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
16
Q
histochemistry
A
- deals with the identification of specific components of the cells and tissue by microscopic methods (LM): type of fibers (diff darkness)
- cytochemistry: location of enzyme in fiber(EM)
17
Q
immunochemistry
A
- immunological techniwues employ antibodies that react with a specific molecule, an antigen
- can bind specific molecules within tissues, cells, sub-cellular structures
17
Q
direct immunofluorescence
A
- a fluorochrome-labelled primary antibody is chosen on the basis of the antigen of interest
- low sensitivity
- 1 step
1. treated with labelled antibody
2. unbound washed away
3. UV: fluorescence where antigen is located
- 1 step
18
Q
indirect immunofluorescence
A
- better than direct
- higher sensitivity
- does not need many individual labelled Abs
1. primary antibody directed against antigen (rabbit)
2. secondary marker-coupled antibodies directed against primary
19
Q
artifacts
A
- can be defined as an artificial structure or tissue alteration on a prepared microscopic slide as a result of an extraneous factor
- preparative techniques are often harsh and can traumatize and change tissue: not real things seen
- impossible to eliminate but should be kept to a minimum and be compatible with the specific technique used
20
Q
other optical microscopy techniques
A
- bright field microscopy
- phase contrast microscopy
- nomarski differential-interference-contrast microscopy
- dark field microscopy
21
Q
phase contrast microscopy
A
- cells in culture are almost invisible when examined by bright field microscopy
- phase contrast allows to appreciate several details
22
Q
dark field microscopy
A
- light comes from the side and the specimen is observed against a dark background
23
Q
fluorescent microscopy
A
- based on use of FL molecules: absorbing light at one wavelength and emitting it at another
- mols are made to glow on dark background increasing sensitivity of detection
- exciting and emission spectra show the change in absorbance of a sample as a function of the wavelength of incident light
24
confocal microscope
- allows imaging complex 3D objects by focusing on a chosen plane in a thick specimen
- rejecting light coming from out-of-focus planes above and below
- optical sections can be recombined to reconstruct 3D image
- can be used to differentiate wether a diffused staining is membrane or pan-cytoplasm staining
25
one angstrom microscope
- TEM to image lithium atoms
26
cryogenic electron microscopy
- apoferritin is a protein in intestinal mucosa membrane
- able to bind and store iron by combining with ferric hydroxide-phosphate to form ferritin
- cooled to cryogenic temperatures
27
preparation for EM (kinda similar)
- fixation in glutaraldehyde
- postfixation in OsO4
- dehydration
- inclusion in acrylic plastics or epoxy resins
- ultramicrotome sectioning with diamond knives (30-60nm)
- increasing contrast with heavy metal ions
28
TEM
- black and white images
- different densities
- very high resolution
- seeing organelles
29
SEM
- lower resolution than TEM
- higher depth of field
- allows 3D
- allows surface visualisation
30
STEM
TEM + SEM
31
atomic force microscopy (AFM)
- non-optical
- works like a fingertip
- exploring the surface topography at molecular and atomic resolution
- force measurement, topographic imaging, manipulation
32
GFP expression
- fluorescent markers of gene expression and for determination of protein localization and motility in living cells
- GFP can attach to and mark another protein with fluorescence
33
tissue and cell culture
- growth and maintenance of tissues or cells under controlled conditions for experimental purposes
34
molecular biology techniques
- PCR
- DNA sequencing
- gene expression analysis
- studying molecular mechanisms in histology and embryology
