds to basic structures because is acidic (cytoplasm, collagen - intra and extra cellular proteins)
Wash with more alcohol because mounting
How do you mount a specimen?
On a non-aqueous mounting medium
Apply a cover slip
Wait for the solvents to evaporate
What do immunohistochemistry and immunofluorescence use?
Labelled antibodies
Targeting specific antigens
What are the antibodies in immunofluorescence bound to?
Dyes
What can you add to the after adding a primary antibody?
What is this called?
Enzyzme activated secondary antibody - precipitate a coloured product at the site of interaction
Indirect immunohistochemistry (even more sensitive)
Why are antigen retrieval methods used?
Protein structure may have been altered by tissue processing procedures
Describe antigen retrieval
Heated - partial digestion
In the presence of a weak acid
Why does antigen retrieval work on less robust tissue samples?
So what would you use?
They cannot be heated
A frozen section
Why can more components be seen using an electron microscope?
Higher resolution
What is the limit resolution mean?
Minimum distance at which two objects can be distinguished
Five comparisons of light and electron microscopes
L - colour E - black and white
L - cheaper and easier preparation
L - living, moving
L - lower magnification
L - lower resolution
What type of microscope is confocal?
Scanning
How does a confocal microscope work?
- Laser excites dye - electrons raised to higher energy level
- Electrons relax back to ground state
- Light with a higher wavelength emitted
- Emitted light sent to mirrors and a pinhole screens to CMOS detector
Why can a confocal microscope give a 3D image?
Multiple images taken
Computer places them in order
Creating a Z stack
What are confocal microscopes used for?
Evaluating eye diseases
What are the five steps on preparing live cells?
- Cutting and dicing
- Collagenase and DNAse (enzymes used to ‘break’ the cells apart)
- Centrifugation
- Put in appropriate growth medium
- Culture cells
What sort of microscope is used to look at live cells?
Phase contrast
Three advantages of cell culture
- control over physical environment
- homogeneity (one type)
- reduced need for animals
Three disadvantages of cell cultures
- hard to maintain (sterile)
- small amount at a high cost
- dedifferentiation
What is dark field?
Illuminate sample of live cells
Light not collected by objective lens
- black background with bright objects
What type of electron microscope gives a 3D image?
Scanning
Why can you see an oocyte?
Bigger than the resolving power of the eye
What is H&E?
Haematoxylin
Eosin
Smallest organelle to see with a light microscope?
Nucleolus
Why is hard to look at fat under the microscope?
Fat dissolves in the alcohol during the preparation method
When using H&E
What is an erythrocyte?
Red blood cell
