lab Flashcards
1
Q
aims of tissue analysis
A
diagnostics and basic knowledge
2
Q
morphological methods
A
- light microscopy: used for assessing cell and tissue morphology
- S/TEM: visualise fine details at high resolution
- atomic force microscopy (AFM): examining surface topography and mechanical properties at nanoscale level
3
Q
other methods
A
- radiosotopoc methods
- biochemical techniques
- cell fractionation methods
- immunological techniques
- tissue and cell culture
- molecular biology techniques
4
Q
what is magnification
A
- obtained through a microscope allows to observe object we do not see w bare eyes
- magnification allows to calculate size of a given object
5
Q
resolution
A
- shortest distance between two points on a specimen that can still be distinguished by the observer or camera system as separate entities
- quality and cost of an optical instrument depends rather on resolution than magnification
6
Q
sizes
A
plant cell - animal cell - bacterium- virus - ribosome - globular protein - small molecule - atom
7
Q
histological technique
FDESMSE
A
- preparation of tissues and examination with a microscope
1. fix tissue (!% paraformaldehyde + buffer)
2. dehydrate tissue (alcohol series followed by toluene)
3. embed tissue in hard medium (wax)
4. section embedded tissue on a microtome
5. mount sections on a supportive structure (slide) that can be placed on a stage
6. stain tissue (hematoxylin-eosin
7. examine tissue with microscope
8
Q
scope of fixation
A
- preserve structure: chemical and morphological
- fixed material is dead
- fixatives: formaldehyde, acetic acid, ethanol, glutaraldehyde, methanol, picric acid
- by denaturing proteins, fixatives prevent lysosomal enzymes to cause autolysis (self- digestion
9
Q
how and why dehydrate fixed tissue?
A
- removal of water (natural solvent) followed by its substitution with a non-polar medium (toluene) that is miscible with the embedding medium
- 1: ethanol, methanol, acetone (miscible with second solvent)
- 2: sylene, toluene (miscible w medium)
- to allow for complete penetration of the tissue by the paraffin wax.
10
Q
embedding
A
- tissue needs to be solid enough to withstand sectioning process
- want components of tissue in natural position
- wax, plastic immobilises structural components
11
Q
sectioning
A
- allows light to pass through tissue making it visible
- allows to see internal structure
- some cases tissues are stained without sectioning e.g smears
- blood or bone marrow smear
- light microscopy: few micrometer thick
- special instrument to obtain these sections: microtome
- a section is 2D, reality is 3D
12
Q
frozen sections
A
- can be obtained in a few minutes
- freezing by spraying with compressed CO2
- sectioning with a cryostat (refrigerated microtome)
- staining
- a routinely prepared section is then made to confirm diagnosis and permanent record
13
Q
staining
A
- to examine tissue with a microscope you need natural contrast among components
- does not cover structures with pigment
- specific reactions between a organelle or molecule with the stain
- depends on cell structure and functional status
14
Q
staining affinities
A
- acid component of the cell (nucleic acids, nucleus, ribosome, RER) are basophilic
- readily stained by basic dyes like hematoxin
- basic components (cytoplasm) are acidophilic
- readily stained by acid dyes like eosin
- different staining solutions have preferential and not absolute affinities for some cellular components: H and E used together
- lipids stain with soluble dyes like oil red or sudan black
- the feulgen reaction is used to stain DNA
- glycoproteins and polysaccharides are stained by the PAS (periodic acid schiff) reaction
15
Q
threechromic staining
A
- highlights extracellular matrix
- uses two or more acid dyes in conjunction with a polyacid
